Comparison of media for detection of fungi on spacecraft

NASA Technical Reports Server (NTRS)

Herring, C. M.; Brandsberg, J. W.; Oxborrow, G. S.; Puleo, J. R.

1974-01-01

Five media, including Trypticase soy agar (TSA; BBL) pour plates, spread plates of TSA, Mycophil agar with chloromycetin, Mycophil agar with chloromycetin and Actidione, and cornmeal agar with chloromycetin were quantitatively and qualitatively compared for the detection of fungi on spacecraft. Cornmeal agar with chloromycetin yielded the highest number of fungal colonies, although not always significantly higher than Mycophil agar with chloromycetin or TSA spread plates. Cornmeal agar with chloromycetin also gave the best qualitative representation of fungi on the spacecraft, recovering 68% of the genera found from all media. This medium yielded 10 times the number of fungal colonies and 3 times the number of genera found on TSA pour plates as currently used for spacecraft assay.

Comparison of media for detection of fungi on spacecraft.

PubMed

Herring, C M; Brandsberg, J W; Oxborrow, G S; Puleo, J R

1974-03-01

Five media, including Trypticase soy agar (TSA; BBL) pour plates, spread plates of TSA, Mycophil agar with chloromycetin, Mycophil agar with chloromycetin and Actidione, and cornmeal agar with chloromycetin were quantitatively and qualitatively compared for the detection of fungi on spacecraft. Cornmeal agar with chloromycetin yielded the highest number of fungal colonies, although not always significantly higher than Mycophil agar with chloromycetin or TSA spread plates. Cornmeal agar with chloromycetin also gave the best qualitative representation of fungi on the spacecraft, recovering 68% of the genera found from all media. This medium yielded 10 times the number of fungal colonies and 3 times the number of genera found on TSA pour plates as currently used for spacecraft assay.

Screening for Indian isolates of egg-parasitic fungi for use in biological control of fascioliasis and amphistomiasis in ruminant livestock.

PubMed

De, S; Sanyal, P K; Sarkar, A K; Patel, N K; Pal, S; Mandal, S C

2008-09-01

Wild isolates of the egg-parasitic fungi Paecilomyces lilacinus and Verticillium chlamydosporium, obtained from the organic environment of Durg, Chhattisgarh, India, were subjected to screening for in vitro growth using different media types, range of incubation temperature and pH, and their predatory activity to the eggs of Fasciola gigantica and Gigantocotyle explanatum. Maximum growth of P. lilacinus was obtained in corn-meal agar compared to any other media types. The preferred medium for growth of V. chlamydosporium was corn-meal agar, followed by potato-dextrose agar. After initial growth for 16 h of incubation, no growth was observed in water agar for both the fungi. Six different temperatures--4 degrees C, 10 degrees C, 18 degrees C, 26 degrees C, 34 degrees C and 40 degrees C--were used to observe growth profiles of the fungi in corn-meal agar medium. While no and very little growth of P. lilacinus and V. chlamydosporium was observed at 4 degrees C and 10 degrees C, respectively, growth profiles of both the fungi were optimal at 26-40 degrees C. A range of pH (pH 4-8) supported growth of both P. lilacinus and V. chlamydosporium. Full-grown plates of the fungi baited with viable eggs of F. gigantica and G. explanatum revealed that V. chlamydosporium was more vigorous in its egg-parasitic ability compared to P. lilacinus. Distortion of the eggs started on day 2-3 of egg baiting in culture plates of V. chlamydosporium, with complete distortion by day 7. On the contrary, P. lilacinus exhibited very limited egg-parasitic ability and some of the baited eggs even showed development of miracidia.

Beyond Agar: Gel Substrates with Improved Optical Clarity and Drug Efficiency and Reduced Autofluorescence for Microbial Growth Experiments

PubMed Central

McElfresh, Cameron; Wong, Lily R.

2015-01-01

Agar, a seaweed extract, has been the standard support matrix for microbial experiments for over a century. Recent developments in high-throughput genetic screens have created a need to reevaluate the suitability of agar for use as colony support, as modern robotic printing systems now routinely spot thousands of colonies within the area of a single microtiter plate. Identifying optimal biophysical, biochemical, and biological properties of the gel support matrix in these extreme experimental conditions is instrumental to achieving the best possible reproducibility and sensitivity. Here we systematically evaluate a range of gelling agents by using the yeast Saccharomyces cerevisiae as a model microbe. We find that carrageenan and Phytagel have superior optical clarity and reduced autofluorescence, crucial for high-resolution imaging and fluorescent reporter screens. Nutrient choice and use of refined Noble agar or pure agarose reduce the effective dose of numerous selective drugs by >50%, potentially enabling large cost savings in genetic screens. Using thousands of mutant yeast strains to compare colony growth between substrates, we found no evidence of significant growth or nutrient biases between gel substrates, indicating that researchers could freely pick and choose the optimal gel for their respective application and experimental condition. PMID:26070672

Beyond Agar: Gel Substrates with Improved Optical Clarity and Drug Efficiency and Reduced Autofluorescence for Microbial Growth Experiments.

PubMed

Jaeger, Philipp A; McElfresh, Cameron; Wong, Lily R; Ideker, Trey

2015-08-15

Agar, a seaweed extract, has been the standard support matrix for microbial experiments for over a century. Recent developments in high-throughput genetic screens have created a need to reevaluate the suitability of agar for use as colony support, as modern robotic printing systems now routinely spot thousands of colonies within the area of a single microtiter plate. Identifying optimal biophysical, biochemical, and biological properties of the gel support matrix in these extreme experimental conditions is instrumental to achieving the best possible reproducibility and sensitivity. Here we systematically evaluate a range of gelling agents by using the yeast Saccharomyces cerevisiae as a model microbe. We find that carrageenan and Phytagel have superior optical clarity and reduced autofluorescence, crucial for high-resolution imaging and fluorescent reporter screens. Nutrient choice and use of refined Noble agar or pure agarose reduce the effective dose of numerous selective drugs by >50%, potentially enabling large cost savings in genetic screens. Using thousands of mutant yeast strains to compare colony growth between substrates, we found no evidence of significant growth or nutrient biases between gel substrates, indicating that researchers could freely pick and choose the optimal gel for their respective application and experimental condition. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Comparison of Media for Detection of Fungi on Spacecraft

PubMed Central

Herring, C. M.; Brandsberg, J. W.; Oxborrow, G. S.; Puleo, J. R.

1974-01-01

Five media, including Trypticase soy agar (TSA; BBL) pour plates, spread plates of TSA, Mycophil agar with chloromycetin, Mycophil agar with chloromycetin and Actidione, and cornmeal agar with chloromycetin were quantitatively and qualitatively compared for the detection of fungi on spacecraft. Cornmeal agar with chloromycetin yielded the highest number of fungal colonies, although not always significantly higher than Mycophil agar with chloromycetin or TSA spread plates. Cornmeal agar with chloromycetin also gave the best qualitative representation of fungi on the spacecraft, recovering 68% of the genera found from all media. This medium yielded 10 times the number of fungal colonies and 3 times the number of genera found on TSA pour plates as currently used for spacecraft assay. PMID:4151044

Application of Fourier Transform Infrared (FTIR) Spectroscopy for Rapid Detection of Fumonisin B2 in Raisins.

PubMed

Heperkan, Dilek; Gökmen, Ece

2016-07-01

The aim of this study was to investigate the potential use of FTIR spectroscopy as a rapid screening method to detect fumonisin produced by Aspergillus niger. A. niger spore suspensions isolated from raisins were inoculated in Petri dishes prepared with sultana raisin or black raisin extracts containing agar and malt extract agar (MEA). After 9 days of incubation at 25°C, fumonisin B2 (FB2) production on each agar plate was determined by subjecting the agar plugs to IR spectroscopy. The presence of amino group (at 1636-1639 cm(-1)) was especially indicative of fumonisin production in MEA and the raisin extracts containing agar. The results were confirmed by HPLC analysis of the agar sample extracts after immunoaffinity column cleanup. It was determined that A. niger produced more FB2 in sultana raisins than in MEA, with no FB2 being produced in black raisin extract agar. This study demonstrated that proper sample preparation procedure followed by FTIR analysis is a useful technique for identifying toxigenic molds and their mycotoxin production in agricultural commodities.

Use of benzimidazole agar plates to assess fall armyworm (Lepidoptera: Noctuidae) feeding on excised maize and sorghum leaves

USDA-ARS?s Scientific Manuscript database

The fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is an economically significant pest of sorghum and maize. To screen sorghum and maize germplasm for resistance to fall armyworm feeding, field, greenhouse, or lab bioassays are often utilized individually or in combinatio...

An interlaboratory study on efficient detection of Shiga toxin-producing Escherichia coli O26, O103, O111, O121, O145, and O157 in food using real-time PCR assay and chromogenic agar.

PubMed

Hara-Kudo, Yukiko; Konishi, Noriko; Ohtsuka, Kayoko; Iwabuchi, Kaori; Kikuchi, Rie; Isobe, Junko; Yamazaki, Takumiko; Suzuki, Fumie; Nagai, Yuhki; Yamada, Hiroko; Tanouchi, Atsuko; Mori, Tetsuya; Nakagawa, Hiroshi; Ueda, Yasufumi; Terajima, Jun

2016-08-02

To establish an efficient detection method for Shiga toxin (Stx)-producing Escherichia coli (STEC) O26, O103, O111, O121, O145, and O157 in food, an interlaboratory study using all the serogroups of detection targets was firstly conducted. We employed a series of tests including enrichment, real-time PCR assays, and concentration by immunomagnetic separation, followed by plating onto selective agar media (IMS-plating methods). This study was particularly focused on the efficiencies of real-time PCR assays in detecting stx and O-antigen genes of the six serogroups and of IMS-plating methods onto selective agar media including chromogenic agar. Ground beef and radish sprouts samples were inoculated with the six STEC serogroups either at 4-6CFU/25g (low levels) or at 22-29CFU/25g (high levels). The sensitivity of stx detection in ground beef at both levels of inoculation with all six STEC serogroups was 100%. The sensitivity of stx detection was also 100% in radish sprouts at high levels of inoculation with all six STEC serogroups, and 66.7%-91.7% at low levels of inoculation. The sensitivity of detection of O-antigen genes was 100% in both ground beef and radish sprouts at high inoculation levels, while at low inoculation levels, it was 95.8%-100% in ground beef and 66.7%-91.7% in radish sprouts. The sensitivity of detection with IMS-plating was either the same or lower than those of the real-time PCR assays targeting stx and O-antigen genes. The relationship between the results of IMS-plating methods and Ct values of real-time PCR assays were firstly analyzed in detail. Ct values in most samples that tested negative in the IMS-plating method were higher than the maximum Ct values in samples that tested positive in the IMS-plating method. This study indicates that all six STEC serogroups in food contaminated with more than 29CFU/25g were detected by real-time PCR assays targeting stx and O-antigen genes and IMS-plating onto selective agar media. Therefore, screening of stx and O-antigen genes followed by isolation of STECs by IMS-plating methods may be an efficient method to detect the six STEC serogroups. Copyright © 2016 Elsevier B.V. All rights reserved.

The Resazurin-Agar Method - a Quick Test to Determine Water Quality

NASA Astrophysics Data System (ADS)

Huckfeldt, J.; Westphal, B.; Claußen, L.

2015-12-01

Rezasurin has been used as a smart tracer in stream ecosystems to indicate metabolic activity, specifically aerobic respiration by heterotrophic bacteria. Resazurin is a blue compound which is irreversibly reduced to the pink resorufin in the presence of aerobic bacteria. The degree and speed of colour change from blue to pink is a measure of the degree of oxygen consumption and thus an indirect indication of the concentration of aerobic bacteria in a given medium. A high concentration of bacteria in water indicates a bad water quality. In our work a method was developed using resazurin agar plates to find a quick and easy way for testing water quality and comparing concentrations of bacteria in freshwater and seawater samples. The theory was to concentrate bacteria from a defined volume of water sample onto polycarbonate filters (0.2 μm), which are then placed onto the resazurin agar plate. The presence of aerobic bacteria on the filter will reduce the resazurin in the agar and the compound changes its colour. First tests conducted with different dilutions of a pure culture of yoghurt bacteria showed promising results and confirmed the feasibility of the method. In a further assay, we used water samples from different water layers and different temperatures and were also able to observe differences in the concentration of bacteria, depending on these different environmental conditions.The assay was also successfully used with seawater samples, collected from 2 different stations at 3 different depths in the Baltic Sea (salinity=15). The discolouration of the plates showed good correlation with the oxygen concentrations in the water. The resazurin-agar plate method is economical and fast. Several samples could be investigated at the same time without sacrificing the reliability of the results. Thus it is a good pre-screening test for a quantitative evaluation of bacteria in a water sample.

Light‐scattering sensor for real‐time identification of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae colonies on solid agar plate

PubMed Central

Huff, Karleigh; Aroonnual, Amornrat; Littlejohn, Amy E. Fleishman; Rajwa, Bartek; Bae, Euiwon; Banada, Padmapriya P.; Patsekin, Valery; Hirleman, E. Daniel; Robinson, J. Paul; Richards, Gary P.; Bhunia, Arun K.

2012-01-01

Summary The three most common pathogenic species of Vibrio, Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus, are of major concerns due to increased incidence of water‐ and seafood‐related outbreaks and illness worldwide. Current methods are lengthy and require biochemical and molecular confirmation. A novel label‐free forward light‐scattering sensor was developed to detect and identify colonies of these three pathogens in real time in the presence of other vibrios in food or water samples. Vibrio colonies grown on agar plates were illuminated by a 635 nm laser beam and scatter‐image signatures were acquired using a CCD (charge‐coupled device) camera in an automated BARDOT (BActerial Rapid Detection using Optical light‐scattering Technology) system. Although a limited number of Vibrio species was tested, each produced a unique light‐scattering signature that is consistent from colony to colony. Subsequently a pattern recognition system analysing the collected light‐scatter information provided classification in 1−2 min with an accuracy of 99%. The light‐scattering signatures were unaffected by subjecting the bacteria to physiological stressors: osmotic imbalance, acid, heat and recovery from a viable but non‐culturable state. Furthermore, employing a standard sample enrichment in alkaline peptone water for 6 h followed by plating on selective thiosulphate citrate bile salts sucrose agar at 30°C for ∼ 12 h, the light‐scattering sensor successfully detected V. cholerae, V. parahaemolyticus and V. vulnificus present in oyster or water samples in 18 h even in the presence of other vibrios or other bacteria, indicating the suitability of the sensor as a powerful screening tool for pathogens on agar plates. PMID:22613192

One-week 96-well soft agar growth assay for cancer target validation.

PubMed

Ke, Ning; Albers, Aaron; Claassen, Gisela; Yu, De-hua; Chatterton, Jon E; Hu, Xiuyuan; Meyhack, Bernd; Wong-Staal, Flossie; Li, Qi-Xiang

2004-05-01

Soft agar growth, used to measure cell anchorage-independent proliferation potential, is one of the most important and most commonly used assays to detect cell transformation. However, the traditional soft agar assay is time-consuming, labor-intensive, and plagued with inconsistencies due to individual subjectivity. It does not, therefore, meet the increasing demands of today's oncology drug target screening or validation processes. This report describes an alternative 96-well soft agar growth assay that can function as a replacement for the traditional method and overcomes the aforementioned limitations. It offers the following advantages: a shortened assay duration (1 week instead of 4 weeks) that makes transient transfection or treatment possible; plate reader quantification of soft agar growth (measuring cloning efficiency and colony size); and a significant reduction in required labor. Higher throughput also makes it possible to process large numbers of samples and treatments simultaneously and in a much more efficient manner, while saving precious workspace and overall cost.

Comparison of Guizotia abyssinica seed extract (birdseed) agar with conventional media for selective identification of Cryptococcus neoformans in patients with acquired immunodeficiency syndrome.

PubMed Central

Denning, D W; Stevens, D A; Hamilton, J R

1990-01-01

Growth of Cryptococcus neoformans from the sputum of patients with acquired immunodeficiency syndrome may be obscured by oral contamination with Candida albicans on conventional media. We prospectively compared direct plating of sputum and urine onto birdseed agar and compared birdseed agar plating with plating onto Mycosel and Sabouraud dextrose agar cultures. Thirty-two sputum and three urine specimens were compared. C. neoformans was isolated from five specimens. In two specimens, one of sputum and one of urine, C. neoformans was detected only on the birdseed agar plate because of overgrowth on the conventional media by C. albicans. C. neoformans produced dark colonies on birdseed agar, unlike C. albicans, which produces white colonies. The use of birdseed agar as the primary culture medium for sputum and urine specimens from patients with acquired immunodeficiency syndrome increases sensitivity for C. neoformans. Images PMID:2254431

Studies on prevalence of Strongyloides infection in Holambra and Maceió, Brazil, by the agar plate faecal culture method.

PubMed

Kobayashi, J; Hasegawa, H; Soares, E C; Toma, H; Dacal, A R; Brito, M C; Yamanaka, A; Foli, A A; Sato, Y

1996-01-01

Prevalence of Strongyloides stercoralis infection in three areas of Brazil was surveyed by a recently developed faecal culture method (an agar plate culture). The Strongyloides infection was confirmed in 11.3% of 432 subjects examined. The diagnostic efficacy of the agar plate culture was as high as 93.9% compared to only 28.5% and 26.5% by the Harada-Mori filter paper culture and faecal concentration methods, when faecal samples were examined simultaneously by these three methods. Among the 49 positive samples, about 60% were confirmed to be positive only by the agar plate culture. These results indicate that the agar plate culture is a sensitive new tool for the correct diagnosis of chronic Strongyloides infection.

RELATIONSHIPS BETWEEN LEVELS OF HETEROTROPHIC BACTERIA AND WATER QUALITY PARAMETERS IN A DRINKING WATER DISTRIBUTION SYSTEM

EPA Science Inventory

Conventional plating methods were used to quantify heterotrophic bacteria from a drinking water distribution system. Three media, plate count agar (PCA), R2A agar and sheep blood agar (TSA-SB) were used to determine heterotrophic plate count (HPC) levels. Grab samples were collec...

Evaluation of the Premi Test and comparison with the One-Plate Test for the detection of antimicrobials in kidney.

PubMed

Cantwell, H; O'Keeffe, M

2006-02-01

The Premi Test, a test kit designed for the rapid screening of antimicrobial residues in meat, fish and eggs, was evaluated and compared with the (modified) One-Plate Test, an agar diffusion assay. The performance characteristics described for qualitative, screening methods in Commission Decision 2002/657/EC were used for the evaluation. The Premi Test was found to detect a range of antimicrobials to MRL levels in kidney fluid but to have poorer sensitivity for some antimicrobials such as tetracyclines, sulphonamides, flumequine and streptomycin. The test was found not to be sensitive for the banned antimicrobial chloramphenicol. The One-Plate Test was found to detect most tetracyclines and flumequine to MRL levels but to be less sensitive than the Premi Test for most of the other classes of antimicrobials. Neither test alone provides a comprehensive screening test for antimicrobial residues in kidney at MRL levels. However, the Premi Test is fast, easy to use and rugged and, in combination with other antimicrobial tests, may be used to provide a comprehensive screening system for antimicrobials in tissues.

Evaluation of Caenorhabditis elegans as an acute lethality and a neurotoxicity screening model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Williams, P.L.

1988-01-01

This investigation evaluated C. elegans as a lethality and neurotoxicity screening model. The lethality experiments were performed in both agar and an aquatic medium. The salts of 8 metals (Hg, Be, Al, Cu, Zn, Pb, Cd, and Sr) were used in the agar studies and the salts of 14 metals (Ag, Hg, Cu, Be, Al, Pb, Cr, As, Tl, Zn, Cd, Ni, Sr, and Sb) were used in the aquatic tests. In each of these tests an LC50 value was determined. The data from the agar plates were compared to the published mammalian oral LD50 values for salts of themore » same metals. Within this set of chemicals C. elegans was found to be a predictor of mammalian acute lethality, generating LC50 values parallel to the rat and mouse LD50 values. The aquatic data were compared to data from EPA Ambient Water Quality Criteria documents. C. elegans was found to be less sensitive than Daphnia but generally more sensitive than the other invertebrate organisms that are presently used. The neurotoxicity testing also was performed in both agar and an aquatic media. The testing in agar was conducted with the salts of 4 metals (Cu, Be, Pb, and Hg) and 2 organophosphate pesticides (malathion and vapona). The studies in an aquatic medium tested the salts of 4 metals (Cu, Be, Pb, and Hg).« less

Rapid detection and differentiation of Staphylococcus colonies using an optical scattering technology.

PubMed

Alsulami, Tawfiq S; Zhu, Xingyue; Abdelhaseib, Maha Usama; Singh, Atul K; Bhunia, Arun K

2018-05-24

Staphylococcus species are a major pathogen responsible for nosocomial infections and foodborne illnesses. We applied a laser-based BARDOT (bacterial rapid detection using optical scattering technology) for rapid colony screening and detection of Staphylococcus on an agar plate and differentiate these from non-Staphylococcus spp. Among the six growth media tested, phenol red mannitol agar (PRMA) was found most suitable for building the Staphylococcus species scatter image libraries. Scatter image library for Staphylococcus species gave a high positive predictive value (PPV 87.5-100%) when tested against known laboratory strains of Staphylococcus spp., while the PPV against non-Staphylococcus spp. was 0-38%. A total of nine naturally contaminated bovine raw milk and ready-to-eat chicken salad samples were tested, and BARDOT detected Staphylococcus including Staphylococcus aureus with 80-100% PPV. Forty-five BARDOT-identified bacterial isolates from naturally contaminated foods were further confirmed by tuf and nuc gene-specific PCR and 16S rRNA gene sequence. This label-free, non-invasive on-plate colony screening technology can be adopted by the food industries, biotechnology companies, and public health laboratories for Staphylococcus species detection including S. aureus from various samples for food safety and public health management. Graphical abstract.

Comparison of seven plating media for enumeration of Listeria spp.

PubMed Central

Loessner, M J; Bell, R H; Jay, J M; Shelef, L A

1988-01-01

The suitability of seven media for the enumeration of Listeria spp. was evaluated at 30 degrees C for 48 h. The media tested were (i) the original McBride Listeria agar formulation (with glycine); (ii) modified McBride agar containing glycine anhydride; (iii) LiCl-phenylethanol-moxalactam (LPM) agar; (iv) acriflavine-ceftazidime agar; (v) Rodriguez isolation agar (RISA); (vi) modified Vogel-Johnson (MVJ) agar; (vii) cyclohexanedione-nalidixic acid-phenylethanol agar; and tryptose agar as control. A total of 66 organisms were used including 11 Listeria monocytogenes strains and 5 other Listeria spp. For L. monocytogenes strains only, all media performed highly similarly. Of the other Listeria spp., only two grew on MVJ agar and three each grew on LPM and RISA. Only LPM agar inhibited the 50 non-listeriae, including five yeasts, while MVJ agar inhibited all but one yeast. The McBride Listeria agar formulation that contained glycine anhydride was less selective than the original. When pure cultures of 10 bacteria (including one L. monocytogenes strain) were combined and plated on four media, L. monocytogenes colonies were easiest to enumerate on MVJ agar, followed by LPM and RISA. These media ranked in the same order when plated with homogenates of various foods to which was added L. monocytogenes Scott A, but LPM agar was the best overall since Scott A was inhibited by MVJ. Upon microscopic examination of listerial colonies from the plating media, atypical cell morphology was noted with cells being about twofold in size on LPM, MVJ, and acriflavine-ceftazidime agars. Overall, LPM agar was the most suitable of the media tested even though it was inhibitory to Listeria grayi and Listeria murrayi. PMID:3146947

Method for in vitro screening of aquatic fungicides

USGS Publications Warehouse

Bailey, T.A.

1983-01-01

Methods were developed for in vitro screening of candidate aquatic fungicides for efficacy against Achlya fiagellata, A. racemosa, Saprolegnia hypogyna and S. megasperma. Agar plugs containing fungal hyphae, removed from the edge of actively growing colonies, were placed in the depressions of spot plates containing 1a??0, 10a??0 and 100 mg/I of the candidate compounds for 15 or 60 min. After exposure, the plugs were transferred on to filter papers (0a??45-A?m pore) in a holder, rinsed, and then placed on cornmeal agar medium in tri-petri dishes. The plates were checked for mycelial growth after 48, 96 and 168 h of incubation in a lighted (400-800 A?m) environmental control chamber at 20A?2A?C. Criteria for the acceptance or rejection of candidate aquatic fungicides for further study were based on the antifungal spectrum index (ASI) comparisons between respective compounds and malachite green after 48 h and the concentration level producing complete growth inhibition. Candidate compounds whose ASI was less than 50% that of malachite green after 48 h or did not inhibit growth at levels less than 100 mg/l were rejected. This method provides a base from which in vivo and definitive test regimens can be developed. Preliminary in vitro screening of candidate fungicides reduces the need for costly in vivo tests on compounds that have low antifungal activity.

Demonstrating Effectiveness of Antibiotics Against Known Bacteria Strains

ERIC Educational Resources Information Center

Keefe, Lois M.

1977-01-01

Procedures are described for showing the effectiveness of antibiotics (penicillin, ampicillin, and tetracycline) against a nonpathogenic bacteria strain (Bacillus cereus). Methods are outlined for preparing nutrient agar, sterilizing tubes, pouring agar plates, preparing antibiotic discs, and transferring antibiotic discs to agar plates. (CS)

Direct identification and recognition of yeast species from clinical material by using albicans ID and CHROMagar Candida plates.

PubMed

Baumgartner, C; Freydiere, A M; Gille, Y

1996-02-01

Two chromogenic media, Albicans ID and CHROMagar Candida agar plates, were compared with a reference medium, Sabouraud-chloramphenicol agar, and standard methods for the identification of yeast species. This study involved 951 clinical specimens. The detection rates for the two chromogenic media for polymicrobial specimens were 20% higher than that for the Sabouraud-chloramphenicol agar plates. The rates of identification of Candida albicans for Albicans ID and CHROMagar Candida agar plates were, respectively, 37.0 and 6.0% after 24 h of incubation and 93.6 and 92.2% after 72 h of incubation, with specificities of 99.8 and 100%. Furthermore, CHROMagar Candida plates identified 13 of 14 Candida tropicalis and 9 of 12 Candida krusei strains after 48 h of incubation.

A Hidden Pitfall in the Preparation of Agar Media Undermines Microorganism Cultivability

PubMed Central

Tanaka, Tomohiro; Kawasaki, Kosei; Daimon, Serina; Kitagawa, Wataru; Yamamoto, Kyosuke; Tamaki, Hideyuki; Tanaka, Michiko; Nakatsu, Cindy H.

2014-01-01

Microbiologists have been using agar growth medium for over 120 years. It revolutionized microbiology in the 1890s when microbiologists were seeking effective methods to isolate microorganisms, which led to the successful cultivation of microorganisms as single clones. But there has been a disparity between total cell counts and cultivable cell counts on plates, often referred to as the “great plate count anomaly,” that has long been a phenomenon that still remains unsolved. Here, we report that a common practice microbiologists have employed to prepare agar medium has a hidden pitfall: when phosphate was autoclaved together with agar to prepare solid growth media (PT medium), total colony counts were remarkably lower than those grown on agar plates in which phosphate and agar were separately autoclaved and mixed right before solidification (PS medium). We used a pure culture of Gemmatimonas aurantiaca T-27T and three representative sources of environmental samples, soil, sediment, and water, as inocula and compared colony counts between PT and PS agar plates. There were higher numbers of CFU on PS medium than on PT medium using G. aurantiaca or any of the environmental samples. Chemical analysis of PT agar plates suggested that hydrogen peroxide was contributing to growth inhibition. Comparison of 454 pyrosequences of the environmental samples to the isolates revealed that taxa grown on PS medium were more reflective of the original community structure than those grown on PT medium. Moreover, more hitherto-uncultivated microbes grew on PS than on PT medium. PMID:25281372

Evaluation of the Granada agar plate for detection of vaginal and rectal group B streptococci in pregnant women.

PubMed

Gil, E G; Rodríguez, M C; Bartolomé, R; Berjano, B; Cabero, L; Andreu, A

1999-08-01

Granada medium was evaluated for the detection of group B streptococci (GBS) in vaginal and rectal swabs compared with selective Columbia blood agar and selective Lim broth. From May 1996 to March 1998, 702 pregnant women (35 to 37 weeks of gestation) participated in this three-phase study; 103 (14.7%) of these women carried GBS. In the first phase of the experiment (n = 273 women), vaginorectal specimens were collected on the same swab; the sensitivities of Granada tube, selective Columbia blood agar, and Lim broth were 31.4, 94.3, and 74.3%, respectively. In the second and third phases (n = 429 women), vaginal and rectal specimens were collected separately; the sensitivities of Granada plate, selective Columbia blood agar, and Lim broth (subcultured at 4 h on selective Columbia agar in the second phase and at 18 to 24 h in Granada plate in the third phase) were 91.1, 83.9, and 75%, respectively, in the second phase and 88.5, 90.4, and 63.5%, respectively, in the third phase. There were no statistically significant differences in GBS recovery between the Granada agar plate and selective Columbia blood agar, but the Granada plate provided a clear advantage; the characteristic red-orange colonies produced overnight by GBS can be identified by the naked eye and is so specific that further identification is unnecessary. The use of the Granada tube and Lim broth did not result in increased isolation of GBS. In conclusion, the Granada agar plate is highly sensitive for detecting GBS in vaginal and rectal swabs from pregnant women and can provide results in 18 to 24 h.

Rapid High-Throughput Assessment of Aerobic Bacteria in Complex Samples by Fluorescence-Based Oxygen Respirometry

PubMed Central

O'Mahony, Fiach C.; Papkovsky, Dmitri B.

2006-01-01

A simple method has been developed for the analysis of aerobic bacteria in complex samples such as broth and food homogenates. It employs commercial phosphorescent oxygen-sensitive probes to monitor oxygen consumption of samples containing bacteria using standard microtiter plates and fluorescence plate readers. As bacteria grow in aqueous medium, at certain points they begin to deplete dissolved oxygen, which is seen as an increase in probe fluorescence above baseline signal. The time required to reach threshold signal is used to either enumerate bacteria based on a predetermined calibration or to assess the effects of various effectors on the growth of test bacteria by comparison with an untreated control. This method allows for the sensitive (down to a single cell), rapid (0.5 to 12 h) enumeration of aerobic bacteria without the need to conduct lengthy (48 to 72 h) and tedious colony counts on agar plates. It also allows for screening a wide range of chemical and environmental samples for their toxicity. These assays have been validated with different bacteria, including Escherichia coli, Micrococcus luteus, and Pseudomonas fluorescens, with the enumeration of total viable counts in broth and industrial food samples (packaged ham, chicken, and mince meat), and comparison with established agar plating and optical-density-at-600-nm assays has been given. PMID:16461677

Automated agar plate streaker: a linear plater on Society for Biomolecular Sciences standard plates.

PubMed

King, Gregory W; Kath, Gary S; Siciliano, Sal; Simpson, Neal; Masurekar, Prakash; Sigmund, Jan; Polishook, Jon; Skwish, Stephen; Bills, Gerald; Genilloud, Olga; Peláez, Fernando; Martín, Jesus; Dufresne, Claude

2006-09-01

Several protocols for bacterial isolation and techniques for aerobic plate counting rely on the use of a spiral plater to deposit concentration gradients of microbial suspensions onto a circular agar plate to isolate colony growth. The advantage of applying a gradient of concentrations across the agar surface is that the original microbiological sample can be applied at a single concentration rather than as multiple serial dilutions. The spiral plater gradually dilutes the sample across a compact area and therefore saves time preparing dilutions and multiple agar plates. Commercial spiral platers are not automated and require manual sample loading. Dispensing of the sample volume and rate of gradients are often very limited in range. Furthermore, the spiral sample application cannot be used with rectangular microplates. Another limitation of commercial spiral platers is that they are useful only for dilute, filtered suspensions and cannot plate suspensions of coarse organic particles therefore precluding the use of many kinds of microorganism-containing substrata. An automated agar plate spreader capable of processing 99 rectangular microplates in unattended mode is described. This novel instrument is capable of dispensing discrete volumes of sample in a linear pattern. It can be programmed to dispense a sample suspense at a uniform application rate or across a decreasing concentration gradient.

Identification and discrimination of Pseudomonas aeruginosa bacteria grown in blood and bile by laser-induced breakdown spectroscopy

NASA Astrophysics Data System (ADS)

Rehse, Steven J.; Diedrich, Jonathan; Palchaudhuri, Sunil

2007-10-01

Pseudomonas aeruginosa bacteria colonies have been analyzed by laser-induced breakdown spectroscopy using nanosecond laser pulses. LIBS spectra were obtained after transferring the bacteria from a nutrient-rich culture medium to a nutrient-free agar plate for laser ablation. To study the dependence of the LIBS spectrum on growth and environmental conditions, colonies were cultured on three different nutrient media: a trypticase soy agar (TSA) plate, a blood agar plate, and a medium chosen deliberately to induce bacteria membrane changes, a MacConkey agar plate containing bile salts. Nineteen atomic and ionic emission lines in the LIBS spectrum, which was dominated by inorganic elements such as calcium, magnesium and sodium, were used to identify and classify the bacteria. A discriminant function analysis was used to discriminate between the P. aeruginosa bacteria and two strains of E. coli: a non-pathogenic environmental strain and the pathogenic strain enterohemorrhagic E. coli 0157:H7 (EHEC). Nearly identical spectra were obtained from P. aeruginosa grown on the TSA plate and the blood agar plate, while the bacteria grown on the MacConkey plate exhibited easily distinguishable differences from the other two. All P. aeruginosa samples, independent of initial growth conditions, were readily discriminated from the two E. coli strains.

Determining the Infectious Dose of Influenza Aerosols in a Mouse Model

DTIC Science & Technology

2012-06-20

the growth of F. tularensis to be relatively slow; incubated at 37 °C it reportedly takes up to 14 days to grow on chocolate agar or cysteine heart...TSB) (BD BBL, Becton Dickinson and Company, Franklin Lakes, NJ), then plated in triplicate on BBL chocolate II agar plates (Lot# S100077/2112/20080806...Laboratories, Philadelphia, PA) and recorded at 580, 600 and 625 nm, and differences before and after aerosolization were noted. Chocolate agar plates

Alternative Differential Identification Approaches for 2 Similar Bacilli Commonly Studied in Microbiology.

ERIC Educational Resources Information Center

Benathen, Isaiah A.

1991-01-01

Alternatives to the traditional unknown tests that permit a clear and unequivocal differential identification decision between Bacillus subtilis and Bacillus megaterium are presented. Plates of Phenylethyl Alcohol agar with Blood (PEAB), slants of Bile Esculin agar and plates of DNA agar are used. The materials, methods, results, and conclusions…

Advanced Behavioral Analyses Show that the Presence of Food Causes Subtle Changes in C. elegans Movement.

PubMed

Angstman, Nicholas B; Frank, Hans-Georg; Schmitz, Christoph

2016-01-01

As a widely used and studied model organism, Caenorhabditis elegans worms offer the ability to investigate implications of behavioral change. Although, investigation of C. elegans behavioral traits has been shown, analysis is often narrowed down to measurements based off a single point, and thus cannot pick up on subtle behavioral and morphological changes. In the present study videos were captured of four different C. elegans strains grown in liquid cultures and transferred to NGM-agar plates with an E. coli lawn or with no lawn. Using an advanced software, WormLab, the full skeleton and outline of worms were tracked to determine whether the presence of food affects behavioral traits. In all seven investigated parameters, statistically significant differences were found in worm behavior between those moving on NGM-agar plates with an E. coli lawn and NGM-agar plates with no lawn. Furthermore, multiple test groups showed differences in interaction between variables as the parameters that significantly correlated statistically with speed of locomotion varied. In the present study, we demonstrate the validity of a model to analyze C. elegans behavior beyond simple speed of locomotion. The need to account for a nested design while performing statistical analyses in similar studies is also demonstrated. With extended analyses, C. elegans behavioral change can be investigated with greater sensitivity, which could have wide utility in fields such as, but not limited to, toxicology, drug discovery, and RNAi screening.

Antibiotic Resistance Patterns of Enterococci and Occurrence of Vancomycin-Resistant Enterococci in Raw Minced Beef and Pork in Germany

PubMed Central

Klein, Günter; Pack, Alexander; Reuter, Gerhard

1998-01-01

The food chain, especially raw minced meat, is thought to be responsible for an increase in the incidence of vancomycin-resistant enterococci (VRE) in human nosocomial infections. Therefore, 555 samples from 115 batches of minced beef and pork from a European Union-licensed meat-processing plant were screened for the occurrence of VRE. The processed meat came from 45 different slaughterhouses in Germany. Enterococci were isolated directly from Enterococcosel selective agar plates and also from Enterococcosel selective agar plates supplemented with 32 mg of vancomycin per liter. In addition, peptone broth was used in a preenrichment procedure, and samples were subsequently plated onto Enterococcosel agar containing vancomycin. To determine resistance, 209 isolates from 275 samples were tested with the glycopeptides vancomycin, teicoplanin, and avoparcin and 19 other antimicrobial substances by using a broth microdilution test. When the direct method was used, VRE were found in 3 of 555 samples (0.5%) at a concentration of 1.0 log CFU/g of minced meat. When the preenrichment procedure was used, 8% of the samples were VRE positive. Our findings indicate that there is a low incidence of VRE in minced meat in Germany. In addition, the resistance patterns of the VRE isolates obtained were different from the resistance patterns of clinical isolates. A connection between the occurrence of VRE in minced meat and nosocomial infections could not be demonstrated on the basis of our findings. PMID:9572958

Selective vs. nonselective media and direct plating vs. enrichment technique in isolation of Vibrio cholerae: recommendations for clinical laboratories.

PubMed

Rennels, M B; Levine, M M; Daya, V; Angle, P; Young, C

1980-09-01

The occurrence of human cholera along the Gulf of Mexico and the isolation of Vibrio cholerae O1 from the Gulf and Chesapeake Bay make it imperative that microbiology laboratories along estuaries develop the capabilities to culture for these pathogens. In attempts to devise a simplified but efficient culture procedure, a selective medium, thiosulfate-citrate-bile salts-sucrose (TCBS) agar, was compared with a nonselective medium, gelatin agar (GA), and the utility of enrichment was examined. TCBS agar detected 99% of the stools found to be positive by all techniques combined, whereas GA identified only 80%. Of acute diarrheal stools, 96% were positive on direct plating, whereas only 66% of formed stools containing V. cholerae were detected by direct plating. Stools from patients with acute diarrhea can be plated directly into TCBS agar alone; stools from persons shedding low numbers of organisms (such as contacts, carriers, or patients receiving antibiotics) should be incubated first in an enrichment broth and then on TCBS agar.

Direct plating technique for enumeration of Listeria monocytogenes in foods.

PubMed

Golden, D A; Beuchat, L R; Brackett, R E

1988-01-01

The advantages and disadvantages of various techniques for detecting and enumerating Listeria monocytogenes in foods are reviewed, and results from a study designed to compare 14 direct plating media for their suitability to recover uninjured cells of L. monocytogenes from 4 foods are summarized. McBride Listeria agar (MLA), gum base nalidixic acid tryptone soy agar (GBNTSA), modified Despierres agar (MDA), and modified MLA (MMLA) performed best for recovering all inoculum populations from milk and ice cream mix. For Brie cheese, MLA, MDA, MMLA, and Dominguez Rodriguez isolation agar were superior for recovering L. monocytogenes; GBNTSA, MDA, MMLA, and Donnelly's Listeria enrichment agar were best for recovering the organism from cabbage. Direct plating procedures without prior enrichment can be utilized successfully for recovering L. monocytogenes from foods such as pasteurized milk and ice cream mix, which contain low populations of background microflora. However, recovery of L. monocytogenes from foods such as raw cabbage and Brie cheese, which contain high populations of other microorganisms, was not satisfactory using direct plating procedures.

Chromogenic agar medium for detection and isolation of Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 from fresh beef and cattle feces.

PubMed

Kalchayanand, Norasak; Arthur, Terrance M; Bosilevac, Joseph M; Wells, James E; Wheeler, Tommy L

2013-02-01

Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains are clinically important foodborne pathogens. Unlike E. coli O157:H7, these foodborne pathogens have no unique biochemical characteristics to readily distinguish them from other E. coli strains growing on plating media. In this study, a chromogenic agar medium was developed in order to differentiate among non-O157 STEC strains of serogroups O26, O45, O103, O111, O121, and O145 on a single agar medium. The ability of this chromogenic agar medium to select and distinguish among these pathogens is based on a combination of utilization of carbohydrates, b -galactosidase activity, and resistance to selective agents. The agar medium in combination with immunomagnetic separation was evaluated and successfully allowed for the detection and isolation of these six serogroups from artificially contaminated fresh beef. The agar medium in combination with immunomagnetic separation also allowed successful detection and isolation of naturally occurring non-O157 STEC strains present in cattle feces. Thirty-five strains of the top six non-O157 STEC serogroups were isolated from 1,897 fecal samples collected from 271 feedlot cattle. This chromogenic agar medium could help significantly in routine screening for the top six non-O157 STEC serogroups from beef cattle and other food.

Compressed-air power tools in orthopaedic surgery: exhaust air is a potential source of contamination.

PubMed

Sagi, H C; DiPasquale, Thomas; Sanders, Roy; Herscovici, Dolfi

2002-01-01

To determine if the exhaust from surgical compressed-air power tools contains bacteria and if the exhaust leads to contamination of sterile surfaces. Bacteriologic study of orthopaedic power tools. Level I trauma center operative theater. None. Part I. Exhaust from two sterile compact air drills was sampled directly at the exhaust port. Part II. Exhaust from the drills was directed at sterile agar plates from varying distances. The agar plates represented sterile surfaces within the operative field. Part III. Control cultures. A battery-powered drill was operated over open agar plates in similar fashion as the compressed-air drills. Agar plates left open in the operative theater served as controls to rule out atmospheric contamination. Random cultures were taken from agar plates, gloves, drills, and hoses. Incidence of positive cultures. In Part I, all filters from both compressed-air drill exhausts were culture negative ( = 0.008). In Part II, the incidence of positive cultures for air drills number one and number two was 73% and 82%, respectively. The most commonly encountered organisms were, coagulase-negative Staphylococcus, and Micrococcus species. All control cultures from agar plates, battery-powered drill, gloves, and hoses were negative ( < 0.01). Exhaust from compressed-air power tools in orthopaedic surgery may contribute to the dissemination of bacteria onto the surgical field. We do not recommend the use of compressed-air power tools that do not have a contained exhaust.

Agar dilution and agar screen with cefoxitin and oxacillin: what is known and what is unknown in detection of meticillin-resistant Staphylococcus aureus.

PubMed

Perez, Leandro Reus Rodrigues; Dias, Cícero; d'Azevedo, Pedro Alves

2008-08-01

In this study we evaluated the performance of the oxacillin agar screen test, and agar dilution tests using cefoxitin and oxacillin antimicrobials, to detect meticillin resistance in Staphylococcus aureus isolates. The presence of the mecA gene, detected by PCR, was used as the standard to which agar screen and agar dilution tests were compared. The best performance was obtained using the agar dilution test (99.4 % accuracy) with breakpoints of 4 mug ml(-1) for oxacillin and 8 mug ml(-1) for cefoxitin, and using the oxacillin agar screen test. Also, a strong correlation between MIC values of cefoxitin and oxacillin permits the use of either drug for detection of meticillin resistance.

Optimisation of a direct plating method for the detection and enumeration of Alicyclobacillus acidoterrestris spores.

PubMed

Henczka, Marek; Djas, Małgorzata; Filipek, Katarzyna

2013-01-01

A direct plating method for the detection and enumeration of Alicyclobacillus acidoterrestris spores has been optimised. The results of the application of four types of growth media (BAT agar, YSG agar, K agar and SK agar) regarding the recovery and enumeration of A. acidoterrestris spores were compared. The influence of the type of applied growth medium, heat shock conditions, incubation temperature, incubation time, plating technique and the presence of apple juice in the sample on the accuracy of the detection and enumeration of A. acidoterrestris spores was investigated. Among the investigated media, YSG agar was the most sensitive medium, and its application resulted in the highest recovery of A. acidoterrestris spores, while K agar and BAT agar were the least suitable media. The effect of the heat shock time on the recovery of spores was negligible. When there was a low concentration of spores in a sample, the membrane filtration method was superior to the spread plating method. The obtained results show that heat shock carried out at 80°C for 10 min and plating samples in combination with membrane filtration on YSG agar, followed by incubation at 46°C for 3 days provided the optimal conditions for the detection and enumeration of A. acidoterrestris spores. Application of the presented method allows highly efficient, fast and sensitive identification and enumeration of A. acidoterrestris spores in food products. This methodology will be useful for the fruit juice industry for identifying products contaminated with A. acidoterrestris spores, and its practical application may prevent economic losses for manufacturers. Copyright © 2012 Elsevier B.V. All rights reserved.

The visual assessment of broth cultures for tissue bank samples.

PubMed

Varettas, Kerry

2017-09-01

The bioburden screening process of allograft musculoskeletal tissue samples received at the South Eastern Area Laboratory Services includes the routine use of solid agar and cooked meat (CM) broth media. CM has been routinely sub-cultured onto solid agar plates after aerobic incubation at 35 °C. This study will evaluate whether a visual assessment of CM can replace sub-culture by an in vitro inoculation and a prospective study. Eight challenge organisms were serially diluted and inoculated into CM. The average inoculum of 0.5-5.5 CFU produced visible turbidity of CM after 24-h incubation for 7 of the challenge organisms with one organism producing turbidity after 48-h incubation. The prospective study evaluated 222 CM of which 213 were visually clear and no-growth on sub-culture and 9 turbid CM which were culture positive. Broth cultures are an integral part of the bioburden screening process of allograft musculoskeletal tissue and swab samples and visual assessment of CM can replace sub-culture.

Trace Amounts of Furan-2-Carboxylic Acids Determine the Quality of Solid Agar Plates for Bacterial Culture

PubMed Central

Hara, Shintaro; Isoda, Reika; Tahvanainen, Teemu; Hashidoko, Yasuyuki

2012-01-01

Background Many investigators have recognised that a significant proportion of environmental bacteria exist in a viable but non-culturable state on agar plates, and some researchers have also noticed that some of such bacteria clearly recover their growth on matrices other than agar. However, the reason why agar is unsuitable for the growth of some bacteria has not been addressed. Methodology/Principal Findings According to the guide of a bioassay for swarming inhibition, we identified 5-hydroxymethylfuran-2-carboxylic acid (5-HMFA) and furan-2-carboxylic acid (FA) as factors that inhibit bacterial swarming and likely inhibit extracellular polysaccharide production on agar. The furan-2-carboxylic acids 5-HMFA and FA effectively inhibited the swarming and swimming of several environmental bacteria at concentrations of 1.8 and 2.3 µg L−1 (13 and 21 nmol L−1), respectively, which are equivalent to the concentrations of these compounds in 0.3% agar. On Luria-Bertani (LB) plates containing 1.0% agar that had been previously washed with MeOH, a mixture of 5-HMFA and FA in amounts equivalent to their original concentrations in the unwashed agar repressed the swarming of Escherichia coli K12 strain W3110, a representative swarming bacterium. Conclusions/Significance Agar that contains trace amounts of 5-HMFA and FA inhibits the proliferation of some slow-growing or difficult-to-culture bacteria on the plates, but it is useful for single colony isolation due to the ease of identification of swarmable bacteria as the non-swarmed colonies. PMID:22848437

Automated Scoring of Chromogenic Media for Detection of Methicillin-Resistant Staphylococcus aureus by Use of WASPLab Image Analysis Software.

PubMed

Faron, Matthew L; Buchan, Blake W; Vismara, Chiara; Lacchini, Carla; Bielli, Alessandra; Gesu, Giovanni; Liebregts, Theo; van Bree, Anita; Jansz, Arjan; Soucy, Genevieve; Korver, John; Ledeboer, Nathan A

2016-03-01

Recently, systems have been developed to create total laboratory automation for clinical microbiology. These systems allow for the automation of specimen processing, specimen incubation, and imaging of bacterial growth. In this study, we used the WASPLab to validate software that discriminates and segregates positive and negative chromogenic methicillin-resistant Staphylococcus aureus (MRSA) plates by recognition of pigmented colonies. A total of 57,690 swabs submitted for MRSA screening were enrolled in the study. Four sites enrolled specimens following their standard of care. Chromogenic agar used at these sites included MRSASelect (Bio-Rad Laboratories, Redmond, WA), chromID MRSA (bioMérieux, Marcy l'Etoile, France), and CHROMagar MRSA (BD Diagnostics, Sparks, MD). Specimens were plated and incubated using the WASPLab. The digital camera took images at 0 and 16 to 24 h and the WASPLab software determined the presence of positive colonies based on a hue, saturation, and value (HSV) score. If the HSV score fell within a defined threshold, the plate was called positive. The performance of the digital analysis was compared to manual reading. Overall, the digital software had a sensitivity of 100% and a specificity of 90.7% with the specificity ranging between 90.0 and 96.0 across all sites. The results were similar using the three different agars with a sensitivity of 100% and specificity ranging between 90.7 and 92.4%. These data demonstrate that automated digital analysis can be used to accurately sort positive from negative chromogenic agar cultures regardless of the pigmentation produced. Copyright © 2016 Faron et al.

Evaluation of Granada agar plate for detection of Streptococcus agalactiae in urine specimens from pregnant women.

PubMed

Tamayo, Javier; Gómez-Garcés, José-Luis; Alós, Juan-Ignacio

2004-08-01

The Granada agar plate (GAP; Biomedics SL, Madrid, Spain) was evaluated for the detection of group B streptococci (GBS) in urine specimens from pregnant women submitted for testing for asymptomatic bacteriuria and was compared with blood agar (BA [Columbia agar with 5% sheep blood]; bioMérieux, Marcy l'Etoile, France). The GAP detected 103 out of 105 GBS, whereas BA detected only 50. Use of the GAP could be a good method for the detection of GBS in urine specimens from pregnant women.

Isolation of Campylobacter from Brazilian broiler flocks using different culturing procedures.

PubMed

Vaz, C S L; Voss-Rech, D; Pozza, J S; Coldebella, A; Silva, V S

2014-11-01

Conventional culturing methods enable the detection of Campylobacter in broiler flocks. However, laboratory culture of Campylobacter is laborious because of its fastidious behavior and the presence of competing nontarget bacteria. This study evaluated different protocols to isolate Campylobacter from broiler litter, feces, and cloacal and drag swabs. Samples taken from commercial Brazilian broiler flocks were directly streaked onto Preston agar (PA), Campy-Line agar (CLA), and modified charcoal cefoperazone deoxycholate agar (mCCDA) and also enriched in blood-free Bolton broth (bfBB) for 24 and 48 h followed by plating onto the different selective media. Higher numbers of Campylobacter-positive cloacal and drag swab samples were observed using either direct plating or enrichment for 24 h before plating onto PA, compared with enrichment for 48 h (P < 0.05). Furthermore, direct plating was a more sensitive method to detect Campylobacter in broiler litter and feces samples. Analysis of directly plated samples revealed that higher Campylobacter levels were detected in feces streaked onto PA (88.8%), cloacal swabs plated onto mCCDA (72.2%), drag swabs streaked onto CLA or mCCDA (69.4%), and litter samples inoculated onto PA (63.8%). Preston agar was the best agar to isolate Campylobacter from directly plated litter samples (P < 0.05), but there was no difference in the efficacies of PA, mCCDA, and CLA in detecting Campylobacter in other samples. The isolated Campylobacter strains were phenotypically identified as Campylobacter jejuni or Campylobacter coli. The predominant contaminant observed in the Campylobacter cultures was Proteus mirabilis, which was resistant to the majority of antimicrobial agents in selective media. Together, these data showed that direct plating onto PA and onto either CLA or mCCDA as the second selective agar enabled the reliable isolation of thermophilic Campylobacter species from broiler samples. Finally, Campylobacter was detected in all broiler flocks sampled. ©2014 Poultry Science Association Inc.

Improved agar diffusion method for detecting residual antimicrobial agents.

PubMed

Tsai, C E; Kondo, F

2001-03-01

The improved agar diffusion method for determination of residual antimicrobial agents was investigated, and the sensitivities of various combinations of test organisms and assay media were determined using 7 organisms, 5 media, and 31 antimicrobial agents. Bacillus stearothermophilus and synthetic assay medium (SAM) showed the greatest sensitivity for screening penicillins (penicillin G and ampicillin). The combination of Bacillus subtilis and minimum medium (MM) was the most sensitive for tetracyclines (oxytetracycline and chlortetracycline), B. stearothermophilus and SAM or Micrococcus luteus and Mueller-Hinton agar (MHA) for detecting tylosin and erythromycin, B. subtilis and MHA for aminoglycosides (streptomycin, kanamycin, gentamicin, and dihydrostreptomycin), B. stearothermophilus and SAM for polyethers (salinomycin and lasalocid), and B. subtilis and MM or Clostridium perfringens and GAM for polypeptides (thiopeptin, enramycin, virginiamycin, and bacitracin). However, gram-negative bacterium Escherichia coli ATCC 27166 and MM were better for screening for colistin and polymixin-B. For detecting the synthetic drugs tested, the best combination was B. subtilis and MM for sulfonamides, E. coli 27166 and MM for quinolones (oxolinic acid and nalidixic acid), B. subtilis and MM for furans (furazolidone), and the bioluminescent bacterium Photobacterium phosphoreum and luminescence assay medium for chloramphenicol and oxolinic acid. The results showed that the use of four assay plates, B. stearothermophilus and SAM, B. subtilis and MM, M. luteus and MHA, and E. coli 27166 and MM, was superior to the currently available techniques for screening for residual antimicrobial agents in edible animal tissues.

Comparison of CHROMagar Salmonella Medium and Hektoen Enteric Agar for Isolation of Salmonellae from Stool Samples

PubMed Central

Gaillot, Olivier; Di Camillo, Patrick; Berche, Patrick; Courcol, René; Savage, Colette

1999-01-01

CHROMagar Salmonella (CAS), a new chromogenic medium, was retrospectively compared to Hektoen enteric agar (HEA) with 501 Salmonella stock isolates and was then prospectively compared to HEA for the detection and presumptive identification of Salmonella spp. with 508 stool samples before and after enrichment. All stock cultures (100%), including cultures of H2S-negative isolates, yielded typical mauve colonies on CAS, while 497 (99%) isolates produced typical lactose-negative, black-centered colonies on HEA. Following overnight incubation at 37°C, a total of 20 Salmonella strains were isolated from the 508 clinical samples. Sensitivities for primary plating and after enrichment were 95% (19 isolates) and 100% (20 isolates), respectively, for CAS and 80% (16 isolates) and 100% (20 isolates), respectively, for HEA. The specificity of CAS (88.9%) was significantly higher than that of HEA (78.5%; P < 0.0001). On the basis of its good sensitivity and specificity, CAS medium can be recommended for use for primary plating when human stool samples are screened for Salmonella spp. PMID:9986847

Correlation between mastitis occurrence and the count of microorganisms in bulk raw milk of bovine dairy herds in four selective culture media.

PubMed

Souto, Luís I M; Minagawa, Clarice Y; Telles, Evelise O; Garbuglio, Márcio A; Amaku, Marcos; Melville, Priscilla A; Dias, Ricardo A; Sakata, Sonia T; Benites, Nilson R

2010-02-01

Milk is the normal secretion of the mammary gland, practically free of colostrum and obtained by the complete milking of one or more healthy animals. Mastitis is an inflammatory process of the mammary gland and it may cause alterations in the milk. The present work aimed to verify whether it is possible, by means of the counts of microorganism in the bulk raw milk in four selective culture media, to establish a correlation with the occurrence of mastitis and therefore, to monitor this disease in bovine dairy herds. The following selective culture media were used: KF Streptococcus Agar, Edwards Agar, Baird-Parker Agar, Blood Agar plus potassium tellurite. Spearman's correlation coefficient was calculated in order to compare the occurrence of mastitis (percentage) in each herd with respective selective culture media counts of microorganisms in bulk raw milk. Thirty-six possibilities were analysed (Tamis and CMT-positive rates were compared with the log-transformed count in four selective culture media) and there was a negative correlation between Tamis 3 and the Baird-Parker Agar plate count. The total results of microbiological tests showed that there were three correlations of the counts in selective culture media. Fifty-two possibilities were analysed and there was a negative correlation between no-bacterial-growth mastitis rates and log10 of KF Streptoccocus Agar plate count and there were two positive correlations between coagulase-positive staphylococci and log10 of Baird-Parker Agar plate count and Blood Agar plus potassium tellurite plate count.

Control of the pattern of perithecium development in Sordaria fimicola on agar medium.

PubMed

Pollock, R T

1975-06-01

In a Sordaria fimicola (Rob.) Ces. and de Not. colony grown on agar medium in a petri plate, perithecia developed in a narrow band around the plate edge after the colony margin reached the edge. Physical wounding of the colony carried out shortly before or during the time perithecia were developing around the plate edge stimulated perithecium development in the wound area. Diffusion barriers were created by cutting small trenches in the agar parallel to the plate edge. The trenches were made at several different positions between the plate center and edge using cultures of several different ages, and the resultant distribution of perithecia along the trench edges suggested that the colony center and periphery produce diffusible inhibitors of perithecium development. These inhibitors may be responsible, in part, for the observed pattern of perithecium development in the colony.

Automatic Digital Analysis of Chromogenic Media for Vancomycin-Resistant-Enterococcus Screens Using Copan WASPLab

PubMed Central

Faron, Matthew L.; Coon, Christopher; Liebregts, Theo; van Bree, Anita; Jansz, Arjan R.; Soucy, Genevieve; Korver, John

2016-01-01

Vancomycin-resistant enterococci (VRE) are an important cause of health care-acquired infections (HAIs). Studies have shown that active surveillance of high-risk patients for VRE colonization can aid in reducing HAIs; however, these screens generate a significant cost to the laboratory and health care system. Digital imaging capable of differentiating negative and “nonnegative” chromogenic agar can reduce the labor cost of these screens and potentially improve patient care. In this study, we evaluated the performance of the WASPLab Chromogenic Detection Module (CDM) (Copan, Brescia, Italy) software to analyze VRE chromogenic agar and compared the results to technologist plate reading. Specimens collected at 3 laboratories were cultured using the WASPLab CDM and plated to each site's standard-of-care chromogenic media, which included Colorex VRE (BioMed Diagnostics, White City, OR) or Oxoid VRE (Oxoid, Basingstoke, United Kingdom). Digital images were scored using the CDM software after 24 or 40 h of growth, and all manual reading was performed using digital images on a high-definition (HD) monitor. In total, 104,730 specimens were enrolled and automation agreed with manual analysis for 90.1% of all specimens tested, with sensitivity and specificity of 100% and 89.5%, respectively. Automation results were discordant for 10,348 specimens, and all discordant images were reviewed by a laboratory supervisor or director. After a second review, 499 specimens were identified as representing missed positive cultures falsely called negative by the technologist, 1,616 were identified as containing borderline color results (negative result but with no package insert color visible), and 8,234 specimens were identified as containing colorimetric pigmentation due to residual matrix from the specimen or yeast (Candida). Overall, the CDM was accurate at identifying negative VRE plates, which comprised 84% (87,973) of the specimens in this study. PMID:27413193

Hyperspectral image reconstruction using RGB color for foodborne pathogen detection on agar plates

NASA Astrophysics Data System (ADS)

Yoon, Seung-Chul; Shin, Tae-Sung; Park, Bosoon; Lawrence, Kurt C.; Heitschmidt, Gerald W.

2014-03-01

This paper reports the latest development of a color vision technique for detecting colonies of foodborne pathogens grown on agar plates with a hyperspectral image classification model that was developed using full hyperspectral data. The hyperspectral classification model depended on reflectance spectra measured in the visible and near-infrared spectral range from 400 and 1,000 nm (473 narrow spectral bands). Multivariate regression methods were used to estimate and predict hyperspectral data from RGB color values. The six representative non-O157 Shiga-toxin producing Eschetichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) were grown on Rainbow agar plates. A line-scan pushbroom hyperspectral image sensor was used to scan 36 agar plates grown with pure STEC colonies at each plate. The 36 hyperspectral images of the agar plates were divided in half to create training and test sets. The mean Rsquared value for hyperspectral image estimation was about 0.98 in the spectral range between 400 and 700 nm for linear, quadratic and cubic polynomial regression models and the detection accuracy of the hyperspectral image classification model with the principal component analysis and k-nearest neighbors for the test set was up to 92% (99% with the original hyperspectral images). Thus, the results of the study suggested that color-based detection may be viable as a multispectral imaging solution without much loss of prediction accuracy compared to hyperspectral imaging.

Screening fungicides for use in fish culture: Evaluation of the agar plug transfer, cellophane transfer, and agar dilution methods

USGS Publications Warehouse

Bailey, Tom A.

1983-01-01

The reliability, reproducibility, and usefulness of three screening methods -- the cellophane transfer, the agar plug transfer, and the agar dilution -- to screen aquatic fungicides were evaluated. Achlya flagellata and Saprolegnia hypogyna were exposed to 1, 10, and 100 mg/L of malachite green to test each method. The cellophane transfer and agar plug transfer techniques had similar reliability and reproducibility in rating fungicidal activity, and were both superior to the agar dilution technique. The agar plug transfer and agar dilution techniques adequately projected in vivo activity of malachite green, but the cellophane transfer technique overestimated its activity. Overall, the agar plug transfer technique most accurately rated the activity of malachite green and was the easiest test to perform. It therefore appears to be the method of choice for testing aquatic fungicides.

Glass bead cultivation of fungi: combining the best of liquid and agar media.

PubMed

Droce, Aida; Sørensen, Jens Laurids; Giese, Henriette; Sondergaard, Teis Esben

2013-09-01

Production of bioactive compounds and enzymes from filamentous fungi is highly dependent on cultivation conditions. Here we present an easy way to cultivate filamentous fungi on glass beads that allow complete control of nutrient supply. Secondary metabolite production in Fusarium graminearum and Fusarium solani cultivated on agar plates, in shaking liquid culture or on glass beads was compared. Agar plate culture and glass bead cultivation yielded comparable results while liquid culture had lower production of secondary metabolites. RNA extraction from glass beads and liquid cultures was easier than from agar plates and the quality was superior. The system allows simple control of nutrient availability throughout fungal cultivation. This combined with the ease of extraction of nucleic acids and metabolites makes the system highly suitable for the study of gene regulation in response to specific nutrient factors. © 2013.

21 CFR 866.4600 - Ouchterlony agar plate.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Ouchterlony agar plate. 866.4600 Section 866.4600 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunology Laboratory Equipment and Reagents § 866...

21 CFR 866.4600 - Ouchterlony agar plate.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Ouchterlony agar plate. 866.4600 Section 866.4600 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunology Laboratory Equipment and Reagents § 866...

21 CFR 866.4600 - Ouchterlony agar plate.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Ouchterlony agar plate. 866.4600 Section 866.4600 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunology Laboratory Equipment and Reagents § 866...

An Agar-Based Method for Plating Marine Protozoan Parasites of the Genus Perkinsus.

PubMed

Cold, Emma R; Freyria, Nastasia J; Martínez Martínez, Joaquín; Fernández Robledo, José A

2016-01-01

The genus Perkinsus includes protozoan parasites of mollusks responsible for losses in the aquaculture industry and hampering the recovery of natural shellfish beds worldwide, and they are a key taxon for understanding intracellular parasitism adaptations. The ability to propagate the parasite in liquid media, in the absence of the host, has been crucial for improving understanding of its biology; however, alternative techniques to grow the parasite are needed to explore other basic aspects of the Perkinsus spp. biology. We optimized a DME: Ham's F12-5% FBS- containing solid agar medium for plating Perkinsus marinus. This solid medium supported trophozoite propagation both by binary fission and schizogony. Colonies were visible to the naked eye 17 days after plating. We tested the suitability of this method for several applications, including the following: 1) Subcloning P. marinus isolates: single discrete P. marinus colonies were obtained from DME: Ham's F12-5% FBS- 0.75% agar plates, which could be further propagated in liquid medium; 2) Subcloning engineered Perkinsus mediterraneus MOE[MOE]: GFP by streaking cultures on plates; 3) Chemical susceptibility: Infusing the DME: Ham's F12-5% FBS- 0.75% agar plates with triclosan resulted in inhibition of the parasite propagation in a dose-dependent manner. Altogether, our plating method has the potential for becoming a key tool for investigating diverse aspects of Perkinsus spp. biology, developing new molecular tools, and for biotechnological applications.

Growth of Desulfovibrio on the surface of agar media.

PubMed

Iverson, W P

1966-07-01

Growth of Desulfovibrio desulfuricans (API strain) was found to take place in an atmosphere of hydrogen on the agar surface of complex media, including yeast extract (Difco), and Trypticase Soy Agar (BBL) without any added reducing agents. For growth on a 2% yeast extract-agar surface in the absence of hydrogen (nitrogen atmosphere), sodium lactate was required in the medium. Growth on the surface of Trypticase Soy Agar (TSA) under nitrogen took place readily in the absence of an added hydrogen donor. A medium (TSA plus salts) is described based upon the addition of sodium lactate (4 ml per liter), magnesium sulfate (2 g per liter), and ferrous ammonium sulfate (0.05%) to TSA, which appears suitable for the isolation and growth of Desulfovibrio on the surface of agar plates in an atmosphere of hydrogen. Sodium lactate does not appear to be essential in this medium for good growth and sulfate reduction in a hydrogen atmosphere, but is essential in a nitrogen atmosphere. Growth of Desulfovibrio (hydrogen atmosphere) on the agar surface of media commonly used for its cultivation as well as on an inorganic medium containing bicarbonate as a source of carbon is poor and erratic unless inoculated (Desulfovibrio) plates of TSA plus salts are incubated in the same container with plates of these media. This stimulatory effect of incubation with inoculated plates of TSA plus salts medium appears to be due to as yet unidentified volatile material produced by D. desulfuricans when growing on this medium. Another volatile material, or possibly the identical material, appears to act similarly to a hydrogen donor.

Development of classification models to detect Salmonella Enteritidis and Salmonella Typhimurium found in poultry carcass rinses by visible-near infrared hyperspectral imaging

NASA Astrophysics Data System (ADS)

Seo, Young Wook; Yoon, Seung Chul; Park, Bosoon; Hinton, Arthur; Windham, William R.; Lawrence, Kurt C.

2013-05-01

Salmonella is a major cause of foodborne disease outbreaks resulting from the consumption of contaminated food products in the United States. This paper reports the development of a hyperspectral imaging technique for detecting and differentiating two of the most common Salmonella serotypes, Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST), from background microflora that are often found in poultry carcass rinse. Presumptive positive screening of colonies with a traditional direct plating method is a labor intensive and time consuming task. Thus, this paper is concerned with the detection of differences in spectral characteristics among the pure SE, ST, and background microflora grown on brilliant green sulfa (BGS) and xylose lysine tergitol 4 (XLT4) agar media with a spread plating technique. Visible near-infrared hyperspectral imaging, providing the spectral and spatial information unique to each microorganism, was utilized to differentiate SE and ST from the background microflora. A total of 10 classification models, including five machine learning algorithms, each without and with principal component analysis (PCA), were validated and compared to find the best model in classification accuracy. The five machine learning (classification) algorithms used in this study were Mahalanobis distance (MD), k-nearest neighbor (kNN), linear discriminant analysis (LDA), quadratic discriminant analysis (QDA), and support vector machine (SVM). The average classification accuracy of all 10 models on a calibration (or training) set of the pure cultures on BGS agar plates was 98% (Kappa coefficient = 0.95) in determining the presence of SE and/or ST although it was difficult to differentiate between SE and ST. The average classification accuracy of all 10 models on a training set for ST detection on XLT4 agar was over 99% (Kappa coefficient = 0.99) although SE colonies on XLT4 agar were difficult to differentiate from background microflora. The average classification accuracy of all 10 models on a validation set of chicken carcass rinses spiked with SE or ST and incubated on BGS agar plates was 94.45% and 83.73%, without and with PCA for classification, respectively. The best performing classification model on the validation set was QDA without PCA by achieving the classification accuracy of 98.65% (Kappa coefficient=0.98). The overall best performing classification model regardless of using PCA was MD with the classification accuracy of 94.84% (Kappa coefficient=0.88) on the validation set.

Detection of Listeria monocytogenes in pork and beef using the VIDAS® LMO2 automated enzyme linked immunoassay method.

PubMed

Meyer, Cornelia; Fredriksson-Ahomaa, Maria; Sperner, Brigitte; Märtlbauer, Erwin

2011-07-01

Listeria (L.) monocytogenes, a foodborne pathogen, is known to be a possible contaminant of foods during production and processing. Samples (n=985) of raw meat and by-products obtained from beef and pork were first screened by the VIDAS system for the presence of Listeria spp., followed by testing for the presence of L. monocytogenes. Positive L. monocytogenes results were confirmed by plating on selective agars: 14% of the samples were positive for Listeria and 4% tested positive for L. monocytogenes, of which 3% were confirmed on selective agars. In by-products (17%) the contamination with listeriae was higher than in meat cuts (10%). Only samples strongly positive for Listeria spp. by VIDAS were positive for L. monocytogenes. Overall, the prevalence of L. monocytogenes in beef and pork samples was rather low in comparison to most previous studies. The VIDAS system was shown to be a suitable method for screening out Listeria-negative samples; the main advantage being a markedly reduced assay time. Copyright © 2011 Elsevier Ltd. All rights reserved.

Hyperspectral imaging for detection of non-O157 Shiga-toxin producing Escherichia coli (STEC) serogroups on spread plates of mixed cultures

NASA Astrophysics Data System (ADS)

Yoon, Seung Chul; Windham, William R.; Ladely, Scott; Heitschmidt, Gerald W.; Lawrence, Kurt C.; Park, Bosoon; Narang, Neelam; Cray, William C.

2012-05-01

We investigated the feasibility of visible and near-infrared (VNIR) hyperspectral imaging for rapid presumptive-positive screening of six representative non-O157 Shiga-toxin producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) on spread plates of mixed cultures. Although the traditional culture method is still the "gold standard" for presumptive-positive pathogen screening, it is time-consuming, labor-intensive, not effective in testing large amount of food samples, and cannot completely prevent unwanted background microflora from growing together with target microorganisms on agar media. A previous study was performed using the data obtained from pure cultures individually inoculated on spot and/or spread plates in order to develop multivariate classification models differentiating each colony of the six non-O157 STEC serogroups and to optimize the models in terms of parameters. This study dealt with the validation of the trained and optimized models with a test set of new independent samples obtained from colonies on spread plates of mixed cultures. A new validation protocol appropriate to a hyperspectral imaging study for mixed cultures was developed. One imaging experiment with colonies obtained from two serial dilutions was performed. A total of six agar plates were prepared, where O45, O111 and O121 serogroups were inoculated into all six plates and each of O45, O103 and O145 serogroups was added into the mixture of the three common bacterial cultures. The number of colonies grown after 24-h incubation was 331 and the number of pixels associated with the grown colonies was 16,379. The best model found from this validation study was based on pre-processing with standard normal variate and detrending (SNVD), first derivative, spectral smoothing, and k-nearest neighbor classification (kNN, k=3) of scores in the principal component subspace spanned by 6 principal components. The independent testing results showed 95% overall detection accuracy at pixel level and 97% at colony level. The developed model was proven to be still valid even for the independent samples although the size of a test set was small and only one experiment was performed. This study was an important first step in validating and updating multivariate classification models for rapid screening of ground beef samples contaminated by non-O157 STEC pathogens using hyperspectral imaging.

Residual Agar Determination in Bacterial Spores by Electrospray Ionization Mass Spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wahl, Karen L.; Colburn, Heather A.; Wunschel, David S.

2010-02-15

Presented here is an analytical method to detect residual agar from a bacterial spore sample as an indication of culturing on an agar plate. This method is based on the resolubilization of agar polysaccharide from a bacterial spore sample, enzymatic digestion, followed by electrospray ionization tandem mass spectrometry (ESI-MSn) analysis for detection of a specific agar fragment ion. A range of Bacillus species and strains were selected to demonstrate the effectiveness of this approach. The characteristic agar fragment ion was detected in the spores grown on agar that were washed from 1 to 5 times, irradiated or non-irradiated and notmore » in the spores grown in broth. A sample containing approximately 108 spores is currently needed for confident detection of residual agar from culture on agar plates in the presence of bacterial spores with a limit of detection of approximately 1 ppm agar spiked into a broth-grown spore sample. The results of a proficiency test with 42 blinded samples are presented demonstrating the utility of this method with no false positives and only 3 false negatives for samples that were below the detection level of the method as documented.« less

cGMP Signalling Mediates Water Sensation (Hydrosensation) and Hydrotaxis in Caenorhabditis elegans

PubMed Central

Wang, Wei; Qin, Li-Wei; Wu, Tai-Hong; Ge, Chang-Li; Wu, Ya-Qian; Zhang, Qiang; Song, Yan-Xue; Chen, Yuan-Hua; Ge, Ming-Hai; Wu, Jing-Jing; Liu, Hui; Xu, Yao; Su, Chun-Ming; Li, Lan-Lan; Tang, Jing; Li, Zhao-Yu; Wu, Zheng-Xing

2016-01-01

Animals have developed the ability to sense the water content in their habitats, including hygrosensation (sensing humidity in the air) and hydrosensation (sensing the water content in other microenvironments), and they display preferences for specific water contents that influence their mating, reproduction and geographic distribution. We developed and employed four quantitative behavioural test paradigms to investigate the molecular and cellular mechanisms underlying sensing the water content in an agar substrate (hydrosensation) and hydrotaxis in Caenorhabditis elegans. By combining a reverse genetic screen with genetic manipulation, optogenetic neuronal manipulation and in vivo Ca2+ imaging, we demonstrate that adult worms avoid the wetter areas of agar plates and hypo-osmotic water droplets. We found that the cGMP signalling pathway in ciliated sensory neurons is involved in hydrosensation and hydrotaxis in Caenorhabditis elegans. PMID:26891989

Ginger Extract Inhibits Biofilm Formation by Pseudomonas aeruginosa PA14

PubMed Central

Kim, Han-Shin; Park, Hee-Deung

2013-01-01

Bacterial biofilm formation can cause serious problems in clinical and industrial settings, which drives the development or screening of biofilm inhibitors. Some biofilm inhibitors have been screened from natural products or modified from natural compounds. Ginger has been used as a medicinal herb to treat infectious diseases for thousands of years, which leads to the hypothesis that it may contain chemicals inhibiting biofilm formation. To test this hypothesis, we evaluated ginger’s ability to inhibit Pseudomonas aeruginosa PA14 biofilm formation. A static biofilm assay demonstrated that biofilm development was reduced by 39–56% when ginger extract was added to the culture. In addition, various phenotypes were altered after ginger addition of PA14. Ginger extract decreased production of extracellular polymeric substances. This finding was confirmed by chemical analysis and confocal laser scanning microscopy. Furthermore, ginger extract formed noticeably less rugose colonies on agar plates containing Congo red and facilitated swarming motility on soft agar plates. The inhibition of biofilm formation and the altered phenotypes appear to be linked to a reduced level of a second messenger, bis-(3′-5′)-cyclic dimeric guanosine monophosphate. Importantly, ginger extract inhibited biofilm formation in both Gram-positive and Gram-negative bacteria. Also, surface biofilm cells formed with ginger extract detached more easily with surfactant than did those without ginger extract. Taken together, these findings provide a foundation for the possible discovery of a broad spectrum biofilm inhibitor. PMID:24086697

Misinterpretation of Gram Stain from the Stationary Growth Phase of Positive Blood Cultures for Brucella and Acinetobacter Species.

PubMed

Bazzi, Ali M; Al-Tawfiq, Jaffar A; Rabaan, Ali A

2017-01-01

Acinetobacter baumannii and Brucella species are Gram-negative organisms that are vulnerable to misinterpretation as Gram-positive or Gram-variable in blood cultures. We assess the random errors in gram stain interpretation to reduce the likelihood of such errors and therefore patient harm. Aerobic and anaerobic blood cultures from two patients in an acute care facility in Saudi Arabia were subjected to preliminary Gram-staining. In case 1, VITEK-2 Anaerobe Identification, repeat Gram staining from a blood agar plate, Remel BactiDrop™ Oxidase test, Urea Agar urease test and real-time PCR were used to confirm presence of Brucella and absence of Coryneform species. In case 2, repeat Gram- staining from the plate and the vials, VITEK-2 Gram-Negative Identification, real-time PCR and subculture on to Columbia agar, blood agar, and MacConkey agar were carried out to identify A. baumannii . In case 1, initially pleomorphic Gram-positive bacteria were identified. Coryneform species were suspected. Tiny growth was observed after 24 h on blood agar plates, and good growth by 48 h. Presence of Brucella species was ultimately confirmed. In case 2, preliminary Gram-stain results suggested giant Gram-positive oval cocci. Further testing over 18-24 h identified A. baumannii . Oxidase test from the plate and urease test from the culture vial is recommended after apparent identification of pleomorphic Gram-positive bacilli from blood culture, once tiny growth is observed, to distinguish Brucella from Corynebacterium species. If giant Gram-positive oval cocci are indicated by preliminary Gram-staining, it is recommended that the Gram stain be repeated from the plate after 4-6 h, or culture should be tested in Triple Sugar Iron (TSI) medium and the Gram stain repeated after 2-4 h incubation.

Simulation of Bacillus subtilis biofilm growth on agar plate by diffusion-reaction based continuum model

NASA Astrophysics Data System (ADS)

Zhang, Xianlong; Wang, Xiaoling; Nie, Kai; Li, Mingpeng; Sun, Qingping

2016-08-01

Various species of bacteria form highly organized spatially-structured aggregates known as biofilms. To understand how microenvironments impact biofilm growth dynamics, we propose a diffusion-reaction continuum model to simulate the formation of Bacillus subtilis biofilm on an agar plate. The extended finite element method combined with level set method are employed to perform the simulation, numerical results show the quantitative relationship between colony morphologies and nutrient depletion over time. Considering that the production of polysaccharide in wild-type cells may enhance biofilm spreading on the agar plate, we inoculate mutant colony incapable of producing polysaccharide to verify our results. Predictions of the glutamate source biofilm’s shape parameters agree with the experimental mutant colony better than that of glycerol source biofilm, suggesting that glutamate is rate limiting nutrient for Bacillus subtilis biofilm growth on agar plate, and the diffusion-limited is a better description to the experiment. In addition, we find that the diffusion time scale is of the same magnitude as growth process, and the common-employed quasi-steady approximation is not applicable here.

Simulation of Bacillus subtilis biofilm growth on agar plate by diffusion-reaction based continuum model.

PubMed

Zhang, Xianlong; Wang, Xiaoling; Nie, Kai; Li, Mingpeng; Sun, Qingping

2016-07-19

Various species of bacteria form highly organized spatially-structured aggregates known as biofilms. To understand how microenvironments impact biofilm growth dynamics, we propose a diffusion-reaction continuum model to simulate the formation of Bacillus subtilis biofilm on an agar plate. The extended finite element method combined with level set method are employed to perform the simulation, numerical results show the quantitative relationship between colony morphologies and nutrient depletion over time. Considering that the production of polysaccharide in wild-type cells may enhance biofilm spreading on the agar plate, we inoculate mutant colony incapable of producing polysaccharide to verify our results. Predictions of the glutamate source biofilm's shape parameters agree with the experimental mutant colony better than that of glycerol source biofilm, suggesting that glutamate is rate limiting nutrient for Bacillus subtilis biofilm growth on agar plate, and the diffusion-limited is a better description to the experiment. In addition, we find that the diffusion time scale is of the same magnitude as growth process, and the common-employed quasi-steady approximation is not applicable here.

[Evaluation of a new medium, eggplant (Solanum melongena) agar as a screening medium for Cryptococcus neoformans in environmental samples].

PubMed

Sengul, Mustafa; Ergin, Cağrı; Kartal, Tuğba

2014-04-01

Cryptococcus neofomans is an encapsulated yeast-like fungus that causes life-threatening infections, especially in immunosuppresive patients. C.neoformans infection is believed to be acquired via inhalation of aerosolized particles from the environment. Avian guano, decaying tree hollows and soil are the related known environmental niches. Brown pigmented yeast growth from the precursors in growth media is an important step for the identification and isolation of C.neoformans. Seeds of plants in nature are preferred owing to easy accessibility and low costs for the preparation of such media. Guizotia abysinicca (Niger seed) as Staib agar, Helianthus annus (Sunflower) as Pal's medium, Brassica nigra (Mustard) agar, tobacco agar, Mucuna pruriens (Velvet bean) seed agar, Perilla frutescens (Beefsteak plant) seed agar, Rubus fruticosus (Blackberry) agar and ground red hot pepper agar are pigment-based selective media for the differentiation of C.neoformans. The aim of this study was to observe the pigment production of C.neoformans in a new medium based on eggplant (Solanum melongena) and also to compare its performance with the simplified Staib, Pal's and tobacco agar for isolation from the environment. Three different eggplant-based medium (S.melongena Melanzaza viserba, S.melongena Pinstripe F1 and S.ovigerum Ivory F1) were included in the study. Pigment-forming eggplant medium, simplified Staib agar, Pal's agar and tobacco agar were used for the cultivation of the environmental swabbed samples from 19 Eucalyptus camaldulensis trunk hollows in continuous colonization region. While pigment formation were observed with S.melongena Melanzaza viserba and S.melongena Pinstripe F1 containing media, S.ovigerum Ivory F1 medium was found to be non-reactive. In colonization area (Gökova-Akyaka, Turkey), 11 (57.9%) out of 19 E.camaldulensis samples were positive with simplified Staib agar, Pal's agar and eggplant agar while 10 (52.6%) of them are positive with tobacco agar. C.neoformans colony forming unit (cfu) per plate were found as 51, 57 and 48 (median values) on simplified Staib agar, Pal's agar and eggplant agar, respectively, while tobacco agar has lower performance with 33 cfu/petri. No statistically significant difference were found between simplified Staib agar, Pal's agar and eggplant agar's performances for C.neoformans isolations from the nature (p=0.71). In conclusion, easily prepared eggplant agar is as functional as widely used media such as simplified Staib agar and Pal's agar for the isolation of C.neoformans from the natural environment.

Frequency of methicillin-resistant Staphylococcus aureus nasal colonization among patients suffering from methicillin resistant Staphylococcus aureus bacteraemia.

PubMed

Aslam, Nadia; Izhar, Mateen; Mehdi, Naima

2013-11-01

To determine rate of nasal colonization in Patients suffering from bacteraemia caused by methicillin resistant Staphylococcus aureus. This descriptive cross sectional study was carried out in a tertiary ca re, University Teaching Hospital (Shaikh Zayed Hospital, Lahore) from October 2010 to August 2011. Nasal swabs were taken from patients suffering from MRSA bacteraemia and were plated on mannitol salt agar plates to isolate Staphylococcus aureus (S. aureus) which were then tested for oxacillin susceptibility. Nasal colonization was present in 52.5% of patients suffering from MRSA bacteraemia. Nasal colonization rates with MRSA were high among patients suffering from MRSA bacteraemia especially in those undergoing dialysis or surgical procedures. Therefore, screening and nasal decolonization should be practiced in hospitals.

Study on E. coli and Salmonella biofilms from fresh fruits and vegetables.

PubMed

Amrutha, Balagopal; Sundar, Kothandapani; Shetty, Prathapkumar Halady

2017-04-01

Foodborne outbreaks associated with fresh fruits and vegetables are on the rise worldwide. Biofilm formation is one of the important traits of pathogens making them strongly attached to substrates as well as express virulence phenotypes. Present study investigates the biofilm forming ability of E. coli and Salmonella sp. isolated from fresh fruits and vegetables. A total of 53 strains, including 35 E. coli and 18 Salmonella sp. isolated from different fruit and vegetable samples were taken into account for the study. Initial screening for biofilm formation was done using Congo Red agar plate test. Results revealed that 22.8% E. coli and 22.2% Salmonella sp. were potential biofilm formers. However, the MTP (Micro-Titre Plate) assay suggested more isolates of both E. coli and Salmonella sp. were moderate to strong biofilm producers. Agar plate diffusion assay with Agrobacterium tumefaciens NTL-4 showed the production of quorum signaling molecules (AHLs) by three isolates of E. coli and one Salmonella sp. Two E. coli isolates showed a significant amount of EPS production indicating higher biofilm forming potential. The Presence of LUX R homologue gene ( sdi A) in two of the Salmonella isolates were confirmed by PCR which demonstrated their potential pathogenicity. Results of the work underline the biofilm forming and potentially virulent capacities of isolates from the surface of fruits and vegetables.

An Agar-Based Method for Plating Marine Protozoan Parasites of the Genus Perkinsus

PubMed Central

Cold, Emma R.; Freyria, Nastasia J.; Martínez Martínez, Joaquín; Fernández Robledo, José A.

2016-01-01

The genus Perkinsus includes protozoan parasites of mollusks responsible for losses in the aquaculture industry and hampering the recovery of natural shellfish beds worldwide, and they are a key taxon for understanding intracellular parasitism adaptations. The ability to propagate the parasite in liquid media, in the absence of the host, has been crucial for improving understanding of its biology; however, alternative techniques to grow the parasite are needed to explore other basic aspects of the Perkinsus spp. biology. We optimized a DME: Ham’s F12–5% FBS- containing solid agar medium for plating Perkinsus marinus. This solid medium supported trophozoite propagation both by binary fission and schizogony. Colonies were visible to the naked eye 17 days after plating. We tested the suitability of this method for several applications, including the following: 1) Subcloning P. marinus isolates: single discrete P. marinus colonies were obtained from DME: Ham’s F12–5% FBS– 0.75% agar plates, which could be further propagated in liquid medium; 2) Subcloning engineered Perkinsus mediterraneus MOE[MOE]: GFP by streaking cultures on plates; 3) Chemical susceptibility: Infusing the DME: Ham’s F12–5% FBS– 0.75% agar plates with triclosan resulted in inhibition of the parasite propagation in a dose-dependent manner. Altogether, our plating method has the potential for becoming a key tool for investigating diverse aspects of Perkinsus spp. biology, developing new molecular tools, and for biotechnological applications. PMID:27149378

Growth of Desulfovibrio on the Surface of Agar Media

PubMed Central

Iverson, Warren P.

1966-01-01

Growth of Desulfovibrio desulfuricans (API strain) was found to take place in an atmosphere of hydrogen on the agar surface of complex media, including yeast extract (Difco), and Trypticase Soy Agar (BBL) without any added reducing agents. For growth on a 2% yeast extract-agar surface in the absence of hydrogen (nitrogen atmosphere), sodium lactate was required in the medium. Growth on the surface of Trypticase Soy Agar (TSA) under nitrogen took place readily in the absence of an added hydrogen donor. A medium (TSA plus salts) is described based upon the addition of sodium lactate (4 ml per liter), magnesium sulfate (2 g per liter), and ferrous ammonium sulfate (0.05%) to TSA, which appears suitable for the isolation and growth of Desulfovibrio on the surface of agar plates in an atmosphere of hydrogen. Sodium lactate does not appear to be essential in this medium for good growth and sulfate reduction in a hydrogen atmosphere, but is essential in a nitrogen atmosphere. Growth of Desulfovibrio (hydrogen atmosphere) on the agar surface of media commonly used for its cultivation as well as on an inorganic medium containing bicarbonate as a source of carbon is poor and erratic unless inoculated (Desulfovibrio) plates of TSA plus salts are incubated in the same container with plates of these media. This stimulatory effect of incubation with inoculated plates of TSA plus salts medium appears to be due to as yet unidentified volatile material produced by D. desulfuricans when growing on this medium. Another volatile material, or possibly the identical material, appears to act similarly to a hydrogen donor. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:5955798

Antimicrobial susceptibility testing of Mycobacterium tuberculosis complex for first and second line drugs by broth dilution in a microtiter plate format.

PubMed

Hall, Leslie; Jude, Kurt P; Clark, Shirley L; Wengenack, Nancy L

2011-06-24

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioMérieux, Hazelwood, MO). The agar proportion method, while considered the "gold" standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is ≥1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Diagnostic value of morphological, physiological and biochemical tests in distinguishing Trichophyton rubrum from Trichophyton mentagrophytes complex.

PubMed

Ates, Aylin; Ozcan, Kadri; Ilkit, Macit

2008-12-01

The two most frequently encountered dermatophyte etiologic agents of glabrous skin and nail dermatophytoses are Trichophyton rubrum and T. mentagrophytes. This study was aimed to discuss the efficacy of morphological, physiological and biochemical diagnostic tests commonly used in the identification of T. rubrum and members of the T. mentagrophytes complex. In this study, we evaluated; hydrolysis of urea in broth and on urea agar slants and Petri plates incubated at 22 degrees C, 28 degrees C and 37 degrees C, in vitro hair perforation (blond child, sheep and goat hair), pigment production on cornmeal dextrose agar (CMDA) and bromcresol purple-milk solids-glucose agar (BCP-MS-G), Tween opacity, sorbitol assimilation, and salt tolerance. Additionally, the production of micro- and macroconidia was investigated by using brain heart infusion agar (BHIA), Christensen's urea agar in Petri plates (UPA), CMDA, Lowenstein-Jensen agar (LJA), malt extract agar, oatmeal agar, Oxoid chromogenic Candida agar, and potato dextrose agar. All cultures were incubated at 28 degrees C, and conidial production was compared on days 5, 10 and 15. It was found that the urea hydrolysis test yielded more rapid and significant results when urea medium was prepared in Petri plates and incubated at 28 degrees C (P<0.01). LJA supported the highest production of microconidia after 15 days (P<0.001). Additionally, it was found that T. rubrum strains produced red pigment on CMDA (P<0.01) and BCP-MS-G, while strains of the T. mentagrophytes species complex did not. A special algorithm containing the various test procedures employed in these studies is presented which was found to be useful in the differentiation of T. rubrum strains from T. mentagrophytes complex. Our results revealed that UPA, CMDA, BCP-MS-G, LJA, and BHIA may be used as common mycological agars in routine practice.

Agar Underlay Method for Recovery of Sublethally Heat-Injured Bacteria

PubMed Central

Kang, D. H.; Siragusa, G. R.

1999-01-01

A method of recovering sublethally heat-injured bacteria was developed. The procedure (termed the agar underlay method) uses a nonselective agar underlaid with a selective medium. In a two-chambered petri dish, the Lutri plate (LP), a nonselective agar is inoculated with a population of sublethally heat-injured bacteria. After a 2-h repair incubation period, selective agar is added to the bottom chamber of the LP and incubated. By diffusing through the nonselective top agar, selective agents from the underlay medium impart selectivity to the system. By the agar underlay method, recovery rates of the heat-injured food-borne pathogens Escherichia coli O157:H7 and Salmonella typhimurium were not different (P > 0.05) from recovery rates determined with nonselective media. Sublethally heat-injured cells (60°C for 1.5 min in buffer or 80°C for 30 s on meat surfaces) grew and produced a typical colony morphology and color reaction when the agar underlay procedure was used with the appropriate respective selective agars. Unlike agar overlay methods for injury repair, the agar underlay procedure allows the typical selective-medium colony morphology to develop and allows colonies to be more easily picked for further characterization. Higher recovery rates of heat-injured fecal enterococci from bovine fecal samples and total coliforms from animal waste lagoons were obtained by the agar underlay method with selective agars than by direct plating on the respective selective media. PMID:10583985

Wrinkly-Spreader Fitness in the Two-Dimensional Agar Plate Microcosm: Maladaptation, Compensation and Ecological Success

PubMed Central

Spiers, Andrew J.

2007-01-01

Bacterial adaptation to new environments often leads to the establishment of new genotypes with significantly altered phenotypes. In the Wrinkly Spreader (WS), ecological success in static liquid microcosms was through the rapid colonisation of the air-liquid interface by the production of a cellulose-based biofilm. Rapid surface spreading was also seen on agar plates, but in this two-dimensional environment the WS appears maladapted and rapidly reverts to the ancestral smooth (SM)-like colony genotype. In this work, the fitness of WS relative to SM in mixed colonies was found to be low, confirming the WS instability on agar plates. By examining defined WS mutants, the maladaptive characteristic was found to be the expression of cellulose. SM-like revertants had a higher growth rate than WS and no longer expressed significant amounts of cellulose, further confirming that the expression of this high-cost polymer was the basis of maladaptation and the target of compensatory mutation in developing colonies. However, examination of the fate of WS-founded populations in either multiple-colony or single mega-colony agar plate microcosms demonstrated that the loss of WS lineages could be reduced under conditions in which the rapid spreading colony phenotype could dominate nutrient and oxygen access more effectively than competing SM/SM-like genotypes. WS-like isolates recovered from such populations showed increased WS phenotype stability as well as changes in the degree of colony spreading, confirming that the WS was adapting to the two-dimensional agar plate microcosm. PMID:17710140

Hyperspectral imaging for differentiating colonies of non-O157 shiga-toxin producing echerichia coli (STEC) serogroups on spread plates of pure cultures

USDA-ARS?s Scientific Manuscript database

Direct plating onto solid agar media has been widely used in microbiology laboratories for presumptive-positive pathogen detection in spite of the fact that it is often subjective, labor intensive and time consuming. Rainbow agar is a selective and chromogenic medium that helps to detect pathogenic ...

Combination of nutrients in a mammalian cell culture medium kills cryptococci.

PubMed

Granger, Donald L; Call, Donna M

2018-06-06

We found that a large inoculum of Cryptococcus gattii cells, when plated on Dulbecco's modified eagle's medium (DMEM) incorporated into agar, died within a few hours provided that DMEM agar plates had been stored in darkness for approximately 3 days after preparation. Standard conditions were developed for quantification of killing. The medium lost its fungicidal activity when exposed to visible light of wave length ∼400 nm. The amount of energy required was estimated at 5.8 × 104 joules @ 550 nm. Liquid DMEM conditioned by incubation over DMEM agar plates stored in darkness was fungicidal. We found that fungicidal activity was heat-stable (100°C). Dialysis tubing with MWC0 < 100 Daltons retained fungicidal activity. Neutral pH was required. Strains of Cryptococcus were uniformly sensitive, but some Candida species were resistant. Components of DMEM required for killing were pyridoxal and cystine. Micromolar amounts of iron shortened the time required for DMEM agar plates to become fungicidal when stored in the dark. Organic and inorganic compounds bearing reduced sulfur atoms at millimolar concentrations inhibited fungicidal activity. Our results point to a light-sensitive antifungal compound formed by reaction of pyridoxal with cystine possibly by Schiff base formation.

Evaluation of cephamycins as supplements to selective agar for detecting Campylobacter spp. in chicken carcass rinses.

PubMed

Chon, Jung-Whan; Kim, Young-Ji; Kim, Hong-Seok; Kim, Dong-Hyeon; Kim, Hyunsook; Song, Kwang-Young; Sung, Kidon; Seo, Kun-Ho

2016-04-16

Although cefoperazone is the most commonly used antibiotic in Campylobacter-selective media, the distribution of cefoperazone-resistant bacteria such as extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli is increasing. Here we evaluated the potential of cephamycins for use as supplements to improve modified charcoal-cefoperazone-deoxycholate agar (mCCDA) by replacing cefoperazone with the same concentrations (32 mg/L) of cefotetan (modified charcoal-cefotetan-deoxycholate agar, mCCtDA) and cefoxitin (modified charcoal-cefoxitin-deoxycholate agar, mCCxDA). In chicken carcass rinse samples, the number of mCCDA plates detecting for Campylobacter (18/70, 26%) was significantly lower than that of mCCtDA (42/70, 60%) or mCCxDA plates (40/70, 57%). The number of mCCDA plates (70/70, 100%) that were contaminated with non-Campylobacter species was significantly higher than that of mCCtDA (20/70, 29%) or mCCxDA plates (21/70, 30%). The most common competing species identified using mCCDA was ESBL-producing E. coli, while Pseudomonas species frequently appeared on mCCtDA and mCCxDA. Copyright © 2016. Published by Elsevier B.V.

Arabidopsis alcohol dehydrogenase expression in both shoots and roots is conditioned by root growth environment

NASA Technical Reports Server (NTRS)

Chung, H. J.; Ferl, R. J.

1999-01-01

It is widely accepted that the Arabidopsis Adh (alcohol dehydrogenase) gene is constitutively expressed at low levels in the roots of young plants grown on agar media, and that the expression level is greatly induced by anoxic or hypoxic stresses. We questioned whether the agar medium itself created an anaerobic environment for the roots upon their growing into the gel. beta-Glucuronidase (GUS) expression driven by the Adh promoter was examined by growing transgenic Arabidopsis plants in different growing systems. Whereas roots grown on horizontal-positioned plates showed high Adh/GUS expression levels, roots from vertical-positioned plates had no Adh/GUS expression. Additional results indicate that growth on vertical plates closely mimics the Adh/GUS expression observed for soil-grown seedlings, and that growth on horizontal plates results in induction of high Adh/GUS expression that is consistent with hypoxic or anoxic conditions within the agar of the root zone. Adh/GUS expression in the shoot apex is also highly induced by root penetration of the agar medium. This induction of Adh/GUS in shoot apex and roots is due, at least in part, to mechanisms involving Ca2+ signal transduction.

Radiation Resistance of Asporogenous Bacteria in Frozen Beef

DTIC Science & Technology

1976-03-01

Salmonella enteritidis , and Escherichia coli were used. Cultures were grown to the maximum stationary phase for use as an inoculum. Ground beef containing...eosin methylene blue agar, Shigella- Salmonella agar, and growth on plate count agar with 2.5% and 6.5% NaCl was observed. Penicillin susceptibility was...selective media as follows: Staphylococcus Medium No. 110 for S. aureus; Violet Red Bile Agar for E. coli; and Bismuth Sulfite Agar for S. enteritidis

The Efficiency of UVC Radiation in the Inactivation of
Listeria monocytogenes on Beef-Agar Food Models.

PubMed

Hamidi-Oskouei, Amir M; James, Christian; James, Stephen

2015-06-01

The aim of this study is to evaluate the effect of meat content and surface smoothness on the deactivation of Listeria monocytogenes in beef-agar food models achieved by shortwave ultraviolet (UVC) light. Food models with various meat contents were made using chopped beef slices and agar solution. Prepared models together with a Listeria selective agar (LSA) plate and a slice of cooked beef were inoculated with L. monocytogenes and then exposed to UVC light. Population of Listeria reduced to below the level of detection on the LSA plates. As the content of beef in the beef-agar models increased, more L. monocytogenes cells survived. Survival was greatest on the treated cooked slice of beef. To better understand the effect of surface irregularities, a white light interferometer was used to analyse the surface smoothness of beef-agar media and LSA plates. No correlation was observed between the surface roughness of seven out of nine types of produced beef-agar media and the degree of inactivation resulting from UVC radiation at the given dose, whereas, less bacterial cells were killed as beef content of the food models increased. The findings of the current study show that the chemical composition of the treated sample also plays an important role in pathogen resistance and survival, meaning that two samples with similar surface irregularities but different chemical composition might produce very different inactivation results when exposed to UVC light.

The Efficiency of UVC Radiation in the Inactivation of
Listeria monocytogenes on Beef-Agar Food Models

PubMed Central

James, Christian; James, Stephen

2015-01-01

Summary The aim of this study is to evaluate the effect of meat content and surface smoothness on the deactivation of Listeria monocytogenes in beef-agar food models achieved by shortwave ultraviolet (UVC) light. Food models with various meat contents were made using chopped beef slices and agar solution. Prepared models together with a Listeria selective agar (LSA) plate and a slice of cooked beef were inoculated with L. monocytogenes and then exposed to UVC light. Population of Listeria reduced to below the level of detection on the LSA plates. As the content of beef in the beef-agar models increased, more L. monocytogenes cells survived. Survival was greatest on the treated cooked slice of beef. To better understand the effect of surface irregularities, a white light interferometer was used to analyse the surface smoothness of beef-agar media and LSA plates. No correlation was observed between the surface roughness of seven out of nine types of produced beef-agar media and the degree of inactivation resulting from UVC radiation at the given dose, whereas, less bacterial cells were killed as beef content of the food models increased. The findings of the current study show that the chemical composition of the treated sample also plays an important role in pathogen resistance and survival, meaning that two samples with similar surface irregularities but different chemical composition might produce very different inactivation results when exposed to UVC light. PMID:27904353

Frequency of methicillin-resistant Staphylococcus aureus nasal colonization among patients suffering from methicillin resistant Staphylococcus aureus bacteraemia

PubMed Central

Aslam, Nadia; Izhar, Mateen; Mehdi, Naima

2013-01-01

Objective: To determine rate of nasal colonization in Patients suffering from bacteraemia caused by methicillin resistant Staphylococcus aureus. Methods: This descriptive cross sectional study was carried out in a tertiary ca re, University Teaching Hospital (Shaikh Zayed Hospital, Lahore) from October 2010 to August 2011. Nasal swabs were taken from patients suffering from MRSA bacteraemia and were plated on mannitol salt agar plates to isolate Staphylococcus aureus (S. aureus) which were then tested for oxacillin susceptibility. Results: Nasal colonization was present in 52.5% of patients suffering from MRSA bacteraemia. Conclusion: Nasal colonization rates with MRSA were high among patients suffering from MRSA bacteraemia especially in those undergoing dialysis or surgical procedures. Therefore, screening and nasal decolonization should be practiced in hospitals. PMID:24550968

Towards a phenotypic screening strategy for emerging β-lactamases in Gram-negative bacilli.

PubMed

Willems, Elise; Verhaegen, Jan; Magerman, Koen; Nys, Sita; Cartuyvels, Reinoud

2013-02-01

The purpose of this manuscript was to review recent literature and guidelines regarding phenotypic detection of emerging β-lactamases [extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases and carbapenemases] in Gram-negative bacilli (GNB) in order to formulate recommendations on best practice to screen for them. We conclude that chromogenic ESBL screening agar plates are suitable to screen for ESBL-producing Enterobacteriaceae directly from clinical samples. Furthermore, ceftazidime (CAZ) and ceftriaxone or cefotaxime (CTX) are the indicator antimicrobial agents of choice for ESBL detection in GNB. In non-inducible Enterobacteriaceae, the combined double-disk synergy test (CDDST) with at least CTX and CAZ and additionally cefepime as indicators is the preferred ESBL confirmation assay. The two most suitable ESBL confirmation strategies in AmpC co-producing Enterobacteriaceae are adapted CDDSTs: (i) with addition of 3-aminophenylboronic acid to CTX and CAZ disks; and (ii) with addition of cloxacillin (CLOX) to Mueller-Hinton agar. Reduced cefoxitin susceptibility and decreased susceptibility to cefotetan are regarded as suitable screening tests for plasmid-mediated and derepressed AmpC production. A CLOX-based CDDST with CTX and CAZ as indicators is considered to be the best AmpC confirmation assay. Finally, in Enterobacteriaceae isolates we suggest to screen for carbapenemases with a 0.5 μg/mL meropenem screening breakpoint. For class A carbapenemase confirmation, the home-prepared as well as the commercially available boronic acid-based CDDST can be considered. For metallo-β-lactamase confirmation, ethylene diamine tetra-acetic-acid-based home-prepared assays are recommended. The most suitable method (CDDST or DDST) and indicator antimicrobial agent(s) vary depending on the bacterial genus. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Predicting the growth situation of Pseudomonas aeruginosa on agar plates and meat stuffs using gas sensors

PubMed Central

Gu, Xinzhe; Sun, Ye; Tu, Kang; Dong, Qingli; Pan, Leiqing

2016-01-01

A rapid method of predicting the growing situation of Pseudomonas aeruginosa is presented. Gas sensors were used to acquire volatile compounds generated by P. aeruginosa on agar plates and meat stuffs. Then, optimal sensors were selected to simulate P. aeruginosa growth using modified Logistic and Gompertz equations by odor changes. The results showed that the responses of S8 or S10 yielded high coefficients of determination (R2) of 0.89–0.99 and low root mean square errors (RMSE) of 0.06–0.17 for P. aeruginosa growth, fitting the models on the agar plate. The responses of S9, S4 and the first principal component of 10 sensors fit well with the growth of P. aeruginosa inoculated in meat stored at 4 °C and 20 °C, with R2 of 0.73–0.96 and RMSE of 0.25–1.38. The correlation coefficients between the fitting models, as measured by electronic nose responses, and the colony counts of P. aeruginosa were high, ranging from 0.882 to 0.996 for both plate and meat samples. Also, gas chromatography–mass spectrometry results indicated the presence of specific volatiles of P. aeruginosa on agar plates. This work demonstrated an acceptable feasibility of using gas sensors—a rapid, easy and nondestructive method for predicting P. aeruginosa growth. PMID:27941841

Identification of fecal input sites in spring water by selection and genotyping of multiresistant Escherichia coli.

PubMed

Wicki, Melanie; Karabulut, Fatma; Auckenthaler, Adrian; Felleisen, Richard; Tanner, Marcel; Baumgartner, Andreas

2011-12-01

The localization of fecal input sites is important for water quality management. For this purpose, we have developed a new approach based on a three-step procedure, including a preparatory phase, the screening of multiresistant bacteria using selective agar plates, and a typing phase where selected Escherichia coli isolates are characterized by antibiotic resistance profiles and molecular fingerprinting techniques (pulsed-field gel electrophoresis [PFGE]). These two well-known source tracking methods were combined in order to reduce cost and effort. This approach was successfully applied under field conditions in a study area located in the north-western part of Switzerland. E. coli isolates from spring water and surface water samples collected in this area were screened with selective agar plates. In this way, 21 different groups, each consisting of strains with the same pattern of antibiotic resistance, were found. Of these, four groups were further analyzed using PFGE. Strains with identical PFGE profiles were detected repeatedly, demonstrating the suitability of this method for the localization of fecal input sites over an extended period of time. Identical PFGE patterns of strains detected in water from two different springs were also found in the stream flowing through the study area. These results demonstrated the applicability of the new approach for the examination of incidents of fecal contamination in drinking water. The advantages of the described approach over genotyping methods currently being used to identify sources of fecal contaminants are a reduction in time, costs, and the effort required. Identical isolates could be identified without the construction of large libraries.

A simple agar plate preparation for effective transfer of Ureaplasma colonies onto nitrocellulose membranes for colony immunoblotting.

PubMed

Zimmerman, Carl-Ulrich R; Stiedl, Thomas; Spergser, Joachim; Rosengarten, Renate

2014-09-01

A simple method for preparing agar plates is presented, which allows an efficient transfer of Ureaplasma colonies to nitrocellulose membranes for subsequent immunological detection. This simple and reproducible procedure was used to demonstrate antigenic variation in the phase-variable mba-locus of Ureaplasma parvum serovar 3. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of a selective agar plate for the detection of Campylobacter spp. in fresh produce.

PubMed

Yoo, Jin-Hee; Choi, Na-Young; Bae, Young-Min; Lee, Jung-Su; Lee, Sun-Young

2014-10-17

This study was conducted to develop a selective medium for the detection of Campylobacter spp. in fresh produce. Campylobacter spp. (n=4), non-Campylobacter (showing positive results on Campylobacter selective agar) strains (n=49) isolated from fresh produce, indicator bacteria (n=13), and spoilage bacteria isolated from fresh produce (n=15) were plated on four Campylobacter selective media. Bolton agar and modified charcoal cefoperazone deoxycholate agar (mCCDA) exhibited higher sensitivity for Campylobacter spp. than did Preston agar and Hunt agar, although certain non-Campylobacter strains isolated from fresh produce by using a selective agar isolation method, were still able to grow on Bolton agar and mCCDA. To inhibit the growth of non-Campylobacter strains, Bolton agar and mCCDA were supplemented with 5 antibiotics (rifampicin, polymyxin B, sodium metabisulfite, sodium pyruvate, ferrous sulfate) and the growth of Campylobacter spp. (n=7) and non-Campylobacter strains (n=44) was evaluated. Although Bolton agar supplemented with rifampicin (BR agar) exhibited a higher selectivity for Campylobacter spp. than did mCCDA supplemented with antibiotics, certain non-Campylobacter strains were still able to grow on BR agar (18.8%). When BR agar with various concentrations of sulfamethoxazole-trimethoprim were tested with Campylobacter spp. (n=8) and non-Campylobacter (n=7), sulfamethoxazole-trimethoprim was inhibitory against 3 of 7 non-Campylobacter strains. Finally, we validated the use of BR agar containing 50mg/L sulfamethoxazole (BRS agar) or 0.5mg/L ciprofloxacin (BRCS agar) and other selective agars for the detection of Campylobacter spp. in chicken and fresh produce. All chicken samples were positive for Campylobacter spp. when tested on mCCDA, BR agar, and BRS agar. In fresh produce samples, BRS agar exhibited the highest selectivity for Campylobacter spp., demonstrating its suitability for the detection of Campylobacter spp. in fresh produce. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid automated method for screening of enteric pathogens from stool specimens.

PubMed Central

Villasante, P A; Agulla, A; Merino, F J; Pérez, T; Ladrón de Guevara, C; Velasco, A C

1987-01-01

A total of 800 colonies suggestive of Salmonella, Shigella, or Yersinia species isolated on stool differential agar media were inoculated onto both conventional biochemical test media (triple sugar iron agar, urea agar, and phenylalanine agar) and Entero Pathogen Screen cards of the AutoMicrobic system (Vitek Systems, Inc., Hazelwood, Mo.). Based on the conventional tests, the AutoMicrobic system method yielded the following results: 587 true-negatives, 185 true-positives, 2 false-negatives, and 26 false-positives (sensitivity, 99%; specificity, 96%). Both true-positive and true-negative results were achieved considerably earlier than false results (P less than 0.001). The Entero Pathogen Screen card method is a fast, easy, and sensitive method for screening for Salmonella, Shigella, or Yersinia species. The impossibility of screening for oxidase-positive pathogens is a minor disadvantage of this method. PMID:3553230

Identification of non-streptococcal organisms from human dental plaque grown on the Streptococcus-selective medium mitis-salivarius agar.

PubMed

Kim, Yeon-Hee; Lee, Si Young

2015-02-01

Mitis-salivarius (MS) agar has been used widely in microbial epidemiological studies because oral viridans streptococci can be selectively grown on this medium. Even though the previous findings reported the limited selecting power of MS agar for streptococcus strains, the identities of non-streptococcal strains from human oral samples which can grow on this medium are not clear yet. In this study, we identified non-streptococcal organisms grown on MS agar plates by polymerase chain reaction (PCR) amplification and sequencing of the 16S ribosomal RNA (rRNA) gene. Eighty bacterial colonies on MS plates were isolated from plaque samples, and bacterial identification was achieved with the rapid ID 32 Strep system and mini API reader. The bacterial colonies identified as non-streptococci by the API system were selected for further identification. The 16S rRNA gene was amplified by PCR and verified using DNA sequencing analysis for identification. Sequences were compared with those of reference organisms in the genome database of the National Center for Biotechnology Information using the Basic Local Alignment Search Tool (BLAST). Among the 11 isolated non-streptococcal strains on MS plates, 3 strains were identified as Actinomyces naeslundii, 7 strains were identified as Actinomyces oris and 1 strain were identified as Actinomyces sp. using Blastn. In this study, we showed that some oral Actinomyces species can grow on Streptococcus-selective MS agar plates. Copyright © 2014 Elsevier Ltd. All rights reserved.

Evaluation of the ability of four ESBL-screening media to detect ESBL-producing Salmonella and Shigella.

PubMed

Sturød, Kjersti; Dahle, Ulf R; Berg, Einar Sverre; Steinbakk, Martin; Wester, Astrid L

2014-09-04

The aim of this study was to compare the ability of four commercially available media for screening extended-spectrum beta-lactamase (ESBL) to detect and identify ESBL-producing Salmonella and Shigella in fecal samples. A total of 71 Salmonella- and 21 Shigella-isolates producing ESBL(A) and/or AmpC, were received at Norwegian Institute of Public Health between 2005 and 2012. The 92 isolates were mixed with fecal specimens and tested on four ESBL screening media; ChromID ESBL (BioMèrieux), Brilliance ESBL (Oxoid), BLSE agar (AES Chemunex) and CHROMagar ESBL (CHROMagar). The BLSE agar is a biplate consisting of two different agars. Brilliance and CHROMagar are supposed to suppress growth of AmpC-producing bacteria while ChromID and BLSE agar are intended to detect both ESBL(A) and AmpC. The total sensitivity (ESBL(A)+AmpC) with 95% confidence intervals after 24 hours of incubation were as follows: ChromID: 95% (90.4-99.6), Brilliance: 93% (87.6-98.4), BLSE agar (Drigalski): 99% (96.9-100), BLSE agar (MacConkey): 99% (96.9-100) and CHROMagar: 85% (77.5-92.5). The BLSE agar identified Salmonella and Shigella isolates as lactose-negative. The other agars based on chromogenic technology displayed Salmonella and Shigella flexneri isolates with colorless colonies (as expected). Shigella sonnei produced pink colonies, similar to the morphology described for E. coli. All four agar media were reliable in screening fecal samples for ESBL(A)-producing Salmonella and Shigella. However, only ChromID and BLSE agar gave reliable detection of AmpC-producing isolates. Identification of different bacterial species based on colony colour alone was not accurate for any of the four agars.

Laboratory Evaluation of Australian Ration Packs.

DTIC Science & Technology

1982-09-01

Monitoring of Freeze Dried Meals , Potato & Onion Powder .......................... 40 Appendix 6 Emergency Flying Ration ............................ 41...Moulds were enumerated using the pour plate method as described in AS 1766 Part 2.1.2 1976. On occasions malt extract agar was used in place of...potato dextrose agar . Coliforms & E. coli were enumerated using the pour plate method as described in AS 1766 Part 2.1.3.7 1976. Violet red bile dextrose

Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation.

PubMed

Kusakawa, Shinji; Yasuda, Satoshi; Kuroda, Takuya; Kawamata, Shin; Sato, Yoji

2015-12-08

Contamination with tumorigenic cellular impurities is one of the most pressing concerns for human cell-processed therapeutic products (hCTPs). The soft agar colony formation (SACF) assay, which is a well-known in vitro assay for the detection of malignant transformed cells, is applicable for the quality assessment of hCTPs. Here we established an image-based screening system for the SACF assay using a high-content cell analyzer termed the digital SACF assay. Dual fluorescence staining of formed colonies and the dissolution of soft agar led to accurate detection of transformed cells with the imaging cytometer. Partitioning a cell sample into multiple wells of culture plates enabled digital readout of the presence of colonies and elevated the sensitivity for their detection. In practice, the digital SACF assay detected impurity levels as low as 0.00001% of the hCTPs, i.e. only one HeLa cell contained in 10,000,000 human mesenchymal stem cells, within 30 days. The digital SACF assay saves time, is more sensitive than in vivo tumorigenicity tests, and would be useful for the quality control of hCTPs in the manufacturing process.

Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation

PubMed Central

Kusakawa, Shinji; Yasuda, Satoshi; Kuroda, Takuya; Kawamata, Shin; Sato, Yoji

2015-01-01

Contamination with tumorigenic cellular impurities is one of the most pressing concerns for human cell-processed therapeutic products (hCTPs). The soft agar colony formation (SACF) assay, which is a well-known in vitro assay for the detection of malignant transformed cells, is applicable for the quality assessment of hCTPs. Here we established an image-based screening system for the SACF assay using a high-content cell analyzer termed the digital SACF assay. Dual fluorescence staining of formed colonies and the dissolution of soft agar led to accurate detection of transformed cells with the imaging cytometer. Partitioning a cell sample into multiple wells of culture plates enabled digital readout of the presence of colonies and elevated the sensitivity for their detection. In practice, the digital SACF assay detected impurity levels as low as 0.00001% of the hCTPs, i.e. only one HeLa cell contained in 10,000,000 human mesenchymal stem cells, within 30 days. The digital SACF assay saves time, is more sensitive than in vivo tumorigenicity tests, and would be useful for the quality control of hCTPs in the manufacturing process. PMID:26644244

Individual based simulations of bacterial growth on agar plates

NASA Astrophysics Data System (ADS)

Ginovart, M.; López, D.; Valls, J.; Silbert, M.

2002-03-01

The individual based simulator, INDividual DIScrete SIMulations (INDISIM) has been used to study the behaviour of the growth of bacterial colonies on a finite dish. The simulations reproduce the qualitative trends of pattern formation that appear during the growth of Bacillus subtilis on an agar plate under different initial conditions of nutrient peptone concentration, the amount of agar on the plate, and the temperature. The simulations are carried out by imposing closed boundary conditions on a square lattice divided into square spatial cells. The simulator studies the temporal evolution of the bacterial population possible by setting rules of behaviour for each bacterium, such as its uptake, metabolism and reproduction, as well as rules for the medium in which the bacterial cells grow, such as concentration of nutrient particles and their diffusion. The determining factors that characterize the structure of the bacterial colony patterns in the presents simulations, are the initial concentrations of nutrient particles, that mimic the amount of peptone in the experiments, and the set of values for the microscopic diffusion parameter related, in the experiments, to the amount of the agar medium.

Screening method for detection of immediate amino acid decarboxylases--producing bacteria implicated in food poisoning.

PubMed

Hussain, Husniza; Mohd Fuat, A R; Vimala, B; Ghazali, H M

2011-08-01

Assessment of amino acid decarboxylase activity can be conducted using tubed broth or plated agar. In this study, the test was carried out in microtitre plates containing lysine, ornithine, arginine, tyrosine, tryptophan, phenylalanine or histidine as biogenic amine precursors. Møller decarboxylase base broth (MDB) with or without 1% of a known amino acid were added to wells of a 96 well-microtitre plate. The wells were inoculated with Escherichia coli, Klebsiella pneumoniae, Acinetobacter anitratus or Staphylococcus aureus to the final concentration of 6.0 x 10(7) cfu/ml and incubated at 35ºC. The absorbance of the culture broth was read at 570 nm at 0, 1.0, 2.0, 3.0, 4.0, 5.5, 6.5 and 7.5 hour. Comparison of means of A'(570) between 0 hour and a specified incubation time was determined statistically. Positive decarboxylase activities were detected in the media inoculated with E. coli and K. pneumoniae in less than 6 hours. The current method is suitable for immediate producers of amino acid decarboxylase enzymes. It costs less as it uses less amino acid and it has the potential to be used for screening aliquots of food materials for amino acid decarboxylase activities.

[Chemical-genetics based screening for furanonaphthoquinone producing endophytic actinomycetes from seeds of Trewia nudiflora].

PubMed

Li, Fang; Kang, Qianjin; Yao, Xiaoling; Li, Yanyan; Wei, Maolong; Cao, Yong; Lin, Shuangjun; Bai, Linquan; Ma, Wei; Deng, Zixin

2012-04-04

The seeds of Trewia nudiflora containing maytansine (an anticancer agent), was investigated to explore the endophytic actinomycetes diversity and screen for naphthoquinones producing strain. The seeds of Trewia nudiflora were sliced and plated on different selective media after surface sterilization. Clones that looked like actinomycetes were selected, and classified according to the 16S rRNA sequences. Isolated strains were screened for furanonaphthoquinone biosynthesis gene by PCR, and tested for antibacterial and antifungal activity using Staphyloccocusaureus, Pseudomon-asaeruginosa, Bacillus subtilis, Rhizoctoniasolani and Gibberellasaubinetii. LC-MS and NMR were used to determine the structure of candidate compounds. More than 100 endophytic bacteria were isolated. Among them 66 were streptomycetes. FNQ6 (polyketide synthase Type III) and FNQ21 (carboxymuconate cycloisomerase) were only detected in Streptomyces sp. HTZ 27. We got 5 mg pure furanonaphthoquinone (FNQI) from 1 liter Streptomyces sp. HTZ 27 agar fermentation medium. The use of chemical-genetics method increased the efficiency of screening for target compound producing bacteria.

Hydrodynamics of bacterial colonies: Phase diagrams

NASA Astrophysics Data System (ADS)

Lega, J.; Passot, T.

2004-09-01

We present numerical simulations of a recent hydrodynamic model describing the growth of bacterial colonies on agar plates. We show that this model is able to qualitatively reproduce experimentally observed phase diagrams, which relate a colony shape to the initial quantity of nutrients on the plate and the initial wetness of the agar. We also discuss the principal features resulting from the interplay between hydrodynamic motions and colony growth, as described by our model.

RAPA: a novel in vitro method to evaluate anti-bacterial skin cleansing products.

PubMed

Ansari, S A; Gafur, R B; Jones, K; Espada, L A; Polefka, T G

2010-04-01

Development of efficacious anti-bacterial skin cleansing products has been limited by the availability of a pre-clinical (in vitro) method to predict clinical efficacy adequately. We report a simple and rapid method, designated as rapid agar plate assay (RAPA), that uses the bacteriological agar surface as a surrogate substrate for skin and combines elements of two widely used in vivo (clinical) methods (Agar Patch and Cup Scrub). To simulate the washing of the human hand or forearm skin with the test product, trypticase soy agar plates were directly washed with the test product and rinsed under running tap water. After air-drying the washed plates, test bacteria (Staphylococcus aureus or Escherichia coli) were applied and the plates were incubated at 37 degrees C for 18-24 h. Using S. aureus as the test organism, anti-bacterial bar soap containing triclocarbanilide showed a strong linear relationship (R(2) = 0.97) between bacterial dose and their per cent reduction. A similar dose-response relationship (R(2) = 0.96) was observed for anti-bacterial liquid hand soap against E. coli. RAPA was able to distinguish between anti-bacterial products based on the nature and level of actives in them. In limited comparative tests, results obtained by RAPA were comparable with the results obtained by clinical agar patch and clinical cup scrub methods. In conclusion, RAPA provides a simple, rugged and reproducible in vitro method for testing the relative efficacy of anti-bacterial skin cleansing products with a likelihood of comparable clinical efficacy. Further testing is warranted to improve the clinical predictability of this method.

Clinical effectiveness of rapid tests for methicillin resistant Staphylococcus aureus (MRSA) in hospitalized patients: a systematic review

PubMed Central

2011-01-01

Background Methicillin resistant Staphylococcus aureus (MRSA) are often resistant to multiple classes of antibiotics. The research objectives of this systematic review were to evaluate the clinical effectiveness of polymerase chain reaction (PCR) versus chromogenic agar for MRSA screening, and PCR versus no screening for several clinical outcomes, including MRSA colonization and infection rates. Methods An electronic literature search was conducted on studies evaluating polymerase chain reaction techniques and methicillin (also spelled meticillin) resistant Staphylococcus aureus that were published from 1993 onwards using Medline, Medline In-Process & Other Non-Indexed Citations, BIOSIS Previews, and EMBASE. Due to the presence of heterogeneity in the selected studies, the clinical findings of individual studies were described. Results Nine studies that compared screening for MRSA using PCR versus screening using chromogenic agar in a hospital setting, and two studies that compared screening using PCR with no or targeted screening were identified. Some studies found lower MRSA colonization and acquisition, infection, and transmission rates in screening with PCR versus screening with chromogenic agar, and the turnaround time for screening test results was lower for PCR. One study reported a lower number of unnecessary isolation days with screening using PCR versus screening with chromogenic agar, but the proportion of patients isolated was similar between both groups. The turnaround time for test results and number of isolation days were lower for PCR versus chromogenic agar for MRSA screening. Conclusions The use of PCR for MRSA screening demonstrated a lower turnaround time and number of isolation days compared with chromogenic agar. Given the mixed quality and number of studies (11 studies), gaps remain in the published literature and the evidence remains insufficient. In addition to screening, factors such as the number of contacts between healthcare workers and patients, number of patients attended by one healthcare worker per day, probability of colonization among healthcare workers, and MRSA status of hospital shared equipment and hospital environment must be considered to control the transmission of MRSA in a hospital setting. PMID:22151575

Comparison of real-time PCR with disk diffusion, agar screen and E-test methods for detection of methicillin-resistant Staphylococcus aureus.

PubMed

Shariati, Laleh; Validi, Majid; Tabatabaiefar, Mohammad Amin; Karimi, Ali; Nafisi, Mohammad Reza

2010-12-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen. Our main objective was to compare oxacillin disk test, oxacillin E-test, and oxacillin agar screen for detection of methicillin resistance in S. aureus, using real-time PCR for mecA as the "gold standard" comparison assay. 196 S. aureus isolates were identified out of 284 Staphylococcus isolates. These isolates were screened for MRSA with several methods: disk diffusion, agar screen (6.0 μg/ml), oxacillin E-test, and real-time PCR for detection of mecA gene. Of the 196 S. aureus isolates tested, 96 isolates (49%) were mecA-positive and 100 isolates (51%) mecA-negative. All methods tested had a statistically significant agreement with real-time PCR. E-test was 100% sensitive and specific for mecA presence. The sensitivity and specificity of oxacillin agar screen method were 98 and 99%, respectively and sensitivity and specificity of oxacillin disk diffusion method were 95 and 93%, respectively. In the present study, oxacillin E-test is proposed as the best phenotypic method. For economic reasons, the oxacillin agar screen method (6.0 μg/ml), which is suitable for the detection of MRSA, is recommended due to its accuracy and low cost.

Comparison of plate counts, Petrifilm, dipslides, and adenosine triphosphate bioluminescence for monitoring bacteria in cooling-tower waters.

PubMed

Mueller, Sherry A; Anderson, James E; Kim, Byung R; Ball, James C

2009-04-01

Effective bacterial control in cooling-tower systems requires accurate and timely methods to count bacteria. Plate-count methods are difficult to implement on-site, because they are time- and labor-intensive and require sterile techniques. Several field-applicable methods (dipslides, Petrifilm, and adenosine triphosphate [ATP] bioluminescence) were compared with the plate count for two sample matrices--phosphate-buffered saline solution containing a pure culture of Pseudomonas fluorescens and cooling-tower water containing an undefined mixed bacterial culture. For the pure culture, (1) counts determined on nutrient agar and plate-count agar (PCA) media and expressed as colony-forming units (CFU) per milliliter were equivalent to those on R2A medium (p = 1.0 and p = 1.0, respectively); (2) Petrifilm counts were not significantly different from R2A plate counts (p = 0.99); (3) the dipslide counts were up to 2 log units higher than R2A plate counts, but this discrepancy was not statistically significant (p = 0.06); and (4) a discernable correlation (r2 = 0.67) existed between ATP readings and plate counts. For cooling-tower water samples (n = 62), (1) bacterial counts using R2A medium were higher (but not significant; p = 0.63) than nutrient agar and significantly higher than tryptone-glucose yeast extract (TGE; p = 0.03) and PCA (p < 0.001); (2) Petrifilm counts were significantly lower than nutrient agar or R2A (p = 0.02 and p < 0.001, respectively), but not statistically different from TGE, PCA, and dipslides (p = 0.55, p = 0.69, and p = 0.91, respectively); (3) the dipslide method yielded bacteria counts 1 to 3 log units lower than nutrient agar and R2A (p < 0.001), but was not significantly different from Petrifilm (p = 0.91), PCA (p = 1.00) or TGE (p = 0.07); (4) the differences between dipslides and the other methods became greater with a 6-day incubation time; and (5) the correlation between ATP readings and plate counts varied from system to system, was poor (r2 values ranged from < 0.01 to 0.47), and the ATP method was not sufficiently sensitive to measure counts below approximately 10(4) CFU/mL.

Comparison of direct selective versus nonselective agar media plus LIM broth enrichment for determination of group B streptococcus colonization status in pregnant women.

PubMed

Elsayed, Sameer; Gregson, Daniel B; Church, Deirdre L

2003-06-01

Group B streptococcus (GBS) is the most common cause of early-onset neonatal sepsis in developed countries, and determination of the GBS colonization status in pregnant patients near term is essential for the provision of prophylactic measures to prevent early-onset disease. To determine if GBS recovery rates and/or result turnaround times for vaginal or combined vaginal/rectal swab specimens from pregnant patients near term are enhanced if swabs are inoculated initially onto selective versus nonselective agar media, in addition to the standard Centers for Disease Control and Prevention method. Prospective laboratory analysis. Urban health region/centralized diagnostic microbiology laboratory. Pregnant women presenting for routine obstetrical care and collection of vaginal or combined vaginal/rectal swab specimens for GBS testing at 35 to 37 weeks' gestation. Culture of specimens directly onto selective (5% sheep blood with colistin and nalidixic acid) or nonselective (5% sheep blood) agar media, in addition to LIM broth enrichment and terminal subculture. Group B streptococcus recovery rate and culture result turnaround time. A total of 639 specimens were tested, with 128 (20%) positive for GBS. Sixty-three isolates were recovered on direct agar media at 24 hours, of which 16 (12.5%) were isolated on selective plates only. An additional 38 isolates were recovered at 48 hours from direct plates. Twenty-seven (21.1%) isolates that failed to grow on direct plates were recovered from the LIM broth subculture only. Three (2.3%) isolates not recovered from LIM broths were detected at 48 hours on the direct selective (2 isolates) and nonselective (1 isolate) agar plates. A 24-hour result turnaround time was achieved for 63 (49.2%) and 47 (36.7%) of the 128 culture-positive specimens for direct selective and nonselective plates, respectively (chi2 = 76.63, P <.001). Use of direct selective agar media, in addition to LIM broth enrichment, for the determination of the GBS colonization status in pregnant patients near term results in decreased turnaround time for reporting positive results.

Validation of an automated colony counting system for group A Streptococcus.

PubMed

Frost, H R; Tsoi, S K; Baker, C A; Laho, D; Sanderson-Smith, M L; Steer, A C; Smeesters, P R

2016-02-08

The practice of counting bacterial colony forming units on agar plates has long been used as a method to estimate the concentration of live bacteria in culture. However, due to the laborious and potentially error prone nature of this measurement technique, an alternative method is desirable. Recent technologic advancements have facilitated the development of automated colony counting systems, which reduce errors introduced during the manual counting process and recording of information. An additional benefit is the significant reduction in time taken to analyse colony counting data. Whilst automated counting procedures have been validated for a number of microorganisms, the process has not been successful for all bacteria due to the requirement for a relatively high contrast between bacterial colonies and growth medium. The purpose of this study was to validate an automated counting system for use with group A Streptococcus (GAS). Twenty-one different GAS strains, representative of major emm-types, were selected for assessment. In order to introduce the required contrast for automated counting, 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) dye was added to Todd-Hewitt broth with yeast extract (THY) agar. Growth on THY agar with TTC was compared with growth on blood agar and THY agar to ensure the dye was not detrimental to bacterial growth. Automated colony counts using a ProtoCOL 3 instrument were compared with manual counting to confirm accuracy over the stages of the growth cycle (latent, mid-log and stationary phases) and in a number of different assays. The average percentage differences between plating and counting methods were analysed using the Bland-Altman method. A percentage difference of ±10 % was determined as the cut-off for a critical difference between plating and counting methods. All strains measured had an average difference of less than 10 % when plated on THY agar with TTC. This consistency was also observed over all phases of the growth cycle and when plated in blood following bactericidal assays. Agreement between these methods suggest the use of an automated colony counting technique for GAS will significantly reduce time spent counting bacteria to enable a more efficient and accurate measurement of bacteria concentration in culture.

A Repeating Sulfated Galactan Motif Resuscitates Dormant Micrococcus luteus Bacteria.

PubMed

Böttcher, Thomas; Szamosvári, Dávid; Clardy, Jon

2018-07-01

Only a small fraction of bacteria can autonomously initiate growth on agar plates. Nongrowing bacteria typically enter a metabolically inactive dormant state and require specific chemical trigger factors or signals to exit this state and to resume growth. Micrococcus luteus has become a model organism for this important yet poorly understood phenomenon. Only a few resuscitation signals have been described to date, and all of them are produced endogenously by bacterial species. We report the discovery of a novel type of resuscitation signal that allows M. luteus to grow on agar but not agarose plates. Fractionation of the agar polysaccharide complex and sulfation of agarose allowed us to identify the signal as highly sulfated saccharides found in agar or carrageenans. Purification of hydrolyzed κ-carrageenan ultimately led to the identification of the signal as a small fragment of a large linear polysaccharide, i.e., an oligosaccharide of five or more sugars with a repeating disaccharide motif containing d-galactose-4-sulfate (G4S) 1,4-linked to 3,6-anhydro-α-d-galactose (DA), G4S-(DA-G4S) n ≥2 IMPORTANCE Most environmental bacteria cannot initiate growth on agar plates, but they can flourish on the same plates once growth is initiated. While there are a number of names for and manifestations of this phenomenon, the underlying cause appears to be the requirement for a molecular signal indicating safe growing conditions. Micrococcus luteus has become a model organism for studying this growth initiation process, often called resuscitation, because of its apparent connection with the persistent or dormant form of Mycobacterium tuberculosis , an important human pathogen. In this report, we identify a highly sulfated saccharide from agar or carrageenans that robustly resuscitates dormant M. luteus on agarose plates. We identified and characterized the signal as a small repeating disaccharide motif. Our results indicate that signals inherent in or absent from the polysaccharide composition of solid growth media can have major effects on bacterial growth. Copyright © 2018 American Society for Microbiology.

Detection of non-O157 Shiga toxin-producing Escherichia coli in 375 grams of beef trim enrichments across multiple commercial PCR detection platforms.

PubMed

Wheeler, Sarita Raengpradub; Heard, Preciaus; Dufour, Christophe; Thevenot-Sergentet, Delphine; Loukiadis, Estelle; Flowers, Russell S; McMahon, Wendy

2015-01-01

Although serotype O157:H7 remains the pathogenic Shiga toxin-producing Escherichia coli (STEC) of primary concern worldwide, some focus in the United States has shifted to six particular non-O157 STEC serogroups (O26, O45, O103, O111, O121, and O145). Some of these serogroups have also emerged as concerns elsewhere around the world, including Europe. The objective of this work was to compare commercial detection methods with the U.S. Department of Agriculture (USDA) reference method for detection of non-O157 STEC in 375 g of beef trim using a limit of detection study design. Overall, the commercial platforms performed well, showing similar levels of sensitivity for detection of presumptive positives for O45, O26, O103, and O121 (PCR screen results only). For O111, one method that utilizes an integrated immunomagnetic separation and PCR approach was more sensitive than a PCR-only screen approach. Additionally, one commercial method showed more presumptive and confirmed positives overall. Use of an immunomagnetic separation tool, such as antibody-coated beads, aided considerably with the confirmation procedures and is an important step when confirming suspect samples. A secondary goal of this study was to evaluate isolation and International Organization for Standardization confirmation protocols used in Europe compared with strategies provided by the USDA Microbiology Laboratory Guidebook (MLG). Generally, results from the USDA confirmation plates (modified Rainbow agar) were better than the European Union confirmation plates (MacConkey agar with or without rhamnose). In summary, detection of non-O157 STEC in 375 g of beef trim can be performed by any of the three methods on the market evaluated in the study.

Antibacterial Effect of Silver Diamine Fluoride on Cariogenic Organisms.

PubMed

Lou, Yali; Darvell, Brain W; Botelho, Michael G

2018-05-01

To screen the possible antimicrobial activity of a range of clinically used, silver-based compounds on cariogenic organisms: silver diamine fluoride (SDF), silver fluoride, and silver nitrate. Preliminary screening disk-diffusion susceptibility tests were conducted on Mueller-Hinton agar plates inoculated with Streptococcus mutans, Lactobacillus acidophilus, and Actinomyces naeslundii, organisms known to be cariogenic. In order to identify which component of the silver compounds was responsible for any antibacterial (AB) effect, and to provide controls, the following were also investigated at high and low concentrations: sodium fluoride, ammonium fluoride, ammonium chloride, sodium fluoride, sodium chloride, and sodium nitrate, as well as deionized water as control. A volume of 10 pL of a test solution was dispensed onto a paper disk resting on the inoculated agar surface, and the plate incubated anaerobically at 37°C for 48 hours. The zones of inhibition were then measured. Silver diamine fluoride, silver fluoride, silver nitrate, and ammonium fluoride had significant AB effect (p < 0.05) on all three test organisms, although ammonium fluoride had no effect at low concentration; the remaining other compounds had no effect. Silver ions appear to be the principal AB agent at both high and low concentration; fluoride ions only have an AB effect at high concentration, while ammonium, nitrate, chloride and sodium ions have none. The anticaries effect of topical silver solutions appears restricted to that of the silver ions. Silver compounds, such as SDF, silver fluoride, and silver nitrate have AB effect against cariogenic organisms and these may have clinical impact in arresting or preventing dental decay. Sodium fluoride did not have AB effect under the conditions tested.

Outbreak of invasive group A streptococcus: investigations using agar settle plates detect perineal shedding from a healthcare worker.

PubMed

Mahida, N; Prescott, K; Yates, C; Spencer, F; Weston, V; Boswell, T

2018-03-29

Outbreaks of group A streptococcus (GAS) infections may occur in healthcare settings. Transmission to patients is sometimes linked to colonized healthcare workers (HCWs) and/or a contaminated environment. To describe the investigation and control of an outbreak of healthcare-associated GAS on an elderly care medical ward, over six months. Four patients developed septicaemia due to GAS infection without a clinically obvious site of infection. The outbreak team undertook an investigation involving a retrospective review of GAS cases, prospective case finding, HCW screening and environmental sampling using both swabs and settle plates. Immediate control measures included source isolation and additional cleaning of the ward environment with a chlorine disinfectant and hydrogen peroxide. Prospective patient screening identified one additional patient with throat GAS carriage. Settle plate positivity for GAS was strongly associated with the presence of one individual HCW on the ward, who was subsequently found to have GAS perineal carriage. Contamination of a fabric-upholstered chair in an office adjacent to the ward, used by the HCW, was also detected. In total, three asymptomatic HCWs had throat GAS carriage and one HCW had both perineal and throat carriage. All isolates were typed as emm 28. This is the first outbreak report demonstrating the use of settle plates in a GAS outbreak investigation on a medical ward, to identify the likely source of the outbreak. Based on this report we recommend that both throat and perineal sites should be sampled if HCW screening is undertaken during an outbreak of GAS. Fabric, soft furnishings should be excluded from clinical areas as well as any adjacent offices because pathogenic bacteria such as GAS may contaminate this environment. Copyright © 2018 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Determination of antitrypsin activity on agar plates: relationship between antitrypsin and biological value of soybean for trout.

PubMed

Sandholm, M; Smith, R R; Shih, J C; Scott, M L

1976-06-01

A new method is described for determination of antitrypsin activity based on inhibition of trypsin solubilization of calcium caseinate in agar plates. The method was applied to analyze soybeans after graded heat treatments for their antitrypsin content. Biological determination of protein and energy values of the soybean samples showed direct correlation of these values with the destruction of antitrypsin as measured by the new method, using rainbow trout.

High-performance thin-layer chromatography screening of multi class antibiotics in animal food by bioluminescent bioautography and electrospray ionization mass spectrometry.

PubMed

Chen, Yisheng; Schwack, Wolfgang

2014-08-22

The world-wide usage and partly abuse of veterinary antibiotics resulted in a pressing need to control residues in animal-derived foods. Large-scale screening for residues of antibiotics is typically performed by microbial agar diffusion tests. This work employing high-performance thin-layer chromatography (HPTLC) combined with bioautography and electrospray ionization mass spectrometry introduces a rapid and efficient method for a multi-class screening of antibiotic residues. The viability of the bioluminescent bacterium Aliivibrio fischeri to the studied antibiotics (16 species of 5 groups) was optimized on amino plates, enabling detection sensitivity down to the strictest maximum residue limits. The HPTLC method was developed not to separate the individual antibiotics, but for cleanup of sample extracts. The studied antibiotics either remained at the start zones (tetracyclines, aminoglycosides, fluoroquinolones, and macrolides) or migrated into the front (amphenicols), while interfering co-extracted matrix compounds were dispersed at hRf 20-80. Only after a few hours, the multi-sample plate image clearly revealed the presence or absence of antibiotic residues. Moreover, molecular information as to the suspected findings was rapidly achieved by HPTLC-mass spectrometry. Showing remarkable sensitivity and matrix-tolerance, the established method was successfully applied to milk and kidney samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Use of agar diffusion assay to evaluate bactericidal activity of formulations of alkaline salts of fatty acids against bacteria associated with poultry processing

USDA-ARS?s Scientific Manuscript database

The agar diffusion assay was used to examine antibacterial activity of alkaline salts of fatty acids (FA). Wells in agar media seeded with bacteria were filled with FA-potassium hydroxide (KOH) solutions, plates were incubated, and zones of inhibition were measured. The relationship between bacteric...

Differentiating non-0157:H7 STEC serogroups from ground beef plated on agar media by hyperspetral imaging

USDA-ARS?s Scientific Manuscript database

Introduction: The development of an assay to detect and confirm a positive non-O157:H7 isolate is challenging when mixed morphologically results are obtained from the serogroups growing on Rainbow agar. Rainbow agar is only claimed by the manufacturer to be very specific for E.coli O157:H7 strain...

Assessing the growth and recovery of Salmonella Enteritidis SE86 after sodium dichloroisocyanurate exposure

PubMed Central

Ferreira, Fernanda Stoduto; Horvath, Mariana Bandeira; Tondo, Eduardo Cesar

2013-01-01

The objective of the present study was to assess the growth and the recovery of Salmonella (S.) Enteritidis SE86 in different diluents, culture media and using different plating methods after the exposure to 200 mg/kg sodium dichloroisocyanurate (NaDCC). Before and after NaDCC exposure, SE86 was cultured at 30 °C and 7 °C in the following diluents: Peptone water (P), Saline solution (SaS), Peptone water+Saline solution (P+SaS), Peptone water+Tween 80+Lecithin+Sodium thiosulfate (P+N) and Saline solution+Tween 80+Lecithin+Sodium thiosulfate (SaS+N). The SaS diluent was chosen because it was able to maintain cells viable without growth and was further used for plating SE86 on non selective medium (Tryptic Soy Agar-TSA) and on selective media (Mannitol Lysine Crystal Violet Brilliant Green Agar-MLCB; Brilliant Green Agar-BGA; Salmonella Shigella Agar-SS and Xylose Lysine Dextrose–XLD). The Thin Agar Layer method (TAL) i.e., selective media overlayed with non selective TSA was also evaluated. Results indicated that SE86 not exposed to NaDCC was able to grow in P, P+N, SaS+N and P+SaS, but not in SaS, that was able to maintain cells viable. SE86 exposed to NaDCC demonstrated similar counts after dilution in SaS and the plating on non selective TSA, selective media MLCB, BGA, SS and XLD and on TAL media. SE86, S. Typhimurium and S. Bredeney, exposed or not exposed to NaDCC, showed no significant differences in counts on TSA, XLD and XLD overlayed with TSA, suggesting that all those media may be used to quantify NaDCC-exposed Salmonella by plating method. PMID:24516446

Inference of Antibiotic Resistance and Virulence Among Diverse Group A Streptococcus Strains Using emm Sequencing and Multilocus Genotyping Methods

DTIC Science & Technology

2009-09-04

apparent GAS-associated conditions were sampled by oropharyn- geal swab. Swabs were streaked on blood agar plates using Table 3. Isolate properties by...testing, samples were re-streaked on blood agar plates (5% sheep blood in TSA base) (Hardy Diagnostics, Santa Maria, CA), and incubated at 35–37uC with 5–10...sensitivity (A-disk method, Hardy Diagnostics) and positive GAS latex agglutination reaction (Hardy Diagnostics). Confirmed GAS isolates were then

Screening for ligninolytic enzymes from autochthonous fungi and applications for decolorization of Remazole Marine Blue

PubMed Central

Erden, Emre; Ucar, M. Cigdem; Gezer, Tekin; Pazarlioglu, Nurdan Kasikara

2009-01-01

This study presents new and alternative fungal strains for the production of ligninolytic enzymes which have great potential to use in industrial and biotechnological processes. Thirty autochthonous fungal strains were harvested from Bornova-Izmir in Turkiye. In the fresh fruitbody extracts laccase, manganese peroxidase and lignin peroxidase activities, which are the principal enzymes responsible for ligninocellulose degradation by Basidiomycetes, were screened. Spores of some of the basidiomycetes species such as Cortinarius sp., Trametes versicolor, Pleurotus ostreatus, Abortiporus biennis, Lyophyllum subglobisporium, Ramaria stricta, Ganoderma carnosum, Lactarius delicious ve Lepista nuda were isolated and investigated optimum cultivation conditions in submerged fermentation for high yields of ligninolytic enzyme production. In addition, isolated fungal strains were monitored on agar plates whether having the capability of decolorization of a textile dye Remazol Marine Blue. PMID:24031371

Screening of some essential oils against Trichosporon species.

PubMed

Uniyal, Veena; Saxena, Seema; Bhatt, R P

2013-01-01

White Piedra is a superficial mycoses characterized by nodules on the hair shaft, caused by the basidiomycetous yeast Trichosporon species. In this study 25 essential oils were extracted and screened against two Trichosporon species i.e. Trichosporon asahii and Trichosporon cutaneum. Both these fungi procured from MTCC Chandigarh were maintained on yeast malt agar plates and tubes at 25 degrees C. Two screening methods viz., agar well diffusion assay and minimum inhibitory concentration were adopted for the study. The results showed that the maximum anti-yeast activity against T. asahii and T. cutaneum was demonstrated by oil of Mentha piperita showing full inhibition of both the fungi, Melaleuca alternifolia with an inhibition zone of 45 and 40 mm, Cymbopogon winterians with inhibition zone of 45 and 45 mm and Cymbopogon flexuosus with 35 and 30 mm inhibition zones. The oil of Trachyspermum ammi exhibited 10 and 20 mm, Abelmoschus moschatus exhibited 30 and 20 mm, Salvia sclarea showed 20 and 18 mm and Jasminum officinale exhibited 25 and 15 mm inhibition zones showing moderate activity. The oil of Cyperus scariosus, Pogostemon patchouli and Rosa damascene showed no inhibition zone against both the fungi while Vetiveria zizanoides exhibited no inhibition in case of T. asahii and inhibition zone of 10 mm in case of T. cutaneum demonstrating comparatively low activity against both the fungi. These results support that the essential oils can be used to cure superficial mycoses and these oils may have significant role as pharmaceuticals and preservatives.

Application of Microbiological Method Direct Epifluorescence Filter Techique/Aerobic Plate Count Agar in the Identification of Irradiated Herbs and Spices

PubMed Central

Di Schiavi, Maria Teresa; Foti, Marina; Mosconi, Maria Cristina; Mattiolo, Giuseppina; Cavallina, Roberta

2014-01-01

Irradiation is a preservation technology used to improve the safety and hygienic quality of food. Aim of this study was to assess the applicability and validity of the microbiological screening method direct epifluorescence filter technique (DEFT)/aerobic plate count (APC) (EN 13783:2001) for the identification of irradiated herbs and spices. Tests on non-irradiated and irradiated samples of dried herbs and spices were performed. The method was based on the comparison of APC and count obtained using DEFT. In accordance with the standard reference, this method is not applicable to samples with APC<103 colony forming units (CFU)/g and this is its main limit. The results obtained in our laboratories showed that in 50% of cases of non-irradiated samples and in 96% of the samples treated with ionising radiation, the method was not applicable due to a value of CFU/g <103. PMID:27800348

In vitro bactericidal effect of Nd:YAG laser on Actinomyces israelii.

PubMed

Vescovi, Paolo; Conti, Stefania; Merigo, Elisabetta; Ciociola, Tecla; Polonelli, Luciano; Manfredi, Maddalena; Meleti, Marco; Fornaini, Carlo; Rocca, Jean-Paul; Nammour, S Amir

2013-07-01

A bactericidal effect has been reported by the use of near-infrared laser light on both Gram-positive and Gram-negative bacteria. The aim of this study was to evaluate the effect of Nd:YAG laser on Actinomyces israelii, filamentous bacteria causing cervicofacial actinomycosis. Experiments were realized on bacterial cells in saline suspension or streaked on Mueller-Hinton (MH) agar plates with or without India ink. Laser application was performed in Eppendorf tubes with different powers and frequencies for 40 s; bacterial suspensions were then streaked on agar plates and incubated at 35 °C in proper conditions for 5 days before colony enumeration. A reduction of colony number variable from 60.13 to 100 % for powers of 2, 4, and 6 W at 25-50 Hz of frequency was observed in comparison with growth control. For agar plates, laser application was performed with different powers at 50 Hz for 60 s. A growth inhibition was observed after 5 days of incubation on MH plates with powers of 6 W and on MH-ink plates with all applied powers. This preliminary study showed a bactericidal effect caused by Nd:YAG laser application worthy to be evaluated in further experiments in vivo.

Isolation and partial characterization of a mutant of Penicillium funiculosum for the saccharification of straw

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoffman, R.M.; Wood, T.M.

1985-01-01

Clearing of agar plates containing ball-milled, delignified straw has been used for screening mutants of Penicillium funiculosum IMI 87160 III. The effects of glycerol and a number of sugars on the clearing were investigated for selecting derepressed mutants. The ..beta..-glucosidase synthesis by one such mutant, C22c, in shake flasks containing straw was not repressed by 5% glycerol, whereas activities on filter paper, CM-cellulose, and p-nitrophenyl-..beta..-xylosidase were only partially derepressed; xylanase was extensively derepressed. The evidence for separate control of the enzymes involved in the solubilization of straw is discussed. 23 references.

Evaluation of potassium-clavulanate-supplemented modified charcoal-cefoperazone-deoxycholate agar for enumeration of Campylobacter in chicken carcass rinse.

PubMed

Chon, Jung-Whan; Kim, Hong-Seok; Kim, Hyunsook; Oh, Deog-Hwan; Seo, Kun-Ho

2014-05-01

Potassium-clavulanate-supplemented modified charcoal-cefoperazone-deoxycholate agar (C-mCCDA) that was described in our previous study was compared with original mCCDA for the enumeration of Campylobacter in pure culture and chicken carcass rinse. The quantitative detection of viable Campylobacter cells from a pure culture, plated on C-mCCDA, is statistically similar (P > 0.05) to mCCDA. In total, 120 chickens were rinsed using 400 mL buffered peptone water. The rinses were inoculated onto C-mCCDA and mCCDA followed by incubation at 42 °C for 48 h. There was no statistical difference between C-mCCDA (45 of 120 plates; mean count, 145.5 CFU/mL) and normal mCCDA (46 of 120 plates; mean count, 160.8 CFU/mL) in the isolation rate and recovery of Campylobacter (P > 0.05) from chicken carcass rinse. The Pearson correlation coefficient value for the number of Campylobacter cells recovered in the 2 media was 0.942. However, the selectivity was much better on C-mCCDA than on mCCDA plates (P < 0.05). Significantly fewer C-mCCDA plates (33 out of 120 plates; mean count, 1.9 CFU/mL) were contaminated with non-Campylobacter cells than the normal mCCDA plates (67 out of 120 plates; mean count, 27.1 CFU/mL). The C-mCCDA may provide improved results for enumeration of Campylobacter in chicken meat alternative to mCCDA with its increased selectivity the modified agar possess. © 2014 Institute of Food Technologists®

Antibacterial potency of V.A.C. GranuFoam Silver(®) Dressing.

PubMed

Sachsenmaier, Saskia; Peschel, Andreas; Ipach, Ingmar; Kluba, Torsten

2013-10-01

V.A.C.(®) GranuFoam™ therapy is regularly used in the surgical therapy of infected wounds and soft tissue injuries. Silver nanoparticles can destroy bacterial cell walls and inhibit enzymes for cell replication. Silver dressings are therefore successfully used for many indications in wound therapy. In this study, we investigated the antimicrobial potency of ionic silver released from the silver-coated V.A.C.(®) GranuFoam™ during vacuum therapy. Silver dressing was exposed to agar plates populated with bacteria to measure silver release. A total of 15 agar plates colonised with either Staphylococcus aureus populations or with Staphylococcus epidermidis, were loaded with V.A.C. GranuFoam Silver(®) Dressing polyurethane foam (KCI, San Antonio, Texas). Each of 13 pieces of silver-coated foam was applied to an agar plate. Two plates were loaded with conventional black foam without any coating. After connecting to a vacuum pump, the vacuum therapy of the 15 plates lasted 5 days. The zone of inhibition of bacterial growth around the foam was measured daily. Silver release was also determined as a function of time. At each time point, there was evidence of silver in the agar independent of bacterial colonisation. The S. aureus agar showed a consecutive increase in silver concentration from baseline upon 48 h after exposure to the negative pressure of V.A.C. therapy. An increasing mean silver level after 48, 72 and 96 h was measured under V.A.C. therapy with a peak value after 120 h. In contrast, the results from the S. epidermidis plates did not follow a linear pattern. At the beginning of vacuum therapy, we documented a rise in silver concentration. After 48-96h, the silver levels fluctuated. A maximum zone of inhibition in both bacterial colonised plates (S. aureus and S. epidermidis) was found 39 h after the start of the V.A.C. GranuFoam Silver(®) therapy. From our results, we confirmed the antimicrobial effect of the silver ions against S. aureus and S. epidermidis under continuous V.A.C. GranuFoam(®) Silver therapy with a negative pressure of 25 mmHg. Furthermore we could quantify the amounts of silver, which were released from the foam under negative pressure as a function of time. Copyright © 2013 Elsevier Ltd. All rights reserved.

Vulnerability of Bacillus spores and of related genera to physical impaction injury with particular reference to spread-plating.

PubMed

Thomas, P; Sekhar, A C; Mujawar, M M

2014-11-01

To examine whether bacterial spores are vulnerable to impaction injury during standard spread-plating or to other modes of physical impaction. Employing heat-challenged spores of Bacillus pumilus, Bacillus subtilis, Bacillus thuringiensis, Lysinibacillus, Paenibacillus and Brevibacillus spp. from day-4 to day-10 nutrient agar (NA) plates in 50% ethanol, plating the spore suspension to the extent of just drying the agar surface on fresh NA (50-60 s; SP-B) was tested in comparison with the spreader-independent approach of spotting-and-tilt-spreading (SATS), or a brief plating (<10 s; SP-A). Spore CFU was significantly reduced with SP-B in different organisms (23-40%) over SATS independent of the spore size. Comparing 4-, 7- and 10-day-old B. pumilus spores, the former two displayed significant CFU reduction in SP-B indicating a spore age-related effect. Continuous plating for 2-5 min showed a reduction in spore CFU in all organisms depending on plating duration. CFU reduction effect with SP-B was less manifest on refrigerated plates where no friction was experienced but acute on prewarmed and surface-dried plates. Spreader movement over agar surface subsequent to the exhaustion of free moisture proved highly detrimental to spores. A simulated plating study by plating the spores over a plastic film till drying showed a significant reduction in spore CFU. DAPI staining and glass bead-vortexing studies confirmed spore disruption through physical impaction. Bacterial spores are vulnerable to injury during spread-plating or with other forms of physical impaction with variable effects on different genotypes independent of the spore size but altered by spore age. Implications during spore CFU estimations employing spread-plating and during spore surveillance, and the recommendation of SATS as an easier and safer alternative for spore CFU enumeration. © 2014 The Society for Applied Microbiology.

Development of the dichlorvos-ammonia (DV-AM) method for the visual detection of aflatoxigenic fungi.

PubMed

Yabe, Kimiko; Hatabayashi, Hidemi; Ikehata, Akifumi; Zheng, Yazhi; Kushiro, Masayo

2015-12-01

Aflatoxins (AFs) are carcinogenic and toxic secondary metabolites produced mainly by Aspergillus flavus and Aspergillus parasiticus. To monitor and regulate the AF contamination of crops, a sensitive and precise detection method for these toxigenic fungi in environments is necessary. We herein developed a novel visual detection method, the dichlorvos-ammonia (DV-AM) method, for identifying AF-producing fungi using DV and AM vapor on agar culture plates, in which DV inhibits the esterase in AF biosynthesis, causing the accumulation of anthraquinone precursors (versiconal hemiacetal acetate and versiconol acetate) of AFs in mycelia on the agar plate, followed by a change in the color of the colonies from light yellow to brilliant purple-red by the AM vapor treatment. We also investigated the appropriate culture conditions to increase the color intensity. It should be noted that other species producing the same precursors of AFs such as Aspergillus nidulans and Aspergillus versicolor could be discriminated from the Aspergillus section Flavi based on the differences of their phenotypes. The DV-AM method was also useful for the isolation of nonaflatoxigenic fungi showing no color change, for screening microorganisms that inhibit the AF production by fungi, and for the characterization of the fungi infecting corn kernels. Thus, the DV-AM method can provide a highly sensitive and visible indicator for the detection of aflatoxigenic fungi.

Soft agar-based selection of spontaneously transformed rat prostate epithelial cells with highly tumorigenic characteristics.

PubMed

Gajdošik, Martina Šrajer; Hixson, Douglas C; Brilliant, Kate E; Yang, DongQin; De Paepe, Monique E; Josić, Djuro; Mills, David R

2018-05-29

The critical molecular and cellular mechanisms involved in the development and progression of prostate cancer remain elusive. In this report, we demonstrate that normal rat prostate epithelial cells (PEC) undergo spontaneous transformation at high passage (p > 85) evidenced by the acquisition of anchorage independent growth when plated on soft agar and tumorigenicity when injected into immunodeficient mice. In addition, we also report the discovery of a minor subpopulation of spontaneously transformed PEC derived from high passage PEC with the ability to migrate through a layer of 1% agar and form expanding colonies on the underlying plastic substratum. Comparison of these soft agar invasive (SAI) cells with low (p < 35), mid (p36-84) and high passage (p > 85) PEC identified marked differences in cell morphology, proliferation and motility. The SAI subpopulation was more tumorigenic than the high passage anchorage independent cultures from which they were isolated, as manifested by a decreased latency period and an increase in the size of tumors arising in immunodeficient mice. In contrast, low and mid passage cells were unable to grow on soft agar and failed to form tumors when injected into immunodeficient mice. Screening with antibody-based signaling arrays identified several differences in the altered expression levels of signaling proteins between SAI-derived cells and low or high passage PEC, including the up-regulation of EGFR and MAPK-related signaling pathways in SAI-selected cells. In summary, these studies suggest that the SAI assay selects for a novel, highly tumorigenic subpopulation of transformed cells that may represent an early step in the progression of slow growing prostatic carcinomas into more rapidly growing and aggressive tumors. Copyright © 2017. Published by Elsevier Inc.

Lipase, protease, and biofilm as the major virulence factors in staphylococci isolated from acne lesions.

PubMed

Saising, Jongkon; Singdam, Sudarat; Ongsakul, Metta; Voravuthikunchai, Supayang Piyawan

2012-08-01

Staphylococci involve infections in association with a number of bacterial virulence factors. Extracellular enzymes play an important role in staphylococcal pathogenesis. In addition, biofilm is known to be associated with their virulence. In this study, 149 staphylococcal isolates from acne lesions were investigated for their virulence factors including lipase, protease, and biofilm formation. Coagulase-negative staphylococci were demonstrated to present lipase and protease activities more often than coagulase-positive staphylococci. A microtiter plate method (quantitative method) and a Congo red agar method (qualitative method) were comparatively employed to assess biofilm formation. In addition, biofilm forming ability was commonly detected in a coagulase-negative group (97.7%, microtiter plate method and 84.7%, Congo red agar method) more frequently than in coagulase-positive organisms (68.8%, microtiter plate method and 62.5%, Congo red agar method). This study clearly confirms an important role for biofilm in coagulasenegative staphylococci which is of serious concern as a considerable infectious agent in patients with acnes and implanted medical devices. The Congo red agar method proved to be an easy method to quickly detect biofilm producers. Sensitivity of the Congo red agar method was 85.54% and 68.18% and accuracy was 84.7% and 62.5% in coagulase-negative and coagulase-positive staphylococci, respectively, while specificity was 50% in both groups. The results clearly demonstrated that a higher percentage of coagulasenegative staphylococci isolated from acne lesions exhibited lipase and protease activities, as well as biofilm formation, than coagulase-positive staphylococci.

Evaluation of different selective media and culturing techniques for the quantification of Campylobacter ssp. from broiler litter.

PubMed

Kiess, A S; Parker, H M; McDaniel, C D

2010-08-01

Poultry is a major reservoir for Campylobacter, the leading cause of foodborne illness in the United States, but how broilers become initially colonized is still under debate. Broiler litter is a potential source, but the best technique for quantifying Campylobacter from litter is still unknown. Therefore, our objectives were to determine if certain media are more selective for quantifying Campylobacter and if enrichment allows for the detection of stressed or viable but nonculturable cells from broiler litter samples. In this trial, 5 media and 2 culturing techniques were used to enumerate Campylobacter from broiler litter. The media used were campy-Line agar (CLA), campy-cefex agar (CCA), modified CCA, Campylobacter agar plates (CAP), and modified charcoal cefoperazone deoxycholate agar. Litter samples were obtained from a commercial broiler house. Each sample was equally divided and diluted 10-fold into peptone, for direct plating, or 4-fold into Campylobacter enrichment broth. Samples diluted in peptone were direct-plated onto each media and incubated under microaerophilic conditions for 48 h at 42 degrees C. Samples diluted in enrichment broth were incubated under the same conditions for 24 h, then further diluted to 10-fold before plating. Plates from enriched samples were incubated for an additional 24 h after plating. After incubation, all plates (direct and enriched) were counted and presumptive positive colonies were confirmed using a Campylobacter latex agglutination kit. Results indicated that there was no difference in the ability of any of the selective media tested to grow Campylobacter. Direct-plated samples had a higher Campylobacter isolation rate compared with enriched samples. The CLA and CAP were able to suppress total bacterial growth better than modified charcoal cefoperazone deoxycholate, modified CCA, and CCA. The CLA and CAP were the only media able to detect total bacterial population shifts over time. In conclusion, it is important before making a final decision on a selective medium to consider the medium's ability to suppress total bacterial growth as well as isolate Campylobacter.

Performance characteristics and estimation of measurement uncertainty of three plating procedures for Campylobacter enumeration in chicken meat.

PubMed

Habib, I; Sampers, I; Uyttendaele, M; Berkvens, D; De Zutter, L

2008-02-01

In this work, we present an intra-laboratory study in order to estimate repeatability (r), reproducibility (R), and measurement uncertainty (U) associated with three media for Campylobacter enumeration, named, modified charcoal cefoperazone deoxycholate agar (mCCDA); Karmali agar; and CampyFood ID agar (CFA) a medium by Biomérieux SA. The study was performed at three levels: (1) pure bacterial cultures, using three Campylobacter strains; (2) artificially contaminated samples from three chicken meat matrixes (total n=30), whereby samples were spiked using two contamination levels; ca. 10(3)cfuCampylobacter/g, and ca. 10(4)cfuCampylobacter/g; and (3) pilot testing in naturally contaminated chicken meat samples (n=20). Results from pure culture experiment revealed that enumeration of Campylobacter colonies on Karmali and CFA media was more convenient in comparison with mCCDA using spread and spiral plating techniques. Based on artificially contaminated samples testing, values of repeatability (r) were comparable between the three media, and estimated as 0.15log(10)cfu/g for mCCDA, 0.14log(10)cfu/g for Karmali, and 0.18log(10)cfu/g for CFA. As well, reproducibility performance of the three plating media was comparable. General R values which can be used when testing chicken meat samples are; 0.28log(10), 0.32log(10), and 0.25log(10) for plating on mCCDA, Karmali agar, and CFA, respectively. Measurement uncertainty associated with mCCDA, Karmali agar, and CFA using spread plating, for combination of all meat matrixes, were +/-0.24log(10)cfu/g, +/-0.28log(10)cfu/g, and +/-0.22log(10)cfu/g, respectively. Higher uncertainty was associated with Karmali agar for Campylobacter enumeration in artificially inoculated minced meat (+/-0.48log(10)cfu/g). The general performance of CFA medium was comparable with mCCDA performance at the level of artificially contaminated samples. However, when tested at naturally contaminated samples, non-Campylobacter colonies gave similar deep red colour as that given by the typical Campylobacter growth on CFA. Such colonies were not easily distinguishable by naked eye. In general, the overall reproducibility, repeatability, and measurement uncertainty estimated by our study indicate that there are no major problems with the precision of the International Organization for Standardization (ISO) 10272-2:2006 protocol for Campylobacter enumeration using mCCDA medium.

[Methicillin resistance detection in Staphylococcus aureus: comparison between conventional methods and MRSA-Screen latex agglutination technique].

PubMed

Soloaga, R; Corso, A; Gagetti, P; Faccone, D; Galas, M

2004-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen that has emerged over the last four decades, causing both nosocomial and community-acquired infections. Rapid and accurate detection of methicillin resistance in S. aureus is important for the use of appropriate antimicrobial therapy and for the control of nosocomial spread of MRSA strains. We evaluated the efficiency of conventional methods for detection of methicillin resistance such as the disk diffusion, agar dilution, oxacillin agar screen test, and the latex agglutination test MRSA-Screen latex, in 100 isolates of S. aureus, 79 mecA positive and 21 mecA negative. The MRSA-Screen latex (Denka Seiken, Niigata, Japón), is a latex agglutination method that detects the presence of PLP-2a, product of mecA gene in S. aureus. The PCR of the mecA gene was used as the "gold standard" for the evaluation of the different methods tested. The percentages of sensitivity and specificity were as follows: disk difusión 97 and 100%, agar dilution 97 and 95%, oxacillin agar screen test 100 and 100%, and MRSA-Screen latex, 100 and 100 %. All methods presented high sensitivity and specificity, but MRSA-Screen latex had the advantage of giving a reliable result, equivalent to PCR, in only 15 minutes.

Recovery of Sublethally Injured Bacteria Using Selective Agar Overlays.

ERIC Educational Resources Information Center

McKillip, John L.

2001-01-01

This experiment subjects bacteria in a food sample and an environmental sample to conditions of sublethal stress in order to assess the effectiveness of the agar overlay method to recover sublethally injured cells compared to direct plating onto the appropriate selective medium. (SAH)

Enumeration of sublethally injured Escherichia coli O157:H7 ATCC 43895 and Escherichia coli strain B-41560 using selective agar overlays versus commercial methods.

PubMed

Smith, Amanda R; Ellison, Alysha L; Robinson, Amanda L; Drake, Maryanne; McDowell, Susan A; Mitchell, James K; Gerard, Patrick D; Heckler, Rachel A; McKillip, John L

2013-04-01

Quality control procedures during food processing may involve direct inoculation of food samples onto appropriate selective media for subsequent enumeration. However, sublethally injured bacteria often fail to grow, enabling them to evade detection and intervention measures and ultimately threaten the health of consumers. This study compares traditional selective and nonselective agar-based overlays versus two commercial systems (Petrifilm and Easygel) for recovery of injured E. coli B-41560 and O157:H7 strains. Bacteria were propagated in tryptic soy broth (TSB), ground beef slurry, and infant milk formula to a density of 10(6) to 10(8) CFU/ml and then were stressed for 6 min either in lactic acid (pH 4.5) or heat shocked for 3 min at 60°C. Samples were pour plated in basal layers of either tryptic soy agar (TSA), sorbitol MacConkey agar (SMAC), or violet red bile agar (VRB) and were resuscitated for 4 h prior to addition of agar overlays. Other stressed bacteria were plated directly onto Petrifilm and Easygel. Results indicate that selective and nonselective agar overlays recovered significantly higher numbers (greater than 1 log) of acid- and heat-injured E. coli O157:H7 from TSB, ground beef, and infant milk formula compared with direct plating onto selective media, Petrifilm, or Easygel, while no significant differences among these media combinations were observed for stressed E. coli B-41560. Nonstressed bacteria from TSB and ground beef were also recovered at densities significantly higher in nonselective TSA-TSA and in VRB-VRB and SMAC-SMAC compared with Petrifilm and Easygel. These data underscore the need to implement food safety measures that address sublethally injured pathogens such as E. coli O157:H7 in order to avoid underestimation of true densities for target pathogens.

[Screening and identification of an endophytic bacterium with 1-aminocyclopropane-1-carboxylate deaminase activity from Panax ginseng and its effect on host growth].

PubMed

Tian, Lei; Jiang, Yun; Chen, Changqing; Zhang, Guanjun; Li, Tong; Tong, Bin; Xu, Peng

2014-07-04

This study aimed to screen endophytic bacteria with 1-aminocyclopropane-1-carboxylate deaminase activity from Panax ginseng and test the capability of growth promotion to its host. In total 120 endophytic bacterial strains isolated from Panax ginseng were screened for 1-aminocyclopropane-1-carboxylate deaminase activity using the qualitative and quantitative methods. The obtained strain was also tested for its ability of nitrogen fixation using the Ashby agar plates and the gene of nifH, for its ability of phosphate solubilization using the Pikovaskaia's plates and quantitative analysis of Mo-Sb-Ascrobiology acid colorimetry, for its ability of producing siderophores using the method of Chrome azurol S detecting, and its effect on promoting growth of Panax ginseng by laboratory and field experiments. The bacterial strain with ACC deaminase was identified based on morphology, physiological and biochemical traits, and 16S rRNA sequence analysis. The bacterial stain JJ8-3 with the ability of producing ACC deaminase activity was obtained through screening, which its ACC deaminase activity was alpha-ketobutyric acid 6.7 micromol/(mg x h). Strain JJ8-3 had other traits of phosphate solubilizing, nitrogen fixation, producing siderophores, and the ability of promoting growth of Panax ginseng. Strain JJ8-3 was identified as Pseudomonas fluorescens. Strain JJ8-3 of endophytic bacterium with ACC deaminase activity from Panax ginseng was obtained and would lay the foundation for its further study and application on plant growth promotion.

Fungitoxicity of lyophilized and spray-dried garlic extracts.

PubMed

Tedeschi, Paola; Maietti, Annalisa; Boggian, Marisa; Vecchiati, Giorgio; Brandolini, Vincenzo

2007-01-01

Among the compounds discussed for anti-microbial and anti-fungal use allicin (allylthiosulfinate, diallyl disulfide-S-monoxide), an active ingredient of garlic, has attracted considerable attention. The objective of this study is to determine the antifungal activity of a local garlic ecotype (Voghiera) extracts against different pathogens. Primary screening was carried out by the agar plates technique using ethanol garlic extract at four final concentrations against the following organisms: Alternaria alternata, Aspergillus spp., Colletotrichum acutatum, Didymella bryoniae, Fusarium culmorum, Fusarium avenaceum, Fusarium gramineareum, Gliocladium roseum 47, Pythium splendens, Rhizoctonia solani, Sclerotium rolfsii, Stemphylium vesicarium, Trichoderma longibranchiatum, and Botrytis cinerea. Secondary screening was carried out using a lyophilized and a spray-dried preparation at different concentrations against the organisms selected for the high inhibition garlic effect in the primary screening and compared with the commercial fungicides mancozeb and iprodione. The best results were observed for the spray-dried garlic compound that showed a good fungicidal activity at the concentration of 1.5 g/10 mL while lyophilized garlic at the same concentration exhibited less inhibition activity against the four fungi analyzed in the second screening.

Enumerating actinomycetes in compost bioaerosols at source—Use of soil compost agar to address plate 'masking'

NASA Astrophysics Data System (ADS)

Taha, M. P. M.; Drew, G. H.; Tamer Vestlund, A.; Aldred, D.; Longhurst, P. J.; Pollard, S. J. T.

Actinomycetes are the dominant bacteria isolated from bioaerosols sampled at composting facilities. Here, a novel method for the isolation of actinomycetes is reported, overcoming masking of conventional agar plates, as well as reducing analysis time and costs. Repeatable and reliable actinomycetes growth was best achieved using a soil compost media at an incubation temperature of 44 °C and 7 days' incubation. The results are of particular value to waste management operators and their advisors undertaking regulatory risk assessments that support environmental approvals for compost facilities.

Detection of Salmonella sp in chicken cuts using immunomagnetic separation

PubMed Central

de Cássia dos Santos da Conceição, Rita; Moreira, Ângela Nunes; Ramos, Roberta Juliano; Goularte, Fabiana Lemos; Carvalhal, José Beiro; Aleixo, José Antonio Guimarães

2008-01-01

The immunomagnetic separation (IMS) is a technique that has been used to increase sensitivity and specificity and to decrease the time required for detection of Salmonella in foods through different methodologies. In this work we report on the development of a method for detection of Salmonella in chicken cuts using in house antibody-sensitized microspheres associated to conventional plating in selective agar (IMS-plating). First, protein A-coated microspheres were sensitized with polyclonal antibodies against lipopolysacharide and flagella from salmonellae and used to standardize a procedure for capturing Salmonella Enteritidis from pure cultures and detection in selective agar. Subsequently, samples of chicken meat experimentally contaminated with S. Enteritidis were analyzed immediately after contamination and after 24h of refrigeration using three enrichment protocols. The detection limit of the IMS-plating procedure after standardization with pure culture was about 2x10 CFU/mL. The protocol using non-selective enrichment for 6-8h, selective enrichment for 16-18h and a post-enrichment for 4h gave the best results of S. Enteritidis detection by IMS-plating in experimentally contaminated meat. IMS-plating using this protocol was compared to the standard culture method for salmonellae detection in naturally contaminated chicken cuts and yielded 100% sensitivity and 94% specificity. The method developed using in house prepared magnetic microespheres for IMS and plating in selective agar was able to diminish by at least one day the time required for detection of Salmonella in chicken products by the conventional culture method. PMID:24031199

Antibacterial properties of aged dental cements evaluated by direct-contact and agar diffusion tests.

PubMed

Lewinstein, Israel; Matalon, Shlomo; Slutzkey, Shimshon; Weiss, Ervin I

2005-04-01

Since failure of fixed partial dentures is most frequently caused by caries, it would be advantageous if cements possessed antibacterial properties. The purpose of this study was to evaluate the antibacterial properties of 3 dental cements using the direct-contact test and agar diffusion test. For the direct-contact test, wells (n = 4) of microtiter plates were coated with the tested cements (Harvard cement, Duralon, and Ketac-Cem) while Streptococcus mutans suspension was placed directly on the cements. Bacterial growth was evaluated by a temperature-controlled microplate spectrophotometer. Eight wells of bacteria without the tested cements served as the positive control. Six wells of the tested cement without bacteria served as the negative control. For the agar diffusion test, triplicate specimens of freshly mixed cements were poured into uniform wells (5 mm in diameter) punched in the agar plates inoculated with Streptococcus mutans . After incubation at 37 degrees C for 24 hours, the agar plates were examined for bacterial growth and the diameter of the halo formed in the bacterial lawn was measured. In both tests, each cement was mixed in 2 different powder/liquid ratios. For the direct-contact test, data were initially recorded after 1 hour of incubation. Additional experiments were performed on specimens that were aged for 24 hours, 1 week, 1 month, and 3 months before assessment by either direct-contact test or agar diffusion test. The data were subjected to 1-way ANOVA with the Tukey post hoc test (alpha=.05). Compared with the control group, Duralon and Harvard cements demonstrated antibacterial properties even after 3 months with the direct-contact test (P <.002), while Ketac-Cem exhibited no antibacterial properties. In the agar diffusion test, no antibacterial activity was observed for any of the tested cements. The different powder/liquid ratios had a negligible effect on the antibacterial properties of the tested cements. Within the limitations of this study, Duralon and Harvard cements possessed prolonged antibacterial properties, while Ketac-Cem exhibited no antibacterial activity. The direct-contact test may be a more suitable test than the agar diffusion test to evaluate antibacterial properties of definitive cements.

Growth of Streptococcus mutans on various selective media.

PubMed

Emilson, C G; Bratthall, D

1976-07-01

The ability of Streptococcus mutans to grow on mitis-salivarius (MS) agar, MC agar, mitis-sucrose-bacitracin (MSB), BCY agar, and MM10 sucrose agar was studied. Batch cultures of S. mutans serotype a demonstrated no growth on MSB agar. Certain serotype d and g strains did not grow on MC agar. The yield for most strains of other serotypes on these selective media was lower compared with that on MS agar. The number of total colony-forming units on BCY and MM10 sucrose agar was similar to the blood agar results. Similar data were obtained when fermenter-grown strains, harvested in the middle or the end of the logarithmic growth phase, were used for inoculation of the various media. Enumeration of S. mutans from plaque samples plated on MC and MSB agar yielded about 75% of the counts obtained on MS or the nonselective medium. When the proportions of S. mutans were expressed as a percentage of the total cultivable flora, the selective media (MC and MSB agar) showed approximately 10% lower values than the MS, BCY, and MM10 sucrose agar.

Improvement of Karmali Agar by Supplementation with Tazobactam for Detecting Campylobacter in Raw Poultry.

PubMed

Kim, Young-Ji; Whan, Chon-Jung; Kim, Hong-Seok; Kim, Kwang-Yeop; Yim, Jin-Hyeok; Cho, Seung-Hak; Seo, Kun-Ho

2016-11-01

In this study, Karmali agar was modified by adding tazobactam (T-Karmali agar) to suppress the growth of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli , which frequently contaminates raw poultry meat. By inoculating 30 Campylobacter spp. strains and 25 ESBL-producing E. coli strains onto Karmali agar and T-Karmali agar containing various concentrations of the antibacterial agent, we determined the optimum concentration of tazobactam to be 4 mg/liter. The Campylobacter spp. isolation rate on T-Karmali agar (13.3%) was higher than that on Karmali agar (8.3%), although the difference was not significant (P > 0.05). However, T-Karmali agar showed a significantly greater selectivity than Karmali agar, as evaluated by comparing the numbers of contaminated agar plates (20.8 versus 82.5%; P < 0.05) and the growth indexes (1.36 versus 2.83) of competing flora. The predominant competing flora on Karmali and T-Karmali agar were identified as ESBL-producing E. coli . Thus, T-Karmali agar might be effective for determining the real prevalence of Campylobacter in raw poultry and, especially, contamination with ESBL-producing E. coli .

21 CFR 866.4800 - Radial immunodiffusion plate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Radial immunodiffusion plate. 866.4800 Section 866.4800 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... is a device that consists of a plastic plate to which agar gel containing antiserum is added. In...

21 CFR 866.4800 - Radial immunodiffusion plate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Radial immunodiffusion plate. 866.4800 Section 866.4800 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... is a device that consists of a plastic plate to which agar gel containing antiserum is added. In...

Machine for Automatic Bacteriological Pour Plate Preparation

PubMed Central

Sharpe, A. N.; Biggs, D. R.; Oliver, R. J.

1972-01-01

A fully automatic system for preparing poured plates for bacteriological analyses has been constructed and tested. The machine can make decimal dilutions of bacterial suspensions, dispense measured amounts into petri dishes, add molten agar, mix the dish contents, and label the dishes with sample and dilution numbers at the rate of 2,000 dishes per 8-hr day. In addition, the machine can be programmed to select different media so that plates for different types of bacteriological analysis may be made automatically from the same sample. The machine uses only the components of the media and sterile polystyrene petri dishes; requirements for all other materials, such as sterile pipettes and capped bottles of diluents and agar, are eliminated. Images PMID:4560475

Cultured Inquiry

ERIC Educational Resources Information Center

Scheppler, Judith A.; Sethakorn, Nan; Styer, Susan

2003-01-01

The Kirby-Bauer assay, also called the disc diffusion assay, is a standard procedure used in clinical laboratories to test the susceptibility of patients' bacterial isolates to antibiotics. In the assay, the bacteria are swabbed onto an agar plate, and paper discs impregnated with antibiotics are placed on the agar. The antibiotic diffuses from…

Alternative to the soft-agar assay that permits high-throughput drug and genetic screens for cellular transformation

PubMed Central

Rotem, Asaf; Janzer, Andreas; Izar, Benjamin; Ji, Zhe; Doench, John G.; Garraway, Levi A.; Struhl, Kevin

2015-01-01

Colony formation in soft agar is the gold-standard assay for cellular transformation in vitro, but it is unsuited for high-throughput screening. Here, we describe an assay for cellular transformation that involves growth in low attachment (GILA) conditions and is strongly correlated with the soft-agar assay. Using GILA, we describe high-throughput screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. Such molecules are unlikely to be found through conventional drug screening, and they include kinase inhibitors and drugs for noncancer diseases. In addition to known oncogenes, the genetic screen identifies genes that contribute to cellular transformation. Lastly, we demonstrate the ability of Food and Drug Administration-approved noncancer drugs to selectively kill ovarian cancer cells derived from patients with chemotherapy-resistant disease, suggesting this approach may provide useful information for personalized cancer treatment. PMID:25902495

Alternative to the soft-agar assay that permits high-throughput drug and genetic screens for cellular transformation.

PubMed

Rotem, Asaf; Janzer, Andreas; Izar, Benjamin; Ji, Zhe; Doench, John G; Garraway, Levi A; Struhl, Kevin

2015-05-05

Colony formation in soft agar is the gold-standard assay for cellular transformation in vitro, but it is unsuited for high-throughput screening. Here, we describe an assay for cellular transformation that involves growth in low attachment (GILA) conditions and is strongly correlated with the soft-agar assay. Using GILA, we describe high-throughput screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. Such molecules are unlikely to be found through conventional drug screening, and they include kinase inhibitors and drugs for noncancer diseases. In addition to known oncogenes, the genetic screen identifies genes that contribute to cellular transformation. Lastly, we demonstrate the ability of Food and Drug Administration-approved noncancer drugs to selectively kill ovarian cancer cells derived from patients with chemotherapy-resistant disease, suggesting this approach may provide useful information for personalized cancer treatment.

Nonrecovery of varying proportions of viable bacteria during spread plating governed by the extent of spreader usage and proposal for an alternate spotting-spreading approach to maximize the CFU.

PubMed

Thomas, P; Sekhar, A C; Mujawar, M M

2012-08-01

To elucidate the cause of high variations and inconsistencies in bacterial CFU observed within and between different experiments while assessing viable bacterial counts through spread plating (SP). Following the inconsistent results, CFU estimations were undertaken through conventional SP using the spreader, or a modified approach that did not use spreader employing four organisms. The latter approach involving spotting-and-tilt-spreading of inoculum on agar surface [spotting spreading (SS)] yielded higher CFU by 11-120% over the weighted average depending on the organism and diluent. The adverse effect owing to the spreader was the most obvious in Escherichia coli followed by Staphylococcus epidermidis, Enterobacter cloacae and Bacillus pumilus. Plate attributes that determined the surface moisture levels of agar medium and the spreading practice adopted by the personnel formed two other major influencing factors. Plating for shorter periods (<60 s) using fresh 15/20 ml plates caused loss of 3-12% CFU owing to inoculum adhesion to spreader irrespective of glass or polypropylene make. On the other hand, prolonging the plating brought down the CFU significantly. Spreader movement on agar surface subsequent to the exhaustion of free moisture, which was marked by the experiencing of some friction to smooth spreader movement, was detrimental to vegetative cells, while Bacillus spores were less affected. The study brings out that the way SP is carried out exerts significant effects on CFU influenced by plate conditions. Prolonged use of spreader on dry agar surface could be highly detrimental to bacterial cells. A mild use of spreader accounting for spreader-adhering inoculum or the practice of SS not involving the spreader is recommended. This study unravels the effects owing to the spreader on bacterial cells and the CFU and recommends an alternate approach of SS to minimize CFU inconsistencies and to maximize the viable bacterial counts. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Interaction of the nematophagous fungus Pochonia chlamydosporia and Parascaris equorum eggs in different culture media.

PubMed

de Carvalho, Lorendane Millena; Braga, Fabio Ribeiro; Domingues, Rafael Reis; Araujo, Juliana Milani; Lelis, Rosane Teixeira; de Paula, Alessandra Teixeira; da Silveira, Wendeo Ferreira; de Araújo, Jackson Victor

2014-07-01

Research involving the use of nematophagous fungi in the biological control of parasites of interest to veterinarians has occurred over recent years, with promising results. This article reports the infection of Parascaris equorum eggs by the fungus Pochonia chlamydosporia (isolates VC1 and VC4). Six groups were formed for each isolate, with six different culture media: 2% water-agar (2% WA); agar-chitin (AC); YPSSA (yeast extract, K2HPO4, MgSO4 ·7H2O, soluble starch); AELA extract (starch + water + agar); 2% corn-meal-agar (2% CMA); and 2% potato dextrose-agar (2% PDA). A total of 1000 eggs of P. equorum were transferred to each plate containing isolates grown for a period of 7 days (treatment group). Also, 1000 eggs were added to each plate without fungus (controlgroup). The plates were kept in an environmental chamber at 25 °C in the dark for 21 days. After, we analyzed the effects on ovicidal activity: effect 1 (accession shell); effect 2 (penetration hyphae); and effect 3 (destruction of the eggs). No differences were observed in the destruction of eggs between the two isolates. The decreasing effectiveness of the different culture media was: PDA (38.9%); CMA (38.3%); WA (36.7%); YPSSA (36.45%); and AC (32.5%). The highest percentage egg destruction was observed when the strains were grown in culture medium AELA (44.9%); this was the best medium. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genetic Interaction Mapping in Schizosaccharomyces pombe Using the Pombe Epistasis Mapper (PEM) System and a ROTOR HDA Colony Replicating Robot in a 1536 Array Format.

PubMed

Roguev, Assen; Xu, Jiewei; Krogan, Nevan

2018-02-01

This protocol describes an optimized high-throughput procedure for generating double deletion mutants in Schizosaccharomyces pombe using the colony replicating robot ROTOR HDA and the PEM (pombe epistasis mapper) system. The method is based on generating high-density colony arrays (1536 colonies per agar plate) and passaging them through a series of antidiploid and mating-type selection (ADS-MTS) and double-mutant selection (DMS) steps. Detailed program parameters for each individual replication step are provided. Using this procedure, batches of 25 or more screens can be routinely performed. © 2018 Cold Spring Harbor Laboratory Press.

High-Throughput Biosensor Discriminates Between Different Algal H 2-Photoproducing Strains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wecker, Matt S. A.; Maria L. Ghirardi

2014-02-27

A number of species of microalgae and cyanobacteria photosynthetically produce H 2 gas by coupling water oxidation with the reduction of protons to molecular hydrogen, generating renewable energy from sunlight and water. Photosynthetic H 2 production, however, is transitory, and there is considerable interest in increasing and extending it for commercial applications. Here we report a Petri-plate version of our previous, microplate-based assay that detects photosynthetic H 2 production by algae. The assay consists of an agar overlay of H 2-sensing Rhodobacter capsulatus bacteria carrying a green fluorescent protein that responds to H 2 produced by single algal colonies inmore » the bottom agar layer. The assay distinguishes between algal strains that photoproduce H 2 at different levels under high light intensities, and it does so in a simple, inexpensive, and high-throughput manner. The assay will be useful for screening both natural populations and mutant libraries for strains having increased H 2 production, and useful for identifying various genetic factors that physiologically or genetically alter algal hydrogen production.« less

Antagonistic potential against pathogenic microorganisms and hydrogen peroxide production of indigenous lactobacilli isolated from vagina of Chinese pregnant women.

PubMed

Xu, Heng-Yi; Tian, Wan-Hong; Wan, Cui-Xiang; Jia, Li-Jun; Wang, Lan-Yin; Yuan, Jing; Liu, Chun-Mei; Zeng, Ming; Wei, Hua

2008-10-01

To investigate the indigenous lactobacilli from the vagina of pregnant women and to screen the isolates with antagonistic potential against pathogenic microorganisms. The strains were isolated from pregnant women's vagina and identified using the API50CH system. The ability of the isolates to produce hydrogen peroxide was analyzed semi-quantitatively using the TMB-HRP-MRS agar. The antagonistic effects of the isolates on pathogenic microorganisms were determined with a double layer agar plate. One hundred and three lactobacilli strains were isolated from 60 samples of vaginal secretion from healthy pregnant women. Among them, 78 strains could produce hydrogen peroxide, in which 68%, 80%, 80%, and 88% had antagonistic effects against Candida albicans CMCC98001, Staphylococcus aureus CMCC26003, Escherichia coli CMCC44113, and Pseudomonas aeruginosa CMCC10110, respectively. The recovery of hydrogen peroxide-producing lactobacilli decreases with the increasing pregnant age and time. The most commonly isolated species from vagina of Chinese pregnant women are Lactobacillus acidophilus and Lactobacillus crispatus. Most of L. acidophilus and L. crispatus produce a high H2O2 level.

A quantitative in vitro assay for the evaluation of phototoxic potential of topically applied materials.

PubMed

Tenenbaum, S; DiNardo, J; Morris, W E; Wolf, B A; Schnetzinger, R W

1984-10-01

A quantitative in vitro method for phototoxic evaluation of chemicals has been developed and validated. The assay uses Saccharomyces cerevisiae, seeded in an agar overlay on top of a plate count agar base. 8-Methoxy psoralen is used as a reference standard against which materials are measured. Activity is quantified by cytotoxicity measured as zones of inhibition. Several known phototoxins (heliotropine, lyral, phantolid, and bergamot oil) and photoallergens (6-methyl coumarin and musk ambrette) are used to validate the assay. An excellent correlation is observed between in vivo studies employing Hartley albino guinea pigs and the in vitro assay for several fragrance raw materials and other chemicals. The in vitro assay exhibits a greater sensitivity from 2-500 fold. For three fragrance oils, the in vitro assay detects low levels of photobiological activity while the in vivo assay is negative. Although the in vitro assay does not discriminate between phototoxins and photoallergens, it can be used for screening of raw materials so that reduction in animal usage can be achieved while maintaining the protection of the consumer.

Growth on solid media.

PubMed

Elbing, Karen; Brent, Roger

2002-08-01

Detailed protocols are provided for titering and isolating bacterial colonies by serial dilutions, or alternatively by streaking or spreading a plate. Support protocols describe replica plating as well as methods for storing strains as agar stabs or frozen glycerol stocks.

Evaluation of Five Chromogenic Agar Media and the Rosco Rapid Carb Screen Kit for Detection and Confirmation of Carbapenemase Production in Gram-Negative Bacilli

PubMed Central

Gilmour, Matthew W.; DeGagne, Pat; Nichol, Kim; Karlowsky, James A.

2014-01-01

An efficient workflow to screen for and confirm the presence of carbapenemase-producing Gram-negative bacilli was developed by evaluating five chromogenic screening agar media and two confirmatory assays, the Rapid Carb screen test (Rosco Diagnostica A/S, Taastrup, Denmark) and the modified Hodge test. A panel of 150 isolates was used, including 49 carbapenemase-producing isolates representing a variety of β-lactamase enzyme classes. An evaluation of analytical performance, assay cost, and turnaround time indicated that the preferred workflow (screening test followed by confirmatory testing) was the chromID Carba agar medium (bioMérieux, Marcy l'Étoile, France), followed by the Rapid Carb screen test, yielding a combined sensitivity of 89.8% and a specificity of 100%. As an optional component of the workflow, a determination of carbapenemase gene class via molecular means could be performed subsequent to confirmatory testing. PMID:25355764

Broth and agar hop-gradient plates used to evaluate the beer-spoilage potential of Lactobacillus and Pediococcus isolates.

PubMed

Haakensen, M; Schubert, A; Ziola, B

2009-03-15

Identification of the beer-spoilage Lactobacillus and Pediococcus bacteria has largely taken two approaches; identification of spoilage-associated genes or identification of specific species of bacteria regardless of ability to grow in beer. The problem with these two approaches is that they are either overly inclusive (i.e., detect all bacteria of a given species regardless of spoilage potential) or overly selective (i.e., rely upon individual, putative spoilage-associated genes). Our goal was to design a method to assess the ability of Lactobacillus and Pediococcus to spoil beer that is independent of speciation or genetic background. In searching for a method by which to differentiate between beer-spoilage bacteria and bacteria that cannot grow in beer, we explored the ability of lactobacilli and pediococci isolates to grow in the presence of varying concentrations of hop-compounds and ethanol in broth medium versus on agar medium. The best method for differentiating between bacteria that can grow in beer and bacteria that do not pose a threat as beer-spoilage organisms was found to be a hop-gradient agar plate containing ethanol. This hop-gradient agar plate technique provides a rapid and simple solution to the dilemma of assessing the ability of Lactobacillus and Pediococcus isolates to grow in beer, and provides new insights into the different strategies used by these bacteria to survive under the stringent conditions of beer.

A simple and convenient microtiter plate assay for the detection of bactericidal antibodies to Vibrio cholerae O1 and Vibrio cholerae O139.

PubMed

Boutonnier, Alain; Dassy, Bruno; Duménil, Rémy; Guénolé, Alain; Ratsitorahina, Maherisoa; Migliani, René; Fournier, Jean-Michel

2003-12-01

It is believed that the correlate of protection for cholera can be determined by the serum vibriocidal assay. The currently available vibriocidal assays, based on the conventional agar plating technique, are labor intensive. We developed a simple and convenient microtiter plate assay for the detection of vibriocidal antibodies that is equally as efficient for Vibrio cholerae O1 and for V. cholerae O139. The addition of succinate and neotetrazolium made it possible to measure the growth of surviving bacterial target cells by monitoring a color change. We evaluated assay parameters (target strains, growth of target cells, complement source and concentration) that may affect the reproducibility of the method for V. cholerae O139. The results obtained with the microtiter plate assay were uniformly similar to those obtained with the conventional agar plating assay, when testing both the Inaba and Ogawa serotypes of V. cholerae O1. The microtiter plate assay was also convenient for measuring the activity of animal sera and mouse monoclonal antibodies.

Screening of Different Media and Substrates for Cultural Variability and Mass Culture of Arthrobotrys dactyloides Drechsler

PubMed Central

Kumar, D.; Jaiswal, R. K.

2005-01-01

Variability in growth and sporulation of five isolates of Arthrobotrys dactyloides was studied on five agar, 6 bran and 5 grain media. Potato dextrose agar (PDA) supported maximum growth of isolate A, C and E, while growth of isolate B and D was significantly lower on this medium. On Czapek's agar and yeast glucose agar media the differentiation in the isolates in relation to growth was poor than PDA. The other two media showed much poorer differentiation. On Czapek's agar medium, sporulation was recorded in isolate B only, whereas other isolates showed rare sporulation. Among the bran media, pea bran agar medium supported maximum growth of all the isolates except isolate B. Gram and rice bran agar media were next best. However, the growth of isolate B on the gram bran agar medium was more or less equal as other isolates. On pigeon pea bran agar medium, isolate E failed to grow while other isolates recorded poor growth. On lentil bran agar medium, only isolate B and D recorded little growth, whereas other isolates failed to grow. All the isolates recorded good sporulation on bran agar media except pigeon pea and lentil bran agar media. The grain agar media supported moderate to very good growth of all the isolates. In general isolate B remained slow growing on these media except gram grain and sorghum grain agar media on which growth of this isolate was comparable to other isolates. Sporulation in general, was good on all the grain agar media. Among different substrates screened, barley grain and pea bran were found superior to others for mass culture of isolate A of A. dactyloides. PMID:24049504

[Analysis of bactericidal material generated by electrical devices advertising bactericidal ability against bacteria on the agar gel plates].

PubMed

Nishimura, Hidekazu

2012-11-01

Several Japanese companies sell electrical devices advertised as effective in inactivating viruses and killing bacteria by releasing special materials, e.g., Plasmacluster ions, Nanoe particle and minus ions, into the air. These companies claim that their devices killed bacteria on plates in their own experiments. We tested device effectiveness using the same experiments from the Plasmacluster ioniser SHARP Co., Japan, the Nanoe generator Panasonic Co., Japan, and the Vion KING JIM Co., Japan, to test their advertising claims. Bactericidal ability on agar plate was tested, using Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus cereus, and Enterococcus faecalis as follows: the medium containing a certain amount of each bacterium was put onto an agar plate and smeared. Plates were kept in a closed chamber (inner volume 14.4 m3) or a glove box (inner volume 0.2 m), with one of the devices run for 2 hours. Plates not exposed to any device were used as controls. Each plate was retrieved and put in an incubator to count the number of bacterial colonies formed on the plate. There was no significant difference in the number of colonies on plates exposed to devices compared to control, in the number for all devices, or in all bacteria tested in experiments in the 14.4 m3 chamber. These results strongly suggest that these devices have almost no bactericidal effect, at least in space exceeding this volume. Colony formation was suppressed in the glove box in all devices and in all bacteria tested except P. aeruginosa, although the degree of suppression differed among experiments. The colony formation suppression mechanism was analyzed, and indicated that:colony formation did not change even after the removal of Plasmacluster ions, Nanoe particles, or negative ions from the air, while colony formation was decreased drastically by the removal of ozone from space, which was revealed to be generated inevitably during device operation. These results strongly suggest that the bactericidal effect seen only on the agar plate in narrow space was explained by ozone released in space as a by-product, not by special materials as advertising claimed. It is thus important to analyze the effect of special materials such as those done in this study and to suggest the involvement of ozone as the true cause, as have been done in this study, in evaluating bactericidal effect or viral inactivation as advertised by these companies.

Electron microscopy of antigen precipitates extracted from gel diffusion plates

PubMed Central

Watson, D. H.; Le Bouvier, G. L.; Tomlinson, J. A.; Walkey, D. G. A.

1966-01-01

A method is described whereby material from virus precipitin lines from agar gel diffusion plates may be examined in the electron microscope by a negative staining technique. ImagesFIGS. 1-2FIGS. 3-4 PMID:4286708

Comparison of 2 chromogenic media for the detection of extended-spectrum β-lactamase producing Enterobacteriaceae stool carriage in nursing home residents.

PubMed

Blane, Beth; Brodrick, Hayley J; Gouliouris, Theodore; Ambridge, Kirsty E; Kidney, Angela D; Ludden, Catherine M; Limmathurotsakul, Direk; Török, M Estée; Peacock, Sharon J

2016-03-01

ChromID ESBL agar and Brilliance ESBL agar were compared for the isolation of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae from 298 stools. These had comparable sensitivity and selectivity for the 116 positive samples. Pre-enrichment with cefpodoxime and extending incubation to 48 hours after direct plating both significantly increased sensitivity but reduced selectivity of both agars. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Spiral Salmonella assay: validation against the standard pour-plate assay.

PubMed

Diehl, M; Fort, F

1996-01-01

The spiral Ames assay, an automated approach to bacterial mutagenicity testing which simplifies the test procedure and reduces the amount of drug required to generate mutagenic dose-response information, has been evaluated and validated for routine screening. The spiral plater delivers the Salmonella bacteria, exogenous metabolic activation system and drug to the surface of a rotating agar plate one on top of another in such a way that a uniform density of bacteria is exposed to a logarithmically decreasing volume of drug. Following an incubation of 48 hr at 37 degrees C, the plates are scanned by a laser counter, and the data are subjected to a computerized analysis. Petri plates of 15 cm diameter were used to provide a concentration range of about 250-fold per plate. The Salmonella were concentrated 20-fold to increase sensitivity. Thirty-eight compounds from a variety of chemical classes, including both pharmaceuticals and known mutagens of moderate to strong potency, were tested in both the spiral and the standard pour-plate assays. There was overall test agreement on positive or negative results for 82% of the compounds tested. When only the results from strains TA98 plus TA100 were considered, the agreement was 87%. When positive results were obtained, the fold increase over vehicle control was on average twice as great for the spiral assay compared to the pour-plate assay. It was concluded that the two assay procedures generally provided comparable results, with the spiral assay being somewhat more sensitive in terms of dose-response than the pour-plate assay.

Rapid surface colony counts determination with three new miniaturised techniques.

PubMed

Malik, K A

1977-01-01

Three different miniaturised methods for the rapid surface viable counting are described. The methods were tried in parallel to seven different existing methods (Table 1) for viable counts and were found to be easier, quicker and insome cases more accurate. The techniques require about 10% of the material and time needed for conventional spread-plates method and the results were in no way inferior to that (Table 1 and 2). Mini agar discs were cut aseptically with an especially designed stainless steel agar disc cutter (25 mm internal and 28 mm external diameter, Fig. 1b) or with a test tube of similar diameter. The area of the resulted mini-agar-disc of 25 mm diameter was kept such (about 1/10th of the normal plate) that the ratio of the colony-bearing area to the inoculm remained the same as on big plates in spread-plate-method (Table 2). In normal Petri dishes (about 90 mm diameter) up to seven mini agar discs were possible to cut. Each small agar disc was seperated from the other mini-disc by a distance of at least 6 mm (Fig. 1a). The empty place around the disc was still enlarged during over drying of the plates and during incubation. This created complete isolation from the neighbouring disc. For micro-determination of surface viable counts 10 micronl from each dilution was delivered on a well-dired mini-disc with a piston micropipette. The inoculm was immediately spread on the whole mini-disc with a specially designed flame sterilizable platinum-Mini-spreader (Fig. 2a). No spinning of the plate was needed. Alternatively the dropping pipette and spreader was replaced by a calibrated platinum wire Loop-spreader (Fig. 2b). A loop of 3 mm internal diameter made from a platinum-iridium wire of 0.75 mm thickness proved most useful and carried a drop of 10 micronl. Differences especially in surface tension of various diluting fluids did not influence to drop of this size and no recalibration was needed for water and nutrient broth. The loop was further shaped to Loop-spreader form. From each bacterial suspension 10 micronl were carried and spread on each mini-disc. The method is useful for pathogenic organisms as the loop can readily be flame sterilized. For routine purposes where only approximate numbers of bacteria need to be known a still rapid semiquantitative method was deviced making use of a calibrated stainless steel Stamping-disc (Fig. 2c). A disc of 25mm diameter and 1 mm thickness delivered approximateyl 10 microlitres of supensions and was found to be most useful to stamp seven dilutions on a single plate. In collections and bacteriology laboratories where by conventional methods large number of plates are to be plated and counted the presented techniques could prove most convenient, rapid and economical.

Bacteria associated with sabellids (Polychaeta: Annelida) as a novel source of surface active compounds.

PubMed

Rizzo, Carmen; Michaud, Luigi; Hörmann, Barbara; Gerçe, Berna; Syldatk, Christoph; Hausmann, Rudolf; De Domenico, Emilio; Lo Giudice, Angelina

2013-05-15

A total of 69 bacteria were isolated from crude oil enrichments of the polychaetes Megalomma claparedei, Sabella spallanzanii and Branchiomma luctuosum, and screened for biosurfactant (BS) production by conventional methods. Potential BS-producers (30 isolates) were primarily selected due to the production of both interesting spots on thin layer chromatography (TLC) plates and highly stable emulsions (E₂₄ ≥ 50%). Only few strains grew on cetyltrimethylammonium bromide and blood agar plates, indicating the probable production of anionic surfactants. The 16S rRNA gene sequencing revealed that selected isolates mainly belonged to the CFB group of Bacteroidetes, followed by Gammaproteobacteria and Alphaproteobacteria. A number of BS-producers belonged to genera (i.e., Cellulophaga, Cobetia, Cohaesibacter, Idiomarina, Pseudovibrio and Thalassospira) that have been never reported as able to produce BSs, even if they have been previously detected in hydrocarbon-enriched samples. Our results suggest that filter-feeding Polychaetes could represent a novel and yet unexplored source of biosurfactant-producing bacteria. Copyright © 2013 Elsevier Ltd. All rights reserved.

Comparison of CHROMagar Salmonella Medium and Xylose-Lysine-Desoxycholate and Salmonella-Shigella Agars for Isolation of Salmonella Strains from Stool Samples

PubMed Central

Maddocks, Susan; Olma, Tom; Chen, Sharon

2002-01-01

The growth and appearance of 115 stock Salmonella isolates on a new formulation of CHROMagar Salmonella (CAS) medium were compared to those on xylose-lysine-desoxycholate agar (XLD), Salmonella-Shigella agar (SS), and Hektoen enteric agar (HEA) media. CAS medium was then compared prospectively to XLD and SS for the detection and presumptive identification of Salmonella strains in 500 consecutive clinical stool samples. All stock Salmonella isolates produced typical mauve colonies on CAS medium. Nine Salmonella strains were isolated from clinical specimens. The sensitivities for the detection of salmonellae after primary plating on CAS medium and the combination of XLD and SS after enrichment were 100%. The specificity for the detection of salmonellae after primary plating on CAS medium (83%) was significantly (P < 0.0001) higher than that after primary plating on the combination of SS and XLD media (55%) (a 28% difference in rates; 95% confidence interval, 23.0 to 34%). Twenty-nine non-Salmonella organisms produced mauve colonies on CAS medium, including 17 Candida spp. (59%) and 8 Pseudomonas spp. (28%). These were easily excluded as salmonellae by colony morphology, microscopic examination of a wet preparation, or oxidase testing. One biochemically inert Escherichia coli isolate required further identification to differentiate it from Salmonella spp. The use of plating on CAS medium demonstrated high levels of sensitivity and specificity and reduced the time to final identification of Salmonella spp., resulting in substantial cost savings. It can be recommended for use for the primary isolation of Salmonella spp. from stool specimens. Other media (e.g., XLD) are required to detect Shigella spp. concurrently. PMID:12149365

Comparing Diagnostic Accuracy of Kato-Katz, Koga Agar Plate, Ether-Concentration, and FLOTAC for Schistosoma mansoni and Soil-Transmitted Helminths

PubMed Central

Glinz, Dominik; Silué, Kigbafori D.; Knopp, Stefanie; Lohourignon, Laurent K.; Yao, Kouassi P.; Steinmann, Peter; Rinaldi, Laura; Cringoli, Giuseppe; N'Goran, Eliézer K.; Utzinger, Jürg

2010-01-01

Background Infections with schistosomes and soil-transmitted helminths exert a considerable yet underappreciated economic and public health burden on afflicted populations. Accurate diagnosis is crucial for patient management, drug efficacy evaluations, and monitoring of large-scale community-based control programs. Methods/Principal Findings The diagnostic accuracy of four copromicroscopic techniques (i.e., Kato-Katz, Koga agar plate, ether-concentration, and FLOTAC) for the detection of Schistosoma mansoni and soil-transmitted helminth eggs was compared using stool samples from 112 school children in Côte d'Ivoire. Combined results of all four methods served as a diagnostic ‘gold’ standard and revealed prevalences of S. mansoni, hookworm, Trichuris trichiura, Strongyloides stercoralis and Ascaris lumbricoides of 83.0%, 55.4%, 40.2%, 33.9% and 28.6%, respectively. A single FLOTAC from stool samples preserved in sodium acetate-acetic acid-formalin for 30 or 83 days showed a higher sensitivity for S. mansoni diagnosis (91.4%) than the ether-concentration method on stool samples preserved for 40 days (85.0%) or triplicate Kato-Katz using fresh stool samples (77.4%). Moreover, a single FLOTAC detected hookworm, A. lumbricoides and T. trichiura infections with a higher sensitivity than any of the other methods used, but resulted in lower egg counts. The Koga agar plate method was the most accurate diagnostic assay for S. stercoralis. Conclusion/Significance We have shown that the FLOTAC method holds promise for the diagnosis of S. mansoni. Moreover, our study confirms that FLOTAC is a sensitive technique for detection of common soil-transmitted helminths. For the diagnosis of S. stercoralis, the Koga agar plate method remains the method of choice. PMID:20651931

Combination of five diagnostic tests to estimate the prevalence of hookworm infection among school-aged children from a rural area of colombia.

PubMed

Barreto, Rafael E; Narváez, Javier; Sepúlveda, Natalia A; Velásquez, Fabián C; Díaz, Sandra C; López, Myriam Consuelo; Reyes, Patricia; Moncada, Ligia I

2017-09-01

Public health programs for the control of soil-transmitted helminthiases require valid diagnostic tests for surveillance and parasitic control evaluation. However, there is currently no agreement about what test should be used as a gold standard for the diagnosis of hookworm infection. Still, in presence of concurrent data for multiple tests it is possible to use statistical models to estimate measures of test performance and prevalence. The aim of this study was to estimate the diagnostic accuracy of five parallel tests (direct microscopic examination, Kato-Katz, Harada-Mori, modified Ritchie-Frick, and culture in agar plate) to detect hookworm infections in a sample of school-aged children from a rural area in Colombia. We used both, a frequentist approach, and Bayesian latent class models to estimate the sensitivity and specificity of five tests for hookworm detection, and to estimate the prevalence of hookworm infection in absence of a Gold Standard. The Kato-Katz and agar plate methods had an overall agreement of 95% and kappa coefficient of 0.76. Different models estimated a sensitivity between 76% and 92% for the agar plate technique, and 52% to 87% for the Kato-Katz technique. The other tests had lower sensitivity. All tests had specificity between 95% and 98%. The prevalence estimated by the Kato-Katz and Agar plate methods for different subpopulations varied between 10% and 14%, and was consistent with the prevalence estimated from the combination of all tests. The Harada-Mori, Ritchie-Frick and direct examination techniques resulted in lower and disparate prevalence estimates. Bayesian approaches assuming imperfect specificity resulted in lower prevalence estimates than the frequentist approach. Copyright © 2017 Elsevier B.V. All rights reserved.

Big data analytics in hyperspectral imaging for detection of microbial colonies on agar plates (Conference Presentation)

NASA Astrophysics Data System (ADS)

Yoon, Seung-Chul; Park, Bosoon; Lawrence, Kurt C.

2017-05-01

Various types of optical imaging techniques measuring light reflectivity and scattering can detect microbial colonies of foodborne pathogens on agar plates. Until recently, these techniques were developed to provide solutions for hypothesis-driven studies, which focused on developing tools and batch/offline machine learning methods with well defined sets of data. These have relatively high accuracy and rapid response time because the tools and methods are often optimized for the collected data. However, they often need to be retrained or recalibrated when new untrained data and/or features are added. A big-data driven technique is more suitable for online learning of new/ambiguous samples and for mining unknown or hidden features. Although big data research in hyperspectral imaging is emerging in remote sensing and many tools and methods have been developed so far in many other applications such as bioinformatics, the tools and methods still need to be evaluated and adjusted in applications where the conventional batch machine learning algorithms were dominant. The primary objective of this study is to evaluate appropriate big data analytic tools and methods for online learning and mining of foodborne pathogens on agar plates. After the tools and methods are successfully identified, they will be applied to rapidly search big color and hyperspectral image data of microbial colonies collected over the past 5 years in house and find the most probable colony or a group of colonies in the collected big data. The meta-data, such as collection time and any unstructured data (e.g. comments), will also be analyzed and presented with output results. The expected results will be novel, big data-driven technology to correctly detect and recognize microbial colonies of various foodborne pathogens on agar plates.

A single-tube screen for Salmonella and Shigella.

PubMed

Procop, Gary W; Wallace, Jacqueline D; Tuohy, Marion J; Lasalvia, Margret M; Addison, Rachel M; Reller, L Barth

2008-08-01

Salmonella and Shigella species are routinely sought in stool specimens submitted for culture. It is a common practice to screen lactose-negative colonies by using triple sugar iron agar, lysine iron agar, and Christensen urea agar to determine if further identification is necessary. We designed and evaluated a novel combination of media, which are layered in a single tube, for screening isolates suspected to possibly represent Salmonella or Shigella. We tested this media combination with 106 Salmonella, 56 Shigella, and 56 other gram-negative bacilli. All Salmonella and Shigella isolates tested were appropriately characterized as possible Salmonella or Shigella by using an algorithm developed for use with this media combination. Similarly, 53 (95%) of 56 other gram-negative bacilli were appropriately screened as non -Salmonella and non -Shigella isolates. This unique media combination provides the most important biochemical reactions needed to screen for Salmonella and Shigella in a single-tube format, which decreases labor by two thirds (ie, 1 tube is inoculated vs 3).

Discolored Red Seaweed Pyropia yezoensis with Low Commercial Value Is a Novel Resource for Production of Agar Polysaccharides.

PubMed

Sasuga, Keiji; Yamanashi, Tomoya; Nakayama, Shigeru; Ono, Syuetsu; Mikami, Koji

2018-04-26

The red seaweed Pyropia yezoensis has been demonstrated to be a novel resource for the production of high-quality agar. P. yezoensis is grown for the food industry in large-scale Japanese mariculture operations. However, discolored P. yezoensis is mostly discarded as an industrial waste, although it has some kind of utility values. Here, we evaluated the utility of discolored P. yezoensis as a resource for agar production. The quality of agar from the discolored seaweed was comparable to that from normal seaweed. In addition, as a distinguishing characteristic, agar yield was higher from discolored seaweeds than from normal types. Moreover, we successfully used agar from discolored P. yezoensis for bacterial plate media and DNA electrophoresis gels without agarose purification. Thus, our results demonstrate that discolored P. yezoensis is suitable for agar production and use in life science research. Diverting discolored P. yezoensis from disposal to agar production provides a solution to the current industrial waste problem in mariculture, as well as a secure source of agar for research purposes.

Study on the application of shear-wave elastography to thin-layered media and tubular structure: Finite-element analysis and experiment verification

NASA Astrophysics Data System (ADS)

Jang, Jun-keun; Kondo, Kengo; Namita, Takeshi; Yamakawa, Makoto; Shiina, Tsuyoshi

2016-07-01

Shear-wave elastography (SWE) enables the noninvasive and quantitative evaluation of the mechanical properties of human soft tissue. Generally, shear-wave velocity (C S) can be estimated using the time-of-flight (TOF) method. Young’s modulus is then calculated directly from the estimated C S. However, because shear waves in thin-layered media propagate as guided waves, C S cannot be accurately estimated using the conventional TOF method. Leaky Lamb dispersion analysis (LLDA) has recently been proposed to overcome this problem. In this study, we performed both experimental and finite-element (FE) analyses to evaluate the advantages of LLDA over TOF. In FE analysis, we investigated why the conventional TOF is ineffective for thin-layered media. In phantom experiments, C S results estimated using the two methods were compared for 1.5 and 2% agar plates and tube phantoms. Furthermore, it was shown that Lamb waves can be applied to tubular structures by extracting lateral waves traveling in the long axis direction of the tube using a two-dimensional window. Also, the effects of the inner radius and stiffness (or shear wavelength) of the tube on the estimation performance of LLDA were experimentally discussed. In phantom experiments, the results indicated good agreement between LLDA (plate phantoms of 2 mm thickness: 5.0 m/s for 1.5% agar and 7.2 m/s for 2% agar; tube phantoms with 2 mm thickness and 2 mm inner radius: 5.1 m/s for 1.5% agar and 7.0 m/s for 2% agar; tube phantoms with 2 mm thickness and 4 mm inner radius: 5.3 m/s for 1.5% agar and 7.3 m/s for 2% agar) and SWE measurements (bulk phantoms: 5.3 m/s ± 0.27 for 1.5% agar and 7.3 m/s ± 0.54 for 2% agar).

Improvement of Karmali agar by addition of polymyxin B for the detection of Campylobacter jejuni and C. coli in whole-chicken carcass rinse.

PubMed

Chon, Jung-Whan; Kim, Hyunsook; Yim, Jin-Hyeok; Song, Kwang-Young; Moon, Jin-San; Kim, Young-Jo; Seo, Kun-Ho

2013-05-01

The Karmali agar was modified by supplementation with a high concentration of polymyxin B. The goal of the study was to evaluate the effect of a high concentration of polymyxin B on the ability and selectivity of the modified Karmali agar to isolate Campylobacter jejuni and Campylobacter coli from whole chicken carcass rinse. A total of 80 whole chickens were rinsed with 400 mL of buffer peptone water. The rinsed samples were incubated with 2× blood-free modified Bolton enrichment broth for 48 h, and then streaked onto unmodified Karmali agar and modified Karmali agar supplemented with 100000 IU/L polymixin B (P-Karmali agar). The suspected colonies were finally confirmed by colony PCR. The P-Karmali agar exhibited a significantly better (P < 0.05) isolation rate than the unmodified Karmali agar (P-Karmali agar, 73.8%; unmodified Karmali agar, 33.8%). Moreover, the selectivity of the P-Karmali agar was also better (P < 0.05) than that of the other selective agar when comparing the number of contaminated plates (P-Karmali agar, 68.8%; unmodified Karmali agar, 87.5%) and growth index of competing flora (P-Karmali agar, 1.4; unmodified Karmali agar, 2.7). The improved selective agar excluded competing flora resistant to antibiotic agents in unmodified Karmali agar, increasing isolation rate and selectivity for C. jejuni and C. coli. © 2013 Institute of Food Technologists®

Screening haematology patients for carbapenem-resistant Klebsiella pneumoniae

PubMed Central

Kilgour, Elizabeth; Dunn, Caroline; Thomas, Linda; Fox, Richard; Mitchell, Lindsay; Paterson, Pamela

2013-01-01

Following a cluster of haematology patients with carbapenem-resistant Klebsiella pneumoniae (CRKP) septicaemia, we initiated screening for rectal carriage of CRKP and multidrug-resistant K. pneumoniae (MDRKP) in this patient group. Haematology inpatients submit a rectal swab once weekly. When plated onto chromogenic Brilliance™ UTI Agar (Oxoid), and incubated overnight with a 10 µg ertapenem disc (Oxoid), K. pneumoniae is identified and semi-automated antibiotic susceptibility testing is performed using the Vitek 2 analyser (Biomerieux). When no zone of inhibition occurs, immediate intervention through patient isolation and enhanced environmental cleaning can be instigated to control further spread while empirical antibiotic prescribing is adapted to take account of identified resistances. Over 2 years, six patients with CRKP and 20 patients with MDRKP were identified. These isolates were resistant to first-line empirical treatment choices for neutropenic sepsis and presented a clinical risk of treatment failure for sepsis post cytotoxic chemotherapy. We describe how this rectal screening methodology was developed and how the results influenced appropriate antibiotic prescribing, patient placement in single rooms and the cleaning of the ward environment to prevent person-to-person transmission of MDRKP and CRKP. PMID:28989355

Biofilm forming ability of bacteria isolated from necrotic roots canals of teeth

NASA Astrophysics Data System (ADS)

Alwan, Merriam Ghadhanfar; Usup, Gires; Heng, Lee Yook; Ahmad, Asmat

2018-04-01

The growth of microbes in biofilms are associated with repeated and chronic human infections and are extremely resistant to antimicrobial agents. The purpose of this study was to determine the diversity of bacteria from necrotic roots canals of teeth and to detect their biofilm formation ability. A total of 42 bacterial isolates were isolated and identified as belonging to 11 genera. These are Enterococcus sp. (21.4%) followed by Streptococcus sp. (16.8%), Bacillus sp. (11.9%), Peptostreptococcus sp. (9.5%), Staphylococcus sp. (9.5%), Bacteroides sp. (7.1%), Clostridium sp. (7.1%), Actinomyces sp. (7.1%), Fusobacterium sp. (4.76%), Provotella sp. (2.4%) and Chromobacterium sp. (2.4%). Three screening methods for biofilm forming ability were used. Congo Red Agar method (CRA), Tube method (TM) and Microtitre Plate (MTP). From the results, MTP method is a more reliable and quantitative method for the screening and detection of microorganism's ability to form biofilm. This method can be recommended and suggested as a general screening method for the detection of biofilm forming bacteria isolated from roots canals of teeth.

Spreading of nonmotile bacteria on a hard agar plate: Comparison between agent-based and stochastic simulations

NASA Astrophysics Data System (ADS)

Rana, Navdeep; Ghosh, Pushpita; Perlekar, Prasad

2017-11-01

We study spreading of a nonmotile bacteria colony on a hard agar plate by using agent-based and continuum models. We show that the spreading dynamics depends on the initial nutrient concentration, the motility, and the inherent demographic noise. Population fluctuations are inherent in an agent-based model, whereas for the continuum model we model them by using a stochastic Langevin equation. We show that the intrinsic population fluctuations coupled with nonlinear diffusivity lead to a transition from a diffusion limited aggregation type of morphology to an Eden-like morphology on decreasing the initial nutrient concentration.

Screening for antimicrobial and proteolytic activities of lactic acid bacteria isolated from cow, buffalo and goat milk and cheeses marketed in the southeast region of Brazil.

PubMed

Tulini, Fabricio L; Hymery, Nolwenn; Haertlé, Thomas; Le Blay, Gwenaelle; De Martinis, Elaine C P

2016-02-01

Lactic acid bacteria (LAB) can be isolated from different sources such as milk and cheese, and the lipolytic, proteolytic and glycolytic enzymes of LAB are important in cheese preservation and in flavour production. Moreover, LAB produce several antimicrobial compounds which make these bacteria interesting for food biopreservation. These characteristics stimulate the search of new strains with technological potential. From 156 milk and cheese samples from cow, buffalo and goat, 815 isolates were obtained on selective agars for LAB. Pure cultures were evaluated for antimicrobial activities by agar antagonism tests and for proteolytic activity on milk proteins by cultivation on agar plates. The most proteolytic isolates were also tested by cultivation in skim milk followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the fermented milk. Among the 815 tested isolates, three of them identified as Streptococcus uberis (strains FT86, FT126 and FT190) were bacteriocin producers, whereas four other ones identified as Weissella confusa FT424, W. hellenica FT476, Leuconostoc citreum FT671 and Lactobacillus plantarum FT723 showed high antifungal activity in preliminary assays. Complementary analyses showed that the most antifungal strain was L. plantarum FT723 that inhibited Penicillium expansum in modified MRS agar (De Man, Rogosa, Sharpe, without acetate) and fermented milk model, however no inhibition was observed against Yarrowia lipolytica. The proteolytic capacities of three highly proteolytic isolates identified as Enterococcus faecalis (strains FT132 and FT522) and Lactobacillus paracasei FT700 were confirmed by SDS-PAGE, as visualized by the digestion of caseins and whey proteins (β-lactoglobulin and α-lactalbumin). These results suggest potential applications of these isolates or their activities (proteolytic activity or production of antimicrobials) in dairy foods production.

Evaluation of the Sensititre MycoTB plate for susceptibility testing of the Mycobacterium tuberculosis complex against first- and second-line agents.

PubMed

Hall, Leslie; Jude, Kurt P; Clark, Shirley L; Dionne, Kim; Merson, Ryan; Boyer, Ana; Parrish, Nicole M; Wengenack, Nancy L

2012-11-01

The Sensititre MycoTB plate (TREK Diagnostic Systems, Cleveland, OH) uses a microtiter plate MIC format for susceptibility testing of Mycobacterium tuberculosis complex isolates against first- and second-line antituberculosis agents. Categorical agreement versus the agar proportion method for 122 M. tuberculosis complex isolates was 94% to 100%.

Comparison of dry sheet media and conventional agar media methods for enumerating yeasts and molds in food.

PubMed

Beuchat, L R; Mann, David A; Gurtler, Joshua B

2007-11-01

A study was done to compare Nissui Compact Dry Yeast and Mold plates (CDYM), 3M Petrifilm Yeast and Mold count plates (PYM), dichloran-rose bengal chloramphenicol (DRBC) agar, and dichloran 18% glycerol (DG18) agar for enumerating yeasts and molds naturally occurring in 97 foods (grains, legumes, raw fruits and vegetables, nuts, dairy products, meats, and miscellaneous processed foods and dry mixes). Correlation coefficients for plates incubated for 5 days were DG18 versus DRBC (0.93), PYM versus DRBC (0.81), CDYM versus DG18 (0.81), PYM versus DG18 (0.80), CDYM versus DRBC (0.79), and CDYM versus PYM (0.75). The number of yeasts and molds recovered from a group of foods (n = 32) analyzed on a weight basis (CFU per gram) was not significantly different (alpha = 0.05) when samples were plated on DRBC, DG18, PYM, or CDYM. However, the order of recovery from foods (n = 65) in a group analyzed on a unit or piece basis, or a composite of both groups (n = 97), was DRBC > DG18 = CDYM > PYM. Compared with PYM, CDYM recovered equivalent, significantly higher (alpha = 0.05) or significantly lower (alpha = 0.05) numbers of yeasts and molds in 51.5, 27.8, and 20.6%, respectively, of the 97 foods tested; respective values were 68.8, 15.6, and 15.6% in the small group (n = 32) and 43.1, 33.8, and 23.1% in the large group (n = 65) of foods. The two groups contained different types of foods, the latter consisting largely (73.8%) of raw fruits (n = 16) and vegetables (n = 32). Differences in efficacy of the four methods in recovering yeasts and molds from foods in the two groups are attributed in part to differences in genera and predominant mycoflora. While DG18 agar, CDYM, and PYM appear to be acceptable for enumerating yeasts and molds in the foods analyzed in this study, overall, DRBC agar recovered higher numbers from the 97 test foods, thereby supporting its recommended use as a general purpose medium for mycological analysis.

Variability of Photodynamic Killing in Escherichia coli and Avoidance of Variability with Agar

PubMed Central

O'Bryan, Corliss; Harrison, Arthur P.

1971-01-01

Photodynamic killing of Escherichia coli in acridine orange is influenced by the composition of the containing vessel, and after high kill the variance between replicate suspensions is greater than attributable solely to sampling and plating. Addition of agar minimizes both phenomena, but a higher illumination dose is required to produce the same degree of killing. PMID:4934057

Influence of composition of diluent on populations of yeasts and moulds recovered from raw fruits.

PubMed

Beuchat, L R; Scouten, A J; Jablonska, J

2002-01-01

The aims of this study were (i) to determine the retention of viability of mycoflora removed from raw fruits, and how this affected diluents used to prepare samples for enumeration of propagules, and (ii) to evaluate the performance of recovery media for supporting colony development. Yeasts and moulds removed from seven types of raw fruit were held in seven diluents for 1 h before plating on dichloran rose bengal chloramphenicol (DRBC) agar and plate count agar supplemented with chloramphenicol (100 micro g ml-1) (PCAC). Significant reductions (P=0.05) in populations of yeasts, moulds, and yeasts plus moulds occurred within the 1 h holding period, regardless of diluent composition. Overall, retention of viability was not influenced by diluent composition, and neither DRBC agar nor PCAC were superior in supporting colony development. The composition of diluents used to prepare food samples for mycological analysis has little affect on the number of yeasts and moulds recovered from seven types of naturally contaminated raw fruit. Both DRBC agar and PCAC are suitable as enumeration media. Diluents and media most often recommended for enumerating yeasts and moulds in foods are appropriate for raw fruits.

Modification of Karmali agar by supplementation with potassium clavulanate for the isolation of Campylobacter from chicken carcass rinses.

PubMed

Chon, Jung-Whan; Kim, Hong-Seok; Kim, Dong-Hyeon; Kim, Hyunsook; Choi, In-Soo; Oh, Deog-Hwan; Seo, Kun-Ho

2014-07-01

The detection ability and selectivity of Karmali agar was improved by supplementation of an extended-spectrum β-lactamase inhibitor, potassium clavulanate. The optimum concentration of potassium clavulanate (0.5 μg/ml) in Karmali agar was determined by inoculation of 50 Campylobacter and 30 extended-spectrum β-lactamase-producing E. coli strains onto normal and modified Karmali agar containing various concentrations of the agent. Eighty retail carcasses were rinsed with 400 ml of buffered peptone water. The rinse samples were enriched in 2 × blood-free Bolton enrichment broth at 42°C for 48 h and then were streaked onto normal and modified Karmali agar containing 0.5 μg/ml potassium clavulanate. The suspicious colonies were subcultured on Columbia blood agar and confirmed by colony PCR. In chicken carcass samples, the modified Karmali agar showed a significantly greater isolation rate than normal Karmali agar (42.5 versus 21.3%; P < 0.05). Furthermore, the selectivity of the modified Karmali agar was also significantly higher (P < 0.05) than that of the normal Karmali agar, as seen by comparison of the number of contaminated agar plates (83.8 versus 97.5%) and the growth index (1.67 versus 2.91) of the non-Campylobacter colonies.

Evaluation of the antibacterial efficacy of diesters of azelaic acid.

PubMed

Charnock, Colin; Brudeli, Bjarne; Klaveness, Jo

2004-04-01

A number of diesters of the topical dermatosis treatment azelaic (nonanedioic) acid were prepared and tested for antibacterial effect. Two esters, bis-[(hexanoyloxy)methyl] nonanedioate and especially bis-[(butanoyloxy)methyl] nonanedioate showed promising activity against acne related bacteria in vitro. No activity of azelaic acid was detected in Mueller Hinton II agar at pH 7.3 when using the agar diffusion method, whereas both esters gave zones of growth inhibition. At pH 5.6, activity of azelaic acid was detected. At this pH, the zones of inhibition and MIC values obtained with azelaic acid were smaller than those of bis-[(butanoyloxy)methyl] nonanedioate for all test organisms. A preparation for topical use containing 20% (w/w) bis-[(butanoyloxy)methyl] nonanedioate, and the commercially available Skinoren (20% (w/w) azelaic acid), were compared for antibacterial effect against cutaneous bacteria using contact plate analyses of the skin. Though Skinoren was usually most effective, the differences were not statistically significant. Furthermore, bacteria surviving contact with the topical preparations were invariably more sensitive to the ester than to azelaic acid upon subculturing onto agar (pH 5.6) containing either preparation at 0.2-0.7 mg/ml. This might indicate that other factors, such as the composition of the cream base, mitigate the antibacterial activity of the ester. It is proposed that the pharmacological and microbiological properties of bis-[(butanoyloxy)methyl] nonanedioate are worthy of further study based on an extended screening of acne sufferers.

Isolation of Thermophilic Lignin Degrading Bacteria from Oil-Palm Empty Fruit Bunch (EFB) Compost

NASA Astrophysics Data System (ADS)

Lai, C. M. T.; Chua, H. B.; Danquah, M. K.; Saptoro, A.

2017-06-01

Empty Fruit Bunch (EFB) is a potential and sustainable feedstock for bioethanol production due to its high cellulosic content and availability in Malaysia. Due to high lignin content of EFB and the lack of effective delignification process, commercial bioethanol production from EFB is presently not viable. Enzymatic delignification has been identified as one of the key steps in utilising EFB as a feedstock for bioethanol conversion. To date, limited work has been reported on the isolation of lignin degrading bacteria. Hence, there is a growing interest to search for new lignin degrading bacteria with greater tolerance to temperature and high level of ligninolytic enzymes for more effective lignin degradation. This study aimed to isolate and screen thermophilic ligninolytic microorganisms from EFB compost. Ten isolates were successfully isolated from EFB compost. Although they are not capable of decolorizing Methylene Blue (MB) dye under agar plate assay method, they are able to utilize lignin mimicked compound - guaiacol as a sole carbon on the agar plate assay. This infers that there is no correlation of ligninolytic enzymes with dye decolourization for all the isolates that have been isolated. However, they are able to produce ligninolytic enzymes (Lignin peroxidase, Manganese peroxidase, Laccase) in Minimal Salt Medium with Kraft Lignin (MSM-KL) with Lignin Peroxidase (LiP) as the predominant enzyme followed by Manganese Peroxidase (MnP) and Laccase (Lac). Among all the tested isolates, CLMT 29 has the highest LiP production up to 8.7673 U/mL following 24 h of growth.

Use of Dehydrated Agar to Estimate Microbial Water Quality for Horticulture Irrigation.

PubMed

Meador, Dustin P; Fisher, Paul R; Guy, Charles L; Harmon, Philip F; Peres, Natalia A; Teplitski, Max

2016-07-01

Petrifilms are dehydrated agar culture plates that have been used to quantify colony forming units (CFU) mL of either aerobic bacteria (Petrifilm-AC) or fungus (Petrifilm-YM), depending on substrate composition. Microbes in irrigation systems can indicate biofilm risk and potential clogging of irrigation emitters. The research objective was to compare counts on Petrifilms versus traditional, hydrated-agar plates using samples collected from recirculated irrigation waters and cultures of isolated known species. The estimated count (in CFU mL) from a recirculated irrigation sample after 7 d of incubation on Petrifilm-YM was only 5.5% of the count quantified using sabouraud dextrose agar (SDA) with chloramphenicol after 14 d. In a separate experiment with a known species, Petrifilm-YM did not successfully culture zoospores of . Isolates of viable zoospores were cultured successfully on potato-dextrose agar (PDA), with comparable counts with a vegetable juice medium supplemented with the antibiotics pimaricin, ampicillin, rifamycin, pentochloronitrobenzene and hymexazol (PARP-H). The quantification of pv. Begoniaceae on Petrifilm-AC was not significantly different ( < 0.05) than on PDA, but was lower than on Reasoner and Goldrich agar (R2A) or with a hemocytometer. The current formulation of Petrifilm-YM is unlikely to be a useful monitoring method for plant pathogens in irrigation water because of the inability to successfully culture oomycetes. However, Petrifilm-AC was an effective method to quantify bacteria and can provide an easy-to-use on-farm tool to monitor biofilm risk and microbial density. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

Mutation of the cyclic di-GMP phosphodiesterase gene in Burkholderia lata SK875 attenuates virulence and enhances biofilm formation.

PubMed

Jung, Hae-In; Kim, Yun-Jung; Lee, Yun-Jung; Lee, Hee-Soo; Lee, Jung-Kee; Kim, Soo-Ki

2017-10-01

Burkholderia sp. is a gram-negative bacterium that commonly exists in the environment, and can cause diseases in plants, animals, and humans. Here, a transposon mutant library of a Burkholderia lata isolate from a pig with swine respiratory disease in Korea was screened for strains showing attenuated virulence in Caenorhabditis elegans. One such mutant was obtained, and the Tn5 insertion junction was mapped to rpfR, a gene encoding a cyclic di-GMP phosphodiesterase that functions as a receptor. Mutation of rpfR caused a reduction in growth on CPG agar and swimming motility as well as a rough colony morphology on Congo red agar. TLC analysis showed reduced AHL secretion, which was in agreement with the results from plate-based and bioluminescence assays. The mutant strain produced significantly more biofilm detected by crystal violet staining than the parent strain. SEM of the mutant strain clearly showed that the overproduced biofilm contained a filamentous structure. These results suggest that the cyclic di-GMP phosphodiesterase RpfR plays an important role in quorum sensing modulation of the bacterial virulence and biofilm formation.

Introducing a Novel Media to Improve the Recovery of Culturable Bacteria from the Fish Parasite Anisakis spp. larvae (Nematoda: Anisakidae).

PubMed

Svanevik, Cecilie S; Lunestad, Bjørn T

2017-09-01

This paper describes a cultivation method to increase the recovery of bacteria from the marine muscle-invading parasitic nematode larvae of Anisakis spp. These larvae hold a high and complex population of accumulated bacteria, originating from seawater, crustaceans, fish, and marine mammals, all involved in the lifecycle of Anisakis. Two in-house agars based on fish juice prepared by either mechanical or enzymatic degradation of the fish tissue, were made. The Anisakis larvae were homogenised prior to cultivation on the in-house fish juice agars and the bacterial numbers and diversity were compared to those obtained applying the commercially available Marine Agar and Iron Agar Lyngby. Bacterial colonies of unique appearance were subcultured and identified by 16S rRNA gene sequencing. Totally three of twenty identified taxa were found on the in-house fish juice agars only. Fish juice agar prepared enzymatically would be the best supplementary agar, as this agar gave significantly higher heterotrophic plate counts, compared to mechanical preparation. The enzymatically prepared fish juice gave more suitable agar quality, was more resource efficient, and had apparently increased nutrient density and availability.

Identification of Bacterial Species in Kuwaiti Waters Through DNA Sequencing

NASA Astrophysics Data System (ADS)

Chen, K.

2017-01-01

With an objective of identifying the bacterial diversity associated with ecosystem of various Kuwaiti Seas, bacteria were cultured and isolated from 3 water samples. Due to the difficulties for cultured and isolated fecal coliforms on the selective agar plates, bacterial isolates from marine agar plates were selected for molecular identification. 16S rRNA genes were successfully amplified from the genome of the selected isolates using Universal Eubacterial 16S rRNA primers. The resulted amplification products were subjected to automated DNA sequencing. Partial 16S rDNA sequences obtained were compared directly with sequences in the NCBI database using BLAST as well as with the sequences available with Ribosomal Database Project (RDP).

Comparative analysis of cryopreservation methods in Chlamydomonas reinhardtii.

PubMed

Scarbrough, Chasity; Wirschell, Maureen

2016-10-01

Chlamydomonas is a model organism used for studies of many important biological processes. Traditionally, strains have been propagated on solid agar, which requires routine passaging for long-term maintenance. Cryopreservation of Chlamydomonas is possible, yet long-term viability is highly variable. Thus, improved cryopreservation methods for Chlamydomonas are an important requirement for sustained study of genetically defined strains. Here, we tested a commercial cryopreservation kit and directly compared it's effectiveness to a methanol-based method. We also tested thaw-back procedures comparing the growth of cells in liquid culture or on solid agar media. We demonstrated that methanol was the superior cryopreservation method for Chlamydomonas compared to the commercial kit and that post-thaw culture conditions dramatically affect viability. We also demonstrated that cryopreserved cells could be successfully thawed and plated directly onto solid agar plates. Our findings have important implications for the long-term storage of Chlamydomonas that can likely be extended to other algal species. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Promoter library-based module combination (PLMC) technology for optimization of threonine biosynthesis in Corynebacterium glutamicum.

PubMed

Wei, Liang; Xu, Ning; Wang, Yiran; Zhou, Wei; Han, Guoqiang; Ma, Yanhe; Liu, Jun

2018-05-01

Due to the lack of efficient control elements and tools, the fine-tuning of gene expression in the multi-gene metabolic pathways is still a great challenge for engineering microbial cell factories, especially for the important industrial microorganism Corynebacterium glutamicum. In this study, the promoter library-based module combination (PLMC) technology was developed to efficiently optimize the expression of genes in C. glutamicum. A random promoter library was designed to contain the putative - 10 (NNTANANT) and - 35 (NNGNCN) consensus motifs, and refined through a three-step screening procedure to achieve numerous genetic control elements with different strength levels, including fluorescence-activated cell sorting (FACS) screening, agar plate screening, and 96-well plate screening. Multiple conventional strategies were employed for further precise characterizations of the promoter library, such as real-time quantitative PCR, sodium dodecyl sulfate polyacrylamide gel electrophoresis, FACS analysis, and the lacZ reporter system. These results suggested that the established promoter elements effectively regulated gene expression and showed varying strengths over a wide range. Subsequently, a multi-module combination technology was created based on the efficient promoter elements for combination and optimization of modules in the multi-gene pathways. Using this technology, the threonine biosynthesis pathway was reconstructed and optimized by predictable tuning expression of five modules in C. glutamicum. The threonine titer of the optimized strain was significantly improved to 12.8 g/L, an approximate 6.1-fold higher than that of the control strain. Overall, the PLMC technology presented in this study provides a rapid and effective method for combination and optimization of multi-gene pathways in C. glutamicum.

A comparison of legionella and other bacteria concentrations in cooling tower water

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cappabianca, R.M.; Jurinski, N.B.; Jurinski, J.B.

1994-05-01

A field study was conducted in which water samples collected from air conditioning cooling water reservoirs of high-rise buildings throughout an urban area were assayed for Legionella and for total bacteria. Buildings included within the study had ongoing biocidal treatment programs for the cooling towers. Separate sample analyses were performed to measure the viable colony concentrations of total bacteria and of Legionella in the process waters. The occurrence and viable counts of Legionella in 304 environmental water samples were determined by inoculating them onto plates of buffered charcoal yeast extract (BCYE) agar medium (a presumptive screening method). The samples weremore » collected during summer months between July and September. BCYE plate cultures of 50 (16.4%) of the samples yielded Legionella with viable counts ranging from 2 to 608 colony forming units per milliliter. In the water samples, 281 (92.4%) yielded viable counts of bacteria that ranged from 9 to 1.2 x 10{sup 6} per milliliter. This study demonstrates that Legionella are commonly present in the water of air conditioning cooling towers and that there is no significant correlation between concurrently sampled culture plate counts of Legionella and total bacteria plate counts. Correspondingly, there is no demonstrated validity for use of total bacterial counts as an inferential surrogate for the concentration of Legionella in the water. 19 refs., 3 figs., 1 tab.« less

Colonization of Enteroaggregative Escherichia coli and Shiga toxin-producing Escherichia coli in chickens and humans in southern Vietnam.

PubMed

Trung, Nguyen Vinh; Nhung, Hoang Ngoc; Carrique-Mas, Juan J; Mai, Ho Huynh; Tuyen, Ha Thanh; Campbell, James; Nhung, Nguyen Thi; Van Minh, Pham; Wagenaar, Jaap A; Mai, Nguyen Thi Nhu; Hieu, Thai Quoc; Schultsz, Constance; Hoa, Ngo Thi

2016-09-09

Enteroaggregative (EAEC) and Shiga-toxin producing Escherichia coli (STEC) are a major cause of diarrhea worldwide. E. coli carrying both virulence factors characteristic for EAEC and STEC and producing extended-spectrum beta-lactamase caused severe and protracted disease during an outbreak of E. coli O104:H4 in Europe in 2011. We assessed the opportunities for E. coli carrying the aggR and stx genes to emerge in 'backyard' farms in south-east Asia. Faecal samples collected from 204 chicken farms; 204 farmers and 306 age- and gender-matched individuals not exposed to poultry farming were plated on MacConkey agar plates with and without antimicrobials being supplemented. Sweep samples obtained from MacConkey agar plates without supplemented antimicrobials were screened by multiplex PCR for the detection of the stx1, stx2 and aggR genes. One chicken farm sample each (0.5 %) contained the stx1 and the aggR gene. Eleven (2.4 %) human faecal samples contained the stx1 gene, 2 samples (0.4 %) contained stx2 gene, and 31 (6.8 %) contained the aggR gene. From 46 PCR-positive samples, 205 E. coli isolates were tested for the presence of stx1, stx2, aggR, wzx O104 and fliC H4 genes. None of the isolates simultaneously contained the four genetic markers associated with E. coli O104:H4 epidemic strain (aggR, stx2, wzx O104 and fliC H4 ). Of 34 EAEC, 64.7 % were resistant to 3(rd)-generation cephalosporins. These results indicate that in southern Vietnam, the human population is a more likely reservoir of aggR and stx gene carrying E. coli than the chicken population. However, conditions for transmission of isolates and/or genes between human and animal reservoirs resulting in the emergence of highly virulent E. coli strains are still favorable, given the nature of'backyard' farms in Vietnam.

Improved soft-agar colony assay in a fluid processing apparatus.

PubMed

Forsman, A D; Herpich, A R; Chapes, S K

1999-01-01

The standard method for quantitating bone marrow precursor cells has been to count the number of colony-forming units that form in semisolid (0.3%) agar. Recently we adapted this assay for use in hardware, the Fluid Processing Apparatus, that is flown in standard payload lockers of the space shuttle. When mouse or rat macrophage colony-forming units were measured with this hardware in ground-based assays, we found significantly more colony growth than that seen in standard plate assays. The improved growth correlates with increased agar thickness but also appears to be due to properties inherent to the Fluid Processing Apparatus. This paper describes an improved method for determining bone marrow macrophage precursor numbers in semisolid agar.

Visualization of Biosurfactant Film Flow in a Bacillus subtilis Swarm Colony on an Agar Plate

PubMed Central

Kim, Kyunghoon; Kim, Jung Kyung

2015-01-01

Collective bacterial dynamics plays a crucial role in colony development. Although many research groups have studied the behavior of fluidic swarm colonies, the detailed mechanics of its motion remains elusive. Here, we developed a visualization method using submicron fluorescent beads for investigating the flow field in a thin layer of fluid that covers a Bacillus subtilis swarm colony growing on an agar plate. The beads were initially embedded in the agar plate and subsequently distributed spontaneously at the upper surface of the expanding colony. We conducted long-term live cell imaging of the B. subtilis colony using the fluorescent tracers, and obtained high-resolution velocity maps of microscale vortices in the swarm colony using particle image velocimetry. A distinct periodic fluctuation in the average speed and vorticity of flow in swarm colony was observed at the inner region of the colony, and correlated with the switch between bacterial swarming and growth phases. At the advancing edge of the colony, both the magnitudes of velocity and vorticity of flow in swarm colony were inversely correlated with the spreading speed of the swarm edge. The advanced imaging tool developed in this study would facilitate further understanding of the effect of micro vortices in swarm colony on the collective dynamics of bacteria. PMID:26343634

Visualization of Biosurfactant Film Flow in a Bacillus subtilis Swarm Colony on an Agar Plate.

PubMed

Kim, Kyunghoon; Kim, Jung Kyung

2015-08-26

Collective bacterial dynamics plays a crucial role in colony development. Although many research groups have studied the behavior of fluidic swarm colonies, the detailed mechanics of its motion remains elusive. Here, we developed a visualization method using submicron fluorescent beads for investigating the flow field in a thin layer of fluid that covers a Bacillus subtilis swarm colony growing on an agar plate. The beads were initially embedded in the agar plate and subsequently distributed spontaneously at the upper surface of the expanding colony. We conducted long-term live cell imaging of the B. subtilis colony using the fluorescent tracers, and obtained high-resolution velocity maps of microscale vortices in the swarm colony using particle image velocimetry. A distinct periodic fluctuation in the average speed and vorticity of flow in swarm colony was observed at the inner region of the colony, and correlated with the switch between bacterial swarming and growth phases. At the advancing edge of the colony, both the magnitudes of velocity and vorticity of flow in swarm colony were inversely correlated with the spreading speed of the swarm edge. The advanced imaging tool developed in this study would facilitate further understanding of the effect of micro vortices in swarm colony on the collective dynamics of bacteria.

Clarithromycin resistance of Helicobacter pylori strains isolated from children' gastric antrum and fundus as assessed by fluorescent in-situ hybridization and culture on four-sector agar plates.

PubMed

Caristo, Elisa; Parola, Andrea; Rapa, Anna; Vivenza, Daniela; Raselli, Barbara; Dondi, Elena; Boldorini, Renzo; Oderda, Giuseppina

2008-12-01

To assess validity of culture on four-sector agar plates and fluorescent in-situ hybridization (FISH) test, and clarithromycin resistance rate in Helicobacter pylori strains isolated from children in the last 10 years. In the last 5 years, gastric biopsy specimens from antrum and fundus were taken from 89 consecutive children (median age 9 years) with H. pylori gastritis and from 21 controls. Culture was performed on 176 gastric biopsies (89 from antrum, 87 from fundus) on four-sector agar plates, and FISH test with DNA ProbeMix. After its validity was evaluated, FISH test was applied on additional 119 biopsies from 68 children (68 from the antrum, 51 from the fundus) stored in the Pathology archive in the previous 5 years. Culture was positive in 157 of 176 biopsies (sensitivity: 89.2%, 95% confidence interval (CI) 85-94). In 33 of 89 children (37%) resistant strains were found in one or both gastric sites. FISH test was positive in 148 of 176 biopsies from infected children (sensitivity 84.1%, 95%CI 79-89) and in none of 42 biopsies from controls (specificity 100%). When applied on archive biopsies, FISH test was positive in 96 of 119 (80.7%, 95%CI 74-88). Total children harboring resistant strains in the last 10 years, as assessed by FISH test, were 66 of 157 (42%). Mixed infection with both sensitive and resistant strains were found in 40 children (25%) and in 12 of them resistant strains were in the fundus only. Culture on four-sector agar plates and FISH test had a high sensitivity and specificity and showed co-presence of sensitive and resistant strains. In one-third of children with mixed infection, the resistant strains were in the fundus only. Clarithromycin resistance should be assessed in biopsies both from the antrum and the fundus, utilizing antral biopsies only can underestimate its prevalence.

Isolation of Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 from ground beef using modified rainbow agar and post-immunomagnetic separation acid treatment.

PubMed

Tillman, Glenn E; Wasilenko, Jamie L; Simmons, Mustafa; Lauze, Todd A; Minicozzi, Joseph; Oakley, Brian B; Narang, Neelam; Fratamico, Pina; Cray, Ailliam C

2012-09-01

It is estimated that at least 70% of human illnesses due to non-O157 Shiga toxin-producing Escherichia coli (STEC) in the United States are caused by strains from the top six serogroups (O26, O45, O103, O111, O121, and O145). Procedures for isolating STEC from food products often use plating media that include antimicrobial supplements at concentrations that inhibit background microflora growth but can also inhibit target STEC growth. In this study, an agar medium with lower supplement concentrations, modified Rainbow agar (mRBA), was evaluated for recovery of STEC serogroups O26, O45, O103, O111, O121, and O145 from ground beef enrichments. A post-immunomagnetic separation (IMS) acid treatment step was additionally used to reduce background microflora and increase recovery of target STEC strains. Ground beef samples (325 g) were artificially contaminated with STEC and confounding organisms and enriched for 15 h. Recovery of the target STEC was attempted on the enrichments using IMS and plating onto mRBA and Rainbow agar (RBA). Additionally, acid treatment was performed on the post-IMS eluate followed by plating onto mRBA. Using the combination of mRBA and acid treatment, target STEC were isolated from 103 (85.8%) of 120 of the low-inoculated samples (1 to 5 CFU/325-g sample) compared with 68 (56.7%) of 120 using no acid treatment and plating onto RBA with higher levels of novobiocin and potassium tellurite. The combination of acid treatment and mRBA provides a significant improvement over the use of RBA for isolation of STEC serogroups O26, O45, O103, O111, O121, and O145 from raw ground beef.

Plating isolation of various catalase-negative microorganisms from soil

NASA Technical Reports Server (NTRS)

Labeda, D. P.; Hunt, C. M.; Casida, L. E., Jr.

1974-01-01

A unique plating procedure was developed that allows isolation, but not enumeration, of representatives of the catalase-negative soil microflora. The numbers recovered, however, are low as compared to the numbers recovered when the modified dilution-to-extinction isolation procedure is used. The latter procedure provides prolonged inoculation in sealed tubes containing a nutritionally rich broth medium over small submerged agar slants. In contrast, the plating procedure utilizes nutritionally minimal media and the shorter incubations mandated by the inherent problems associated with plating.

Research on Candida dubliniensis in a Brazilian yeast collection obtained from cardiac transplant, tuberculosis, and HIV-positive patients, and evaluation of phenotypic tests using agar screening methods.

PubMed

Ribeiro, Patrícia Monteiro; Querido, Silvia Maria Rodrigues; Back-Brito, Graziela Nueremberg; Mota, Adolfo José; Koga-Ito, Cristiane Yumi; Jorge, Antonio Olavo Cardoso

2011-09-01

The aim of this study was to research Candida dubliniensis among isolates present in a Brazilian yeast collection and to evaluate the main phenotypic methods for discrimination between C. albicans and C. dubliniensis from oral cavity. A total of 200 isolates, presumptively identified as C. albicans or C. dubliniensis obtained from heart transplant patients under immunosuppressive therapy, tuberculosis patients under antibiotic therapy, HIV-positive patients under antiretroviral therapy, and healthy subjects, were analyzed using the following phenotypic tests: formation and structural arrangement of chlamydospores on corn meal agar, casein agar, tobacco agar, and sunflower seed agar; growth at 45 °C; and germ tube formation. All strains were analyzed by polymerase chain reaction (PCR). In a preliminary screen for C. dubliniensis, 48 of the 200 isolates on corn meal agar, 30 of the 200 on casein agar, 16 of the 200 on tobacco agar, and 15 of the 200 on sunflower seed agar produced chlamydoconidia; 27 of the 200 isolates showed no or poor growth at 45 °C. All isolates were positive for germ tube formation. These isolates were considered suggestive of C. dubliniensis. All of them were subjected to PCR analysis using C. dubliniensis-specific primers. C. dubliniensis isolates were not found. C. dubliniensis isolates were not recovered in this study done with immunocompromised patients. Sunflower seed agar was the medium with the smallest number of isolates of C. albicans suggestive of C. dubliniensis. None of the phenotypic methods was 100% effective for discrimination between C. albicans and C. dubliniensis. Copyright © 2011 Elsevier Inc. All rights reserved.

[Analysis on the antimicrobial resistance of lactic acid bacteria isolated from the yogurt sold in China].

PubMed

Fan, Qin; Liu, Shuliang; Li, Juan; Huang, Tingting

2012-05-01

To analyze the antimicrobial susceptibility of lactic acid bacteria (LAB) from yogurt, and to provide references for evaluating the safety of LAB and screening safe strains. The sensitivity of 43 LAB strains, including 14 strains of Streptococcus thermophilus, 12 strains of Lactobacillus acidophilus, 9 strains of Lactobacillus bulgaricus and 8 strains of Bifidobacterium, to 22 antibiotics were tested by agar plate dilution method. All 43 LAB strains were resistant to trimethoprim, nalidixic acid, ciprofloxacin, lomefloxacin, danofloxacin and polymyxin E. Their resistances to kanamycin, tetracycline, clindamycin, doxycycline and cephalothin were varied. The sensitivity to other antibiotics were sensitive or moderate. All isolates were multidrug-resistant. The antimicrobial resistance of tested LAB strains was comparatively serious, and continuously monitoring their antimicrobial resistance and evaluating their safety should be strengthened.

High-throughput screening for cellobiose dehydrogenases by Prussian Blue in situ formation.

PubMed

Vasilchenko, Liliya G; Ludwig, Roland; Yershevich, Olga P; Haltrich, Dietmar; Rabinovich, Mikhail L

2012-07-01

Extracellular fungal flavocytochrome cellobiose dehydrogenase (CDH) is a promising enzyme for both bioelectronics and lignocellulose bioconversion. A selective high-throughput screening assay for CDH in the presence of various fungal oxidoreductases was developed. It is based on Prussian Blue (PB) in situ formation in the presence of cellobiose (<0.25 mM), ferric acetate, and ferricyanide. CDH induces PB formation via both reduction of ferricyanide to ferrocyanide reacting with an excess of Fe³⁺ (pathway 1) and reduction of ferric ions to Fe²⁺ reacting with the excess of ferricyanide (pathway 2). Basidiomycetous and ascomycetous CDH formed PB optimally at pH 3.5 and 4.5, respectively. In contrast to the holoenzyme CDH, its FAD-containing dehydrogenase domain lacking the cytochrome domain formed PB only via pathway 1 and was less active than the parent enzyme. The assay can be applied on active growing cultures on agar plates or on fungal culture supernatants in 96-well plates under aerobic conditions. Neither other carbohydrate oxidoreductases (pyranose dehydrogenase, FAD-dependent glucose dehydrogenase, glucose oxidase) nor laccase interfered with CDH activity in this assay. Applicability of the developed assay for the selection of new ascomycetous CDH producers as well as possibility of the controlled synthesis of new PB nanocomposites by CDH are discussed. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparative evaluation of direct plating and most probable number for enumeration of low levels of Listeria monocytogenes in naturally contaminated ice cream products.

PubMed

Chen, Yi; Pouillot, Régis; S Burall, Laurel; Strain, Errol A; Van Doren, Jane M; De Jesus, Antonio J; Laasri, Anna; Wang, Hua; Ali, Laila; Tatavarthy, Aparna; Zhang, Guodong; Hu, Lijun; Day, James; Sheth, Ishani; Kang, Jihun; Sahu, Surasri; Srinivasan, Devayani; Brown, Eric W; Parish, Mickey; Zink, Donald L; Datta, Atin R; Hammack, Thomas S; Macarisin, Dumitru

2017-01-16

A precise and accurate method for enumeration of low level of Listeria monocytogenes in foods is critical to a variety of studies. In this study, paired comparison of most probable number (MPN) and direct plating enumeration of L. monocytogenes was conducted on a total of 1730 outbreak-associated ice cream samples that were naturally contaminated with low level of L. monocytogenes. MPN was performed on all 1730 samples. Direct plating was performed on all samples using the RAPID'L.mono (RLM) agar (1600 samples) and agar Listeria Ottaviani and Agosti (ALOA; 130 samples). Probabilistic analysis with Bayesian inference model was used to compare paired direct plating and MPN estimates of L. monocytogenes in ice cream samples because assumptions implicit in ordinary least squares (OLS) linear regression analyses were not met for such a comparison. The probabilistic analysis revealed good agreement between the MPN and direct plating estimates, and this agreement showed that the MPN schemes and direct plating schemes using ALOA or RLM evaluated in the present study were suitable for enumerating low levels of L. monocytogenes in these ice cream samples. The statistical analysis further revealed that OLS linear regression analyses of direct plating and MPN data did introduce bias that incorrectly characterized systematic differences between estimates from the two methods. Published by Elsevier B.V.

Comparison of culture based methods for the isolation of Clostridium difficile from stool samples in a research setting.

PubMed

Lister, Michelle; Stevenson, Emma; Heeg, Daniela; Minton, Nigel P; Kuehne, Sarah A

2014-08-01

Effective isolation of Clostridium difficile from stool samples is important in the research setting, especially where low numbers of spores/vegetative cells may be present within a sample. In this study, three protocols for stool culture were investigated to find a sensitive, cost effective and timely method of C. difficile isolation. For the initial enrichment step, the effectiveness of two different rich media, cycloserine-cefoxitin fructose broth (CCFB) and cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme (CCMB-TAL) were compared. For the comparison of four different, selective solid media; Cycloserine-cefoxitin fructose agar (CCFA), Cycloserine-cefoxitin egg yolk agar (CCEY), ChromID C. difficile and tryptone soy agar (TSA) with 5% sheep's blood with and without preceding broth enrichment were used. As a means to enable differentiation between C. difficile and other fecal flora, the effectiveness of the inclusion of a pH indictor (1% Neutral Red), was also evaluated. The data derived indicated that CCFB is more sensitive than CCMB-TAL, however, the latter had an improved recovery rate. A broth enrichment step had a reduced sensitivity over direct plating. ChromID C. difficile showed the best recovery rate whereas CCEY egg yolk agar was the most sensitive of the four. The addition of 1% Neutral Red did not show sufficient colour change when added to CCEY egg yolk agar to be used as a differential medium. For a low cost, timely and sensitive method of isolating C. difficile from stool samples we recommend direct plating onto CCEY egg yolk agar after heat shock. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Plating Bacteriophage M13.

PubMed

Green, Michael R; Sambrook, Joseph

2017-10-03

A plaque of bacteriophage M13 derives from infection of a single bacterium by a single virus particle. The progeny particles infect neighboring bacteria, which, in turn, release another generation of daughter virus particles. If the bacteria are growing in semisolid medium (e.g., containing agar or agarose), then the diffusion of the progeny particles is limited. Cells infected with bacteriophage M13 are not killed, but have a longer generation time than uninfected Escherichia coli In consequence, plaques appear as areas of slower-growing cells on a faster-growing lawn of bacterial cells. This protocol describes plating of bacteriophage M13 stocks. Plaques are readily detectable on top agar after 4-8 h of incubation at 37°C. © 2017 Cold Spring Harbor Laboratory Press.

Use of an acidophilic yeast strain to enable the growth of leaching bacteria on solid media.

PubMed

Ngom, Baba; Liang, Yili; Liu, Yi; Yin, Huaqun; Liu, Xueduan

2015-03-01

In this study, a Candida digboiensis strain was isolated from a heap leaching plant in Zambia and used in double-layer agar plate to efficiently isolate and purify leaching bacteria. Unlike Acidiphilium sp., the yeast strain was tetrathionate tolerant and could metabolize a great range of organic compounds including organic acids. These properties allowed the yeast strain to enable and fasten the growth of iron and sulfur oxidizers on double-layer agar plate. The isolates were identified as Acidithiobacillus ferrooxidans FOX1, Leptospirillun ferriphilum BN, and Acidithiobacillus thiooxidans ZMB. These three leaching bacteria were inhibited by organic acids such as acetic and propionic acids; however, their activities were enhanced by Candida digboiensis NB under dissolved organic matter stress.

[Isolation and identification of rumen bacteria for cellulolytic enzyme production].

PubMed

Aihemaiti, Maierhaba; Zhen, Fan; Li, Yuezhong; Aibaidoula, Gulisimayi; Yimit, Wusiman

2013-05-04

We screened aerobic bacteria with cellulolytic activity from ruminal fluid of sheep, cattle and camel in Xinjiang. Fresh ruminal fluid was inoculated on sterilized sodium carboxymethylcellulose agar plates. Highly cellulolytic aerobic bacteria were screened out by using Congo red staining and liquid secondary screening culture media. The combination of morphological and biochemical test with 16SrDNA sequence analysis were used to classify the strains. Enzymatic activities of four strains with strong cellulose-decomposing abilities were studied under different culture conditions. Out 84 isolated cellulolytic strains, 40 exhibited strong abilities in decomposing cellulose. They are including 37 Gram-negative isolates and 3 Gram-positive strains. Identification of these 40 strains shows that they belong to 11 species of 6 genera, 16 strains in Stenotrophomonas maltophilia, 10 Ochrobactrum, 5 Sphingobacterium, 3 Microbacterium, 3 Paracoccus and 2 Pseudomonas. The results of the enzymatic studies of four strains with strong cellulolytic abilities indicates that the strains have the best enzyme producing property when straw powder was chosen as the carbon source; the pH at 5.5 -6.0 and temperature at 37 degrees C. The strains with highly cellulolytic abilities isolated from ruminal fluid show strong abilities in cellulose decomposition.

Genetic transformation system in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

PubMed Central

Worrell, V E; Nagle, D P; McCarthy, D; Eisenbraun, A

1988-01-01

A wild-type strain of Methanobacterium thermoautotrophicum Marburg was transformed by DNA from strains resistant to 5-fluorouracil. Recipient cells were grown without selection on gellan gum (GELRITE) plates with DNA. Drug-resistant cells were recovered by replica plating the resulting colonies onto drug plates. Transformation required high-molecular-weight DNA with appropriate markers and was not observed on agar or in liquid media under a variety of conditions. PMID:3422229

Methicillin-Resistant Staphylococcus aureus: Comparison of Susceptibility Testing Methods and Analysis of mecA-Positive Susceptible Strains

PubMed Central

Sakoulas, George; Gold, Howard S.; Venkataraman, Lata; DeGirolami, Paola C.; Eliopoulos, George M.; Qian, Qinfang

2001-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for an increasing number of serious nosocomial and community-acquired infections. Phenotypic heterogeneous drug resistance (heteroresistance) to antistaphylococcal beta-lactams affects the results of susceptibility testing. The present study compared the MRSA-Screen latex agglutination test (Denka Seiken Co., Ltd., Tokyo, Japan) for detection of PBP 2a with agar dilution, the VITEK-1 and VITEK-2 systems (bioMérieux, St. Louis, Mo.), and the oxacillin agar screen test for detection of MRSA, with PCR for the mecA gene used as the “gold standard” assay. Analysis of 107 methicillin-susceptible S. aureus (MSSA) isolates and 203 MRSA isolates revealed that the MRSA-Screen latex agglutination test is superior to any single phenotype-based susceptibility testing method, with a sensitivity of 100% and a specificity of 99.1%. Only one isolate that lacked mecA was weakly positive by the MRSA-Screen latex agglutination test. This isolate was phenotypically susceptible to oxacillin and did not contain the mecA gene by Southern blot hybridization. The oxacillin agar screen test, the VITEK-1 system, the VITEK-2 system, and agar dilution showed sensitivities of 99.0, 99.0, 99.5, and 99%, respectively, and specificities of 98.1, 100, 97.2, and 100%, respectively. The differences in sensitivity or specificity were not statistically significant. Oxacillin bactericidal assays showed that mecA- and PBP 2a-positive S. aureus isolates that are susceptible to antistaphylococcal beta-lactams by conventional methods are functionally resistant to oxacillin. We conclude that the accuracy of the MRSA-Screen latex agglutination method for detection of PBP 2a approaches the accuracy of PCR and is more accurate than any susceptibility testing method used alone for the detection of MRSA. PMID:11682512

The Clinical Urine Culture: Enhanced Techniques Improve Detection of Clinically Relevant Microorganisms

PubMed Central

Price, Travis K.; Dune, Tanaka; Hilt, Evann E.; Thomas-White, Krystal J.; Kliethermes, Stephanie; Brincat, Cynthia; Brubaker, Linda; Wolfe, Alan J.

2016-01-01

Enhanced quantitative urine culture (EQUC) detects live microorganisms in the vast majority of urine specimens reported as “no growth” by the standard urine culture protocol. Here, we evaluated an expanded set of EQUC conditions (expanded-spectrum EQUC) to identify an optimal version that provides a more complete description of uropathogens in women experiencing urinary tract infection (UTI)-like symptoms. One hundred fifty adult urogynecology patient-participants were characterized using a self-completed validated UTI symptom assessment (UTISA) questionnaire and asked “Do you feel you have a UTI?” Women responding negatively were recruited into the no-UTI cohort, while women responding affirmatively were recruited into the UTI cohort; the latter cohort was reassessed with the UTISA questionnaire 3 to 7 days later. Baseline catheterized urine samples were plated using both standard urine culture and expanded-spectrum EQUC protocols: standard urine culture inoculated at 1 μl onto 2 agars incubated aerobically; expanded-spectrum EQUC inoculated at three different volumes of urine onto 7 combinations of agars and environments. Compared to expanded-spectrum EQUC, standard urine culture missed 67% of uropathogens overall and 50% in participants with severe urinary symptoms. Thirty-six percent of participants with missed uropathogens reported no symptom resolution after treatment by standard urine culture results. Optimal detection of uropathogens could be achieved using the following: 100 μl of urine plated onto blood (blood agar plate [BAP]), colistin-nalidixic acid (CNA), and MacConkey agars in 5% CO2 for 48 h. This streamlined EQUC protocol achieved 84% uropathogen detection relative to 33% detection by standard urine culture. The streamlined EQUC protocol improves detection of uropathogens that are likely relevant for symptomatic women, giving clinicians the opportunity to receive additional information not currently reported using standard urine culture techniques. PMID:26962083

Detection of Group B Streptococcus in Brazilian pregnant women and antimicrobial susceptibility patterns

PubMed Central

Castellano-Filho, Didier Silveira; da Silva, Vânia Lúcia; Nascimento, Thiago César; de Toledo Vieira, Marcel; Diniz, Cláudio Galuppo

2010-01-01

Group B Streptococcus (GBS) is still not routinely screened during pregnancy in Brazil, being prophylaxis and empirical treatment based on identification of risk groups. This study aimed to investigate GBS prevalence in Brazilian pregnant women by culture or polymerase chain reaction (PCR) associated to the enrichment culture, and to determine the antimicrobial susceptibility patterns of isolated bacteria, so as to support public health policies and empirical prophylaxis. After an epidemiological survey, vaginal and anorectal specimens were collected from 221 consenting laboring women. Each sample was submitted to enrichment culture and sheep blood agar was used to isolate suggestive GBS. Alternatively, specific PCR was performed from enrichment cultures. Antimicrobial susceptibility patterns were determined for isolated bacteria by agar diffusion method. No risk groups were identified. Considering the culture-based methodology, GBS was detected in 9.5% of the donors. Twenty five bacterial strains were isolated and identified. Through the culture-PCR methodology, GBS was detected in 32.6% specimens. Bacterial resistance was not detected against ampicillin, cephazolin, vancomycin and ciprofloxacin, whereas 22.7% were resistant to erythromycin and 50% were resistant to clindamycin. GBS detection may be improved by the association of PCR and enrichment culture. Considering that colony selection in agar plates may be laboring and technician-dependent, it may not reflect the real prevalence of streptococci. As in Brazil prevention strategies to reduce the GBS associated diseases have not been adopted, prospective studies are needed to anchor public health policies especially considering the regional GBS antimicrobial susceptibility patterns. PMID:24031585

The Accuracy of the Sysmex UF-1000i in Urine Bacterial Detection Compared With the Standard Urine Analysis and Culture.

PubMed

Erdman, Patrick; Anderson, Brian; Zacko, J Christopher; Taylor, Kirk; Donaldson, Keri

2017-11-01

- Urinary tract infections are characterized by the presence of microbial pathogens within the urinary tract. They represent one of the most common infections in hospitalized and clinic patients. - To model the parameters of the Sysmex UF-1000i to the gold standard, urine culture, and to compare the detection of dipstick leukocyte esterase and nitrates to urine cultures and UF-1000i results. - Data were compared from urine samples collected in sterile containers for bacterial culture and microscopic analysis. One sample was used to inoculate a 5% sheep blood agar and MacConkey agar plate using a 0.001-mL calibrated loop. The second sample was analyzed by urinalysis-associated microscopy. The media plates were investigated for growth after 18 to 24 hours of aerobic incubation at 37°C. The second sample was analyzed for bacteria and leukocytes with the Sysmex UF-1000i according to the manufacturer's guidelines. Three definitions for culture results, sensitivity, and specificity at different cutoff values were calculated for the UF-1000i. - The negative predictive value for any positive culture in the adult population included in the study was 95.5%, and the negative predictive value for positive cultures containing growth of 100 000 or more colony-forming units was 99.3% using the Sysmex UF-1000i. - Sysmex UF-1000i showed 98% sensitivity and 93.7% specificity with a 95.5% negative predictive value. Thus, a negative screen with the UF-1000i using defined thresholds for white blood cell counts and bacteria was likely to be a true negative, decreasing the need for presumptive antibiotics.

Reflected scatterometry for noninvasive interrogation of bacterial colonies

NASA Astrophysics Data System (ADS)

Kim, Huisung; Doh, Iyll-Joon; Sturgis, Jennifer; Bhunia, Arun K.; Robinson, J. Paul; Bae, Euiwon

2016-10-01

A phenotyping of bacterial colonies on agar plates using forward-scattering diffraction-pattern analysis provided promising classification of several different bacteria such as Salmonella, Vibrio, Listeria, and E. coli. Since the technique is based on forward-scattering phenomena, light transmittance of both the colony and the medium is critical to ensure quality data. However, numerous microorganisms and their growth media allow only limited light penetration and render the forward-scattering measurement a challenging task. For example, yeast, Lactobacillus, mold, and several soil bacteria form colorful and dense colonies that obstruct most of the incoming light passing through them. Moreover, blood agar, which is widely utilized in the clinical field, completely blocks the incident coherent light source used in forward scatterometry. We present a newly designed reflection scatterometer and validation of the resolving power of the instrument. The reflectance-type instrument can acquire backward elastic scatter patterns for both highly opaque media and colonies and has been tested with three different bacterial genera grown on blood agar plates. Cross-validation results show a classification rate above 90% for four genera.

Airborne Salmonella and Listeria associated with Irish commercial beef, sheep and pig plants.

PubMed

Okraszewska-Lasica, Wioletta; Bolton, D J; Sheridan, J J; McDowell, D A

2014-06-01

Air samples from lairage, hide/fleece pulling or dehairing/scraping, evisceration and chilling areas in commercial beef, sheep and pig plants were examined for Salmonella spp. and Listeria monocytogenes, by impaction or sedimentation onto selective (Brilliant Green Agar, BSA; Listeria Selective Agar, LSA) and non-selective (Plate Count Agar, PCA) media. Both pathogens were frequently detected in all three plants. Improved recoveries were achieved by combining sedimentation, and broth based resuscitation, suggesting cell injury. Salmonella were recovered from all three plants, with the highest counts on BGA in the pig plant. The most common serotypes were S. Typhimurium in the beef/sheep plants and S. Derby in the pig plant. Very low counts of L. monocytogenes (e.g. 2.6CFUm(2)) were detected at hide removal on LSA sedimentation plates in the beef plant. These included serogroup 1/2a-3a and 1/2b-3b-7. Pathogen counts in the three plants were generally very low, suggesting that air is unlikely to be a significant source of carcass or plant surface contamination. Copyright © 2014 Elsevier Ltd. All rights reserved.

Improved method of screening for aflatoxin with a coconut agar medium.

PubMed Central

Davis, N D; Iyer, S K; Diener, U L

1987-01-01

Nine isolates of Aspergillus flavus and Aspergillus parasiticus were screened for aflatoxin production on a coconut extract agar medium. Aflatoxin-producing colonies were detected under long-wave UV light (365 nm) by blue fluorescence on the reverse side after 2 to 5 days of growth. Aflatoxin production was verified by chemical analysis. Several types of shredded coconut available in the United States were tested and found to be satisfactory. No additives were required. Various parameters affecting the test were investigated. PMID:3116928

A simple modification of the Baermann method for diagnosis of strongyloidiasis.

PubMed

Hernández-Chavarría, F; Avendaño, L

2001-08-01

The diagnosis of Strongyloides stercoralis infections is routinely made by microscopic observation of larvae in stool samples, a low sensitivity method, or by other, most effective methods, such as the Baermann or agar culture plate methods. We propose in this paper a practical modification of Baermann method. One hundred and six stool samples from alcoholic patients were analyzed using the direct smear test, agar culture plate method, the standard Baermann method, and its proposed modification. For this modification the funnel used in the original version of the method is substituted by a test tube with a rubber stopper, perforated to allow insertion of a pipette tip. The tube with a fecal suspension is inverted over another tube containing 6 ml of saline solution and incubated at 37 degrees C for at least 2 h. The saline solution from the second tube is centrifuged and the pellet is observed microscopically. Larva of S. stercoralis were detected in six samples (5.7%) by the two versions of the Baermann method. Five samples were positive using the agar culture plate method, and only in two samples the larva were observed using direct microscopic observation of fecal smears. Cysts of Endolimax nana and Entamoeba histolytica/dyspar were also detected in the modification of Baermann method. Data obtained by the modified Baermann method suggest that this methodology may helps concentrate larvae of S. stercoralis as efficiently as the original method.

Wood and humus decay strategies by white-rot basidiomycetes correlate with two different dye decolorization and enzyme secretion patterns on agar plates.

PubMed

Barrasa, José M; Blanco, María N; Esteve-Raventós, Fernando; Altés, Alberto; Checa, Julia; Martínez, Angel T; Ruiz-Dueñas, Francisco J

2014-11-01

During several forays for ligninolytic fungi in different Spanish native forests, 35 white-rot basidiomycetes growing on dead wood (16 species from 12 genera) and leaf litter (19 species from 10 genera) were selected for their ability to decolorize two recalcitrant aromatic dyes (Reactive Blue 38 and Reactive Black 5) added to malt extract agar medium. In this study, two dye decolorization patterns were observed and correlated with two ecophysiological groups (wood and humus white-rot basidiomycetes) and three taxonomical groups (orders Polyporales, Hymenochaetales and Agaricales). Depending on the above groups, different decolorization zones were observed on the dye-containing plates, being restricted to the colony area or extending to the surrounding medium, which suggested two different decay strategies. These two strategies were related to the ability to secrete peroxidases and laccases inside (white-rot wood Polyporales, Hymenochaetales and Agaricales) and outside (white-rot humus Agaricales) of the fungal colony, as revealed by enzymatic tests performed directly on the agar plates. Similar oxidoreductases production patterns were observed when fungi were grown in the absence of dyes, although the set of enzyme released was different. All these results suggest that the decolorization patterns observed could be related with the existence of two decay strategies developed by white-rot basidiomycetes adapted to wood and leaf litter decay in the field. Published by Elsevier Inc.

Effect of sampling and short isolation methodologies on the recovery of human pathogenic Yersinia enterocolitica from pig tonsils.

PubMed

Van Damme, Inge; Berkvens, Dirk; De Zutter, Lieven

2012-07-01

The objective of this study was to determine the effect of sampling (swab samples compared to destructive samples) on isolation rates of human pathogenic Yersinia enterocolitica from pig tonsils. Moreover, the relative efficiency of different rapid, routinely applicable isolation methods was evaluated. Therefore, swab and destructive samples from tonsils of 120 pigs at slaughter were analyzed in parallel using direct plating and different enrichment methods. Salmonella-Shigella-desoxycholate-calcium chloride (SSDC) agar, cefsulodin-irgasan-novobiocin (CIN) agar, and Yersinia enterocolitica chromogenic medium (YeCM) were used as selective agar media. For enrichment, irgasan-ticarcillin-potassium chlorate (ITC) broth and peptone-sorbitol-bile (PSB) broth were incubated at 25°C for 48 h. Overall, 55 tonsils (45.8%) were positive for Y. enterocolitica bioserotype 4/O:3. Recovery was significantly higher using the destructive method compared to the swabbing method. Direct plating resulted in 47 and 28 Y. enterocolitica-positive destructive and swab samples, respectively. Alkali treatment of PSB and ITC enrichment broths significantly increased recovery of pathogenic Y. enterocolitica from destructive tonsil samples. The performance of YeCM for qualitative and quantitative isolation of pathogenic Y. enterocolitica from pig tonsils was equal to SSDC and CIN. In conclusion, direct plating and ISO 10273: 2003 with minor modifications are suitable and rapid methods for isolation of pathogenic Y. enterocolitica from destructive tonsil samples.

Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe

PubMed Central

Xue, Yong; Wilkes, Jon G.; Moskal, Ted J.; Williams, Anna J.; Cooper, Willie M.; Nayak, Rajesh; Rafii, Fatemeh; Buzatu, Dan A.

2016-01-01

Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts. PMID:26913737

Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe.

PubMed

Xue, Yong; Wilkes, Jon G; Moskal, Ted J; Williams, Anna J; Cooper, Willie M; Nayak, Rajesh; Rafii, Fatemeh; Buzatu, Dan A

2016-01-01

Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts.

Aseptic laboratory techniques: plating methods.

PubMed

Sanders, Erin R

2012-05-11

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) as well as Material Safety Data Sheets (MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection (ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to: Perform plating procedures without contaminating media. Isolate single bacterial colonies by the streak-plating method. Use pour-plating and spread-plating methods to determine the concentration of bacteria. Perform soft agar overlays when working with phage. Transfer bacterial cells from one plate to another using the replica-plating procedure. Given an experimental task, select the appropriate plating method.

Comparison of different sampling techniques and of different culture methods for detection of group B streptococcus carriage in pregnant women.

PubMed

El Aila, Nabil A; Tency, Inge; Claeys, Geert; Saerens, Bart; Cools, Piet; Verstraelen, Hans; Temmerman, Marleen; Verhelst, Rita; Vaneechoutte, Mario

2010-09-29

Streptococcus agalactiae (group B streptococcus; GBS) is a significant cause of perinatal and neonatal infections worldwide. To detect GBS colonization in pregnant women, the CDC recommends isolation of the bacterium from vaginal and anorectal swab samples by growth in a selective enrichment medium, such as Lim broth (Todd-Hewitt broth supplemented with selective antibiotics), followed by subculture on sheep blood agar. However, this procedure may require 48 h to complete. We compared different sampling and culture techniques for the detection of GBS. A total of 300 swabs was taken from 100 pregnant women at 35-37 weeks of gestation. For each subject, one rectovaginal, one vaginal and one rectal ESwab were collected. Plating onto Columbia CNA agar (CNA), group B streptococcus differential agar (GBSDA) (Granada Medium) and chromID Strepto B agar (CA), with and without Lim broth enrichment, were compared. The isolates were confirmed as S. agalactiae using the CAMP test on blood agar and by molecular identification with tDNA-PCR or by 16S rRNA gene sequence determination. The overall GBS colonization rate was 22%. GBS positivity for rectovaginal sampling (100%) was significantly higher than detection on the basis of vaginal sampling (50%), but not significantly higher than for rectal sampling (82%). Direct plating of the rectovaginal swab on CNA, GBSDA and CA resulted in detection of 59, 91 and 95% of the carriers, respectively, whereas subculturing of Lim broth yielded 77, 95 and 100% positivity, respectively. Lim broth enrichment enabled the detection of only one additional GBS positive subject. There was no significant difference between GBSDA and CA, whereas both were more sensitive than CNA. Direct culture onto GBSDA or CA (91 and 95%) detected more carriers than Lim broth enrichment and subculture onto CNA (77%). One false negative isolate was observed on GBSDA, and three false positives on CA. In conclusion, rectovaginal sampling increased the number GBS positive women detected, compared to vaginal and/or rectal sampling. Direct plating on CA and/or GBSDA provided rapid detection of GBS that was at least as sensitive and specific as the CDC recommended method of Lim broth subcultured onto non chromogenic agar.

Phytochemical screening and antibacterial activity of Cyclamen persicum Mill tuber extracts.

PubMed

Alkowni, Raed; Jodeh, Shehdeh; Hussein, Fatima; Jaradat, Nidal

2018-01-01

The emerging drug resistance bacteria increased the demand on the discovery of antibiotics from natural sources. This research was aimed to study the antibacterial reactivity; as well as the phytochemicals, of the wild type of Cyclamen persicum, using nine different extraction methods where four solvents (Methanol, Ethanol, Hexane; and Water) were involved with varied extraction periods ranged from 2 up to 10 hours. The antibacterial activity of crude methanol extract (CME) was found as the best method of extraction, with particular emphasis on the method with prolonged extraction time of (10 hrs). The antibacterial activities of produced CME were determined by using agar diffusion method against two of gram-positive bacteria and two gram-negative ones. The CME treated Mueller-Hinton-Agar plates, were exhibited antibacterial effects against the gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) by showing of inhibition zone after overnight incubation, while nothing was noticed on those of gram negative ones (Pseudomonas aeruginosa and Escherichia coli). These results that proved the antibacterial activity of the Cyclamen persicum tubers were positively tested the Saponin glycosides from plant. In addition to that, methanol solvent could be the useful method for extractions of Cyclamen and can be used in any developing drugs against pathogenic gram positive bacteria.

A one-step matrix application method for MALDI mass spectrometry imaging of bacterial colony biofilms.

PubMed

Li, Bin; Comi, Troy J; Si, Tong; Dunham, Sage J B; Sweedler, Jonathan V

2016-11-01

Matrix-assisted laser desorption/ionization imaging of biofilms cultured on agar plates is challenging because of problems related to matrix deposition onto agar. We describe a one-step, spray-based application of a 2,5-dihydroxybenzoic acid solution for direct matrix-assisted laser desorption/ionization imaging of hydrated Bacillus subtilis biofilms on agar. Using both an optimized airbrush and a home-built automatic sprayer, region-specific distributions of signaling metabolites and cannibalistic factors were visualized from B. subtilis cells cultivated on biofilm-promoting medium. The approach provides a homogeneous, relatively dry coating on hydrated samples, improving spot to spot signal repeatability compared with sieved matrix application, and is easily adapted for imaging a range of agar-based biofilms. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Development of a high-throughput liquid state assay for lipase activity using natural substrates and rhodamine B.

PubMed

Zottig, Ximena; Meddeb-Mouelhi, Fatma; Beauregard, Marc

2016-03-01

A fluorescence-based assay for the determination of lipase activity using rhodamine B as an indicator, and natural substrates such as olive oil, is described. It is based on the use of a rhodamine B-natural substrate emulsion in liquid state, which is advantageous over agar plate assays. This high-throughput method is simple and rapid and can be automated, making it suitable for screening and metagenomics application. Reaction conditions such as pH and temperature can be varied and controlled. Using triolein or olive oil as a natural substrate allows monitoring of lipase activity in reaction conditions that are closer to those used in industrial settings. The described method is sensitive over a wide range of product concentrations and offers good reproducibility. Copyright © 2015 Elsevier Inc. All rights reserved.

Spatiotemporal Patterns Produced by Bacteria

NASA Astrophysics Data System (ADS)

Shimada, Yuji; Nakahara, Akio; Matsushita, Mitsugu; Matsuyama, Tohey

1995-06-01

Spatiotemporal patterns formed by a bacterial colony of Proteus mirabilis on an agar plate were observed. About half or one hour after the colony spread over the entire surface of the agar medium in a petridish, various patterns including target and spiral patterns appeared. They are very similar to those seen in other dissipative systems, such as chemical oscillations and electrohydrodynamic convective systems. Microscopic observations revealed that the collective motion of bacterial cells is responsible for the formation of these spatiotemporal patterns.

Differential plating medium for quantitative detection of histamine-producing bacteria.

PubMed Central

Niven, C F; Jeffrey, M B; Corlett, D A

1981-01-01

A histidine-containing agar medium has been devised for quantitative detection of histamine-producing bacteria that are alleged to be associated with scombroid fish poisoning outbreaks. The responsible bacteria produce a marked pH change in the agar, with attendant color change of pH indicator adjacent to the colonies, thus facilitating their recognition. Proteus morganii and Klebsiella pneumoniae were the two most common histidine-decarboxylating species isolated from scombroid fish and mahi mahi. PMID:7013698

Isolation of nontuberculous mycobacteria from soil using Middlebrook 7H10 agar with increased malachite green concentration.

PubMed

Hu, Yuli; Yu, Xinglong; Zhao, Dun; Li, Runcheng; Liu, Yang; Ge, Meng; Hu, Huican

2017-12-01

Environmental exposure is considered to be responsible for nontuberculous mycobacterial infections in humans. To facilitate the isolation of mycobacteria from soil, Middlebrook 7H10 agar was optimized as an enhanced selective medium by increasing the concentration of malachite green. A series of modified Middlebrook 7H10 agar media with malachite green concentrations ranging from 2.5 to 2500 mg/L was evaluated using 20 soil samples decontaminated with 3% sodium dodecyl sulfate plus 2% NaOH for 30 min. Among these modified Middlebrook 7H10 media, the medium with malachite green at a concentration of 250 mg/L, i.e., at the same concentration as in Löwenstein-Jensen medium, was the most effective in terms of the number of plates with mycobacterial growth. This medium was further evaluated with 116 soil samples. The results showed that 87.1% (101/116) of the samples produced mycobacterial growth, and 15 samples (12.9%) produced no mycobacterial growth. Of the plates inoculated with the soil samples, each in duplicate, 5.2% (12/232) showed late contamination. In total, 19 mycobacterial species were isolated, including seven (36.8%) rapidly growing mycobacteria and 12 (63.2%) slowly growing mycobacteria. Our results demonstrate that the modified Middlebrook 7H10 agar with 250 mg/L malachite green is useful for the primary isolation of nontuberculous mycobacteria from soil.

Influence of media composition on the production of alkaline α-amylase from Bacillus subtilis CB-18.

PubMed

Ogbonnaya, Nwokoro; Odiase, Anthonia

2012-01-01

Starch, a homopolysaccharide is an important and an abundant food reserve and energy source. Starches are processed to yield different products which find many industrial applications. Alpha-amylases hydrolyze starch by cleaving α-1,4-glucosidic bonds and have been used in food, textile and pharmaceutical industries [Sun et al. 2010]. Enzymatic conversion of starch with amylase presents an economically superior alternative to the conventional method of starch gelatinization. Alkaline α-amylase has an important position in the global enzyme market as a constituent of detergent. In this paper, we screened soil bacteria and an isolate, alkalophilic Bacillus subtilis CB-18 was found to produce an alkaline α-amylase in different media. MATERIAL AND METHODS. Screening of the isolates for amylolytic activity was carried out by growing bacteria isolated from the soil in starch agar plates and subsequently staining the plates with iodine solution to reveal zones of hydrolysis of starch. The selected isolate, Bacillus subtlis CB-18 was grown in different media at alkaline pH to evaluate the influence of media composition on alkaline α-amylase production. Enzyme assay was carried out by growing the culture in a broth medium and obtaining cell - free culture supernatant after centrifugation at 2515 × g for 15 minutes Amylase activity was determined by incubating 0.5 ml of crude enzyme solution in 0.1M Tris/HCl buffer (pH 8.5) with 0.5 ml of 1% soluble starch solution. The reaction was terminated by the addition of DNS reagent and reducing sugar produced from the amylolytic reaction was determined. Bacillus subtilis CB-18 used for this work was selected because it produced 7 mm zone diameter on starch agar plate. This organism was cultured in different alkaline broth media containing 2% soluble starch as inducer carbohydrate for α-amylase production. Among the carbon sources used for enzyme production, sorbitol was the best to stimulate enzyme production with α-amylase activity of 758 U/mL after 48 h. Peptone was the best nitrogen source for enzyme production with α-amylase activity of 680 U/mL after 48 h. Metal ions including Ca (2+), Mn(2+) and Mg(2+) stimulated enzyme production while Hg(2+) and Ag(+) repressed enzyme production. The best enzyme yields were observed in basal media containing agro-based substrates. This work reports the production of alkaline α-amylase by Bacillus subtlis CB-18 in different media. Enzyme production was highest when agro-based media were used to formulate the media.

Development of an Immunoassay for Rapid Detection of Ganglioside GM1 Mimicry in Campylobacter jejuni Strains

PubMed Central

Prendergast, Martina M.; Kosunen, Timo U.; Moran, Anthony P.

2001-01-01

Mimicry of peripheral nerve gangliosides by Campylobacter jejuni lipopolysaccharides (LPSs) has been proposed to induce cross-reacting antiganglioside antibodies in Guillain-Barré syndrome (GBS). Because current methods for LPS characterization are labor-intensive and inhibit the screening of large numbers of strains, a rapid GM1 epitope screening assay was developed. Biomass from two agar plates of confluent growth yielded sufficient LPS using a novel phenol-water and ether extraction procedure. Extracts of LPS were reacted with cholera toxin (GM1 ligand), peanut agglutinin (Galβ1→3GalNAc ligand), and anti-GM1 antibodies. After the assay was validated, 12 of 59 (20%) C. jejuni serostrains, including four serotypes that have not previously been associated with GBS, reacted with two or more anti-GM1 ganglioside reagents. Subsequently, LPS extracts from 5 of 7 (71%) C. jejuni isolates and 2 of 3 (67%) C. jejuni culture collection strains bore GM1 structures. Overall, the assay system was reliable, efficient, and reproducible and may be adapted for large-scale epidemiological studies. PMID:11283076

Antibacterial Activities of Jatropha curcas (LINN) on Coliforms Isolated from Surface Waters in Akure, Nigeria

PubMed Central

Dada, E. O.; Ekundayo, F. O.; Makanjuola, O. O.

2014-01-01

This study investigated the antibacterial activities of hot water, ethanol and acetone extracts of Jatropha curcas (LINN) leaves on coliforms isolated from surface waters using growth inhibition indices based on agar plate technique. The percentage recovery of the extracts was 19.17%, 18.10% and 18.80% for hot water, ethanol and acetone respectively. Phytochemical screening of the extracts was also determined. Qualitative phytochemical screening showed that the plant extracts contained steroids, tannins, flavonoids and cardiac glycosides, while alkaloids, phlobatannin, terpenoids and anthraquinones were absent. Only ethanolic extract did not possess saponins. Aqueous extracts of J. curcas compared most favourably with the standard antibiotics (gentamycin) on all the coliform bacteria except on K. pneumoniae and E. coli likely due to a measurably higher antibacterial activity compared to the organic extracts. The minimum inhibitory concentration of the aqueous extract ranged from 3.00 to 7.00 mg/L while minimum bactericidal concentration ranged from 4.00 to 10.00 mg/L. Aqueous extract of J. curcas could be used as antibacterial agents against diseases caused by coliforms. PMID:24711746

Passage of Campylobacter jejuni and Campylobacter coli subtypes through 0.45 and 0.65 µm pore size nitro-cellulose filters

USDA-ARS?s Scientific Manuscript database

Campylobacter can be difficult to recover from complex samples due to overgrowth by background bacteria. A 0.45 or 0.65 µm pore size filter overlaid on agar plates can be used as a means to separate Campylobacter from confounding non-Campylobacter cells, facilitating detection on solid plating medi...

Single-Center Evaluation of an Agar-Based Screening for Azole Resistance in Aspergillus fumigatus by Using VIPcheck

PubMed Central

van der Lee, H. A. L.; Rijs, A. J. M. M.; Zoll, J.; Hovestadt, J. A. M. F.; Melchers, W. J. G.; Verweij, P. E.

2017-01-01

ABSTRACT Antifungal susceptibility testing is an essential tool for guiding therapy, although EUCAST and CLSI reference methods are often available only in specialized centers. We studied the performance of an agar-based screening method for the detection of azole resistance in Aspergillus fumigatus cultures. The VIPcheck consists of four wells containing voriconazole, itraconazole, posaconazole, or a growth control. Ninety-six A. fumigatus isolates were used. Thirty-three isolates harbored a known resistance mechanism: TR34/L98H (11 isolates), TR46/Y121F/T289A (6 isolates), TR53 (2 isolates), and 14 isolates with other cyp51A gene point mutations. Eighteen resistant isolates had no cyp51A-mediated azole resistance. Forty-five isolates had a wild-type (WT) azole phenotype. Four technicians and two inexperienced interns, blinded to the genotype/phenotype, read the plates visually after 24 h and 48 h and documented minimal growth, uninhibited growth, and no growth. The performance was compared to the EUCAST method. After 24 h of incubation, the mean sensitivity and specificity were 0.54 and 1.00, respectively, with uninhibited growth as the threshold. After 48 h of incubation, the performance mean sensitivity and specificity were 0.98 and 0.93, respectively, with minimal growth. The performance was not affected by observer experience in mycology. The interclass correlation coefficient was 0.87 after 24 h and 0.85 after 48 h. VIPcheck enabled the selection of azole-resistant A. fumigatus colonies, with a mean sensitivity and specificity of 0.98 and 0.93, respectively. Uninhibited growth on any azole-containing well after 24 h and minimal growth after 48 h were indicative of resistance. These results indicate that the VIPcheck is an easy-to-use tool for azole resistance screening and the selection of colonies that require MIC testing. PMID:28923874

The degree of bacterial contamination while performing spine surgery.

PubMed

Ahn, Dong Ki; Park, Hoon Seok; Kim, Tae Woo; Yang, Jong Hwa; Boo, Kyung Hwan; Kim, In Ja; Lee, Hye Jin

2013-03-01

Prospective experimental study. To evaluate bacterial contamination during surgery. The participants of surgery and ventilation system have been known as the most significant sources of contamination. Two pairs of air culture blood agar plate for G(+) bacteria and MacConkey agar plate for G(-) bacteria were placed at 3 different locations in a conventional operation room: in the surgical field, under the airflow of local air conditioner, and pathway to door while performing spine surgeries. One pair of culture plates was retrieved after one hour and the other pair was retrieved after 3 hours. The cultured bacteria were identified and number of colonies was counted. There was no G(-) bacteria identified. G(+) bacteria grew on all 90 air culture blood agar plates. The colony count of one hour group was 14.5±5.4 in the surgical field, 11.3±6.6 under the local air conditioner, and 13.1±8.7 at the pathway to the door. There was no difference among the 3 locations. The colony count of 3 hours group was 46.4±19.5, 30.3±12.9, and 39.7±15.2, respectively. It was more at the surgical field than under the air conditioner (p=0.03). The number of colonies of one hour group was 13.0±7.0 and 3 hours group was 38.8±17.1. There was positive correlation between the time and the number of colonies (r=0.76, p=0.000). Conventional operation room was contaminated by G(+) bacteria. The degree of contamination was most high at the surgical field. The number of bacteria increased right proportionally to the time.

The Degree of Bacterial Contamination While Performing Spine Surgery

PubMed Central

Ahn, Dong Ki; Park, Hoon Seok; Yang, Jong Hwa; Boo, Kyung Hwan; Kim, In Ja; Lee, Hye Jin

2013-01-01

Study Design Prospective experimental study. Purpose To evaluate bacterial contamination during surgery. Overview of Literature The participants of surgery and ventilation system have been known as the most significant sources of contamination. Methods Two pairs of air culture blood agar plate for G(+) bacteria and MacConkey agar plate for G(-) bacteria were placed at 3 different locations in a conventional operation room: in the surgical field, under the airflow of local air conditioner, and pathway to door while performing spine surgeries. One pair of culture plates was retrieved after one hour and the other pair was retrieved after 3 hours. The cultured bacteria were identified and number of colonies was counted. Results There was no G(-) bacteria identified. G(+) bacteria grew on all 90 air culture blood agar plates. The colony count of one hour group was 14.5±5.4 in the surgical field, 11.3±6.6 under the local air conditioner, and 13.1±8.7 at the pathway to the door. There was no difference among the 3 locations. The colony count of 3 hours group was 46.4±19.5, 30.3±12.9, and 39.7±15.2, respectively. It was more at the surgical field than under the air conditioner (p=0.03). The number of colonies of one hour group was 13.0±7.0 and 3 hours group was 38.8±17.1. There was positive correlation between the time and the number of colonies (r=0.76, p=0.000). Conclusions Conventional operation room was contaminated by G(+) bacteria. The degree of contamination was most high at the surgical field. The number of bacteria increased right proportionally to the time. PMID:23508998

High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP(+) to NAD(.).

PubMed

Huang, Rui; Chen, Hui; Zhong, Chao; Kim, Jae Eung; Zhang, Yi-Heng Percival

2016-09-02

Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP(+) to NAD(+). Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfate (PMS), NAD(+), and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP(+) to NAD(+). This screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT.

Kills Germs by the Millions!

ERIC Educational Resources Information Center

Swails, Molly

1980-01-01

Described is a science experiment involving the isolation and study of microorganisms. Bacteria from the mouth are cultured on blood agar culture plates and are then exposed to four different mouthwashes to test their effectiveness. (DS)

Probiotic culture survival and implications in fermented frozen yogurt characteristics.

PubMed

Davidson, R H; Duncan, S E; Hackney, C R; Eigel, W N; Boling, J W

2000-04-01

Low-fat ice cream mix was fermented with probiotic-supplemented and traditional starter culture systems and evaluated for culture survival, composition, and sensory characteristics of frozen product. Fermentations were stopped when the titratable acidity reached 0.15% greater than the initial titratable acidity (end point 1) or when the pH reached 5.6 (end point 2). Mix was frozen and stored for 11 wk at -20 degrees C. The traditional yogurt culture system contained the strains Streptococcus salivarius ssp. thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. The probiotic-supplemented system contained the traditional cultures as well as Bifidobacterium longum and Lactobacillus acidophilus. We compared recovery of Bifodobacterium by three methods, a repair-detection system with roll-tubes and plates on modified bifid glucose medium and plates with maltose + galactose reinforced clostridial medium. Culture bacteria in both systems did not decrease in the yogurt during frozen storage. The roll-tube method with modified bifid glucose agar and repair detection system provided at least one-half log10 cfu/ml higher recovery of B. longum compared with recoveries using modified bifid glucose agar or maltose + galactose reinforced clostridial agar on petri plates. No change in concentrations of lactose or protein for products fermented with either culture system occurred during storage. Acid flavor was more intense when product was fermented to pH 5.6, but yogurt flavor was not intensified. The presence of probiotic bacteria in the supplemented system seemed to cause no differences in protein and lactose concentration and sensory characteristics.

Efficacy of High-volume Evacuator in Aerosol Reduction: Truth or Myth? A Clinical and Microbiological Study.

PubMed

Desarda, Hitesh; Gurav, Abhijit; Dharmadhikari, Chandrakant; Shete, Abhijeet; Gaikwad, Subodh

2014-01-01

Background and aims. Basic periodontal treatment aims at eliminating supra- and sub-gingival plaque and establishing conditions which will allow effective self-performed plaque control. This aim is primarily achieved with sonic and ultrasonic scalers. However, generation of bacterial aerosols during these procedures is of great concern to patients, the dentist and the dental assistant. The aim of this study was to compare the reduction in aerosol with and without high-volume evacuator through a microbiological study. Materials and methods. For this clinical study a fumigated closed operatory was selected. Maxillary incisors and canines were selected as an area for scaling. Piezoelectric ultrasonic scaling was performed in the absence and in the presence of a high-volume evacuator at 12 and 20 inches from the patient's oral cavity. In both groups scaling was carried out for 10 minutes. Nutrient agar plates were exposed for a total of 20 minutes. After this procedure, nutrient agar plates were incubated in an incubator at 37°C for 24 hours. The next day the nutrient agar plates were examined for colony forming units by a single microbiologist. Results. The results showed no statistically significant differences in colony forming units (CFU) with and without the use of a high-volume evacuator either at 12 or 20 inches from the patient's oral cavity. Conclusion. It was concluded that high-volume evacuator, when used as a separate unit without any modification, is not effective in reducing aerosol counts and environmental contamination.

Porphyromonas gingivalis can invade periodontal ligament stem cells.

PubMed

Pan, Chunling; Liu, Junchao; Wang, Hongyan; Song, Jia; Tan, Lisi; Zhao, Haijiao

2017-02-17

Porphyromonas gingivalis is strongly associated with the development, progression, severity and recurrence of periodontitis. Periodontal ligament stem cells (PDLSCs) play an important role in the maintenance of periodontal tissue self-renewal and repair. The purpose of this study was to investigate the ability of P. gingivalis to infect PDLSCs using an in vitro monolayer model. We separated and cultured primary PDLSCs using the tissue block with limiting dilution method. The efficiency of P. gingivalis (ATCC 33277) infection of PDLSCs was measured using agar plate culture and quantitative polymerase chain reaction (q-PCR) methods. PDLSCs infected with P. gingivalis were also observed by transmission electron microscopy. We assessed stem cell properties including cell morphology, clone formation, growth activity, cell surface antigens and multiple differentiation capacity. The infection rates of P. gingivalis in PDLSC at MOIs of 50, 100, 200, and 500 were 5.83%, 8.12%, 7.77% and 7.53% according to the agar plate culture method. By q-PCR, the efficiencies of P. gingivalis infection of PDLSCs at MOIs of 50, 100, 200, and 500 were 6.74%, 10.56%, 10.36% and 9.78%, respectively. Overall, the infection efficiency based on q-PCR was higher than that according to agar plate culture. Using transmission electron microscopy, we verified that P. gingivalis (ATCC 33277) could infect and invade PDLSCs after 2 h of incubation, and endocytic vacuoles were not found surrounding the internalized bacteria. In conclusion, our data demonstrate that P. gingivalis can invade PDLSCs.

Occurrence of Strongyloides stercoralis in Yunnan Province, China, and Comparison of Diagnostic Methods

PubMed Central

Steinmann, Peter; Zhou, Xiao-Nong; Du, Zun-Wei; Jiang, Jin-Yong; Wang, Li-Bo; Wang, Xue-Zhong; Li, Lan-Hua; Marti, Hanspeter; Utzinger, Jürg

2007-01-01

Background Strongyloides stercoralis is a neglected soil-transmitted helminth species, and there is a lack of parasitologic and epidemiologic data pertaining to this parasite in China and elsewhere. We studied the local occurrence of S. stercoralis in a village in Yunnan province, China, and comparatively assessed the performance of different diagnostic methods. Methodology/Principal Findings Multiple stool samples from a random population sample were subjected to the Kato-Katz method, an ether-concentration technique, the Koga agar plate method, and the Baermann technique. Among 180 participants who submitted at least 2 stool samples, we found a S. stercoralis prevalence of 11.7%. Males had a significantly higher prevalence than females (18.3% versus 6.1%, p = 0.011), and infections were absent in individuals <15 years of age. Infections were only detected by the Baermann (highest sensitivity) and the Koga agar plate method, but neither with the Kato-Katz nor an ether-concentration technique. The examination of 3 stool samples rather than a single one resulted in the detection of 62% and 100% more infections when employing the Koga agar plate and the Baermann technique, respectively. The use of a mathematical model revealed a ‘true’ S. stercoralis prevalence in the current setting of up to 16.3%. Conclusions/Significance We conclude that S. stercoralis is endemic in the southern part of Yunnan province and that differential diagnosis and integrated control of intestinal helminth infections needs more pointed emphasis in rural China. PMID:17989788

The Effects of EDTA (Ethylenediaminetetraacetic Acid) and Sonication on the Disaggregation of Oral Bacteria,

DTIC Science & Technology

1984-01-01

Virginia) and 0.5 g/ml menadione . The plates were c,,ltured for 72 hours anaerobically (<-140 my) at 37C A . and the number of colony forming units (CFU) per...CONTROL Immol lommol lOOmmol L DISPERSED pVVORTEX ~Thuwuuuuiii~FOR 30 SECONDS4 1 4 4 * MENADIONE SUPPLEMENTED SHAEDLER BLOOD AGAR PLATES lw- 2.50 U2.45

Diversity and dynamics of antibiotic-resistant bacteria in cheese as determined by PCR denaturing gradient gel electrophoresis.

PubMed

Flórez, Ana Belén; Mayo, Baltasar

2015-12-02

This work reports the composition and succession of tetracycline- and erythromycin-resistant bacterial communities in a model cheese, monitored by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Bacterial 16S rRNA genes were examined using this technique to detect structural changes in the cheese microbiota over manufacturing and ripening. Total bacterial genomic DNA, used as a template, was extracted from cultivable bacteria grown without and with tetracycline or erythromycin (both at 25 μg ml(-1)) on a non-selective medium used for enumeration of total and viable cells (Plate Count agar with Milk; PCA-M), and from those grown on selective and/or differential agar media used for counting various bacterial groups; i.e., lactic acid bacteria (de Man, Rogosa and Sharpe agar; MRSA), micrococci and staphylococci (Baird-Parker agar; BPA), and enterobacteria (Violet Red Bile Glucose agar; VRBGA). Large numbers of tetracycline- and erythromycin-resistant bacteria were detected in cheese samples at all stages of ripening. Counts of antibiotic-resistant bacteria varied widely depending on the microbial group and the point of sampling. In general, resistant bacteria were 0.5-1.0 Log10 units fewer in number than the corresponding susceptible bacteria. The PCR-DGGE profiles obtained with DNA isolated from the plates for total bacteria and the different bacterial groups suggested Escherichia coli, Lactococcus lactis, Enterococcus faecalis and Staphylococcus spp. as the microbial types resistant to both antibiotics tested. This study shows the suitability of the PCR-DGGE technique for rapidly identifying and tracking antibiotic resistant populations in cheese and, by extension, in other foods. Copyright © 2015 Elsevier B.V. All rights reserved.

Degradation of polyisoprene rubber by newly isolated Bacillus sp. AF-666 from soil.

PubMed

Shah, A A; Hasan, F; Shah, Z; Mutiullah; Hameed, A

2012-01-01

Various microorganisms were screened for their ability to degrade polyisoprene rubber (natural rubber latex gloves). Strain AF-666, newly isolated from a soil sample, was selected as the best strain having the ability to grow on polyisoprene containing plates. The strain identified as Bacillus sp. AF-666, was found to degrade polyisoprene rubber, both on basal agar plates (latex overlay) as well as in liquid medium. Qualitative analysis of degradation was done through scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy SEM showed changes in surface morphology, like appearance of pits and cracks, and marked difference in transmittance spectra of test and control due to changes in the functional groups, was detected through FTIR. CO2 evolution as a result of rubber degradation, was calculated gravimetrically by Sturm Test. About 4.43 g/1 of CO2 was produced in case of test, whereas, 1.57 g/1 in case of control. The viable number of cells (CFU/ml) was also higher in test than in control. Present study may provide an opportunity for further studies on the applications of biotechnological processes as a tool for rubber waste management.

Examination of the order of incubation for the recovery of bacteria and fungi from pharmaceutical-grade cleanrooms.

PubMed

Sandle, Tim

2014-01-01

A study was undertaken to compare microbial recoveries from pharmaceutical-grade cleanrooms using two different incubation regimes and a general-purpose agar (Tryptone Soy Agar). One temperature regime (A) incubated plates first at 30 degrees C to 35 degrees C, followed by 20 degrees C to 25 degrees C; the second temperature regime (B) began the incubation with plates at 20 degrees C to 25 degrees C, followed by 30 degrees C to 35 degrees C. The experimental outcomes demonstrated that there was no significant difference with the total microbial count when measured using a t-test (0.05 significance level; 95% confidence interval). However, with the recovery of fungi, the second incubation regime (B), which began with the lower 20 degrees C to 25 degrees C temperature, produced higher incidents and numbers of fungi. While this finding might provide the basis for adopting one incubation regime over another, a review of the types of cleanrooms recovering fungi suggests that fungal incidents are low, and they are more often confined to specific areas. Thus, as an alternative, incubation regimes could be varied to suit different cleanroom environments or a selective mycological agar adopted for specific areas.

Isolation and characterization of agar-digesting Vibrio species from the rotten thallus of Gracilariopsis heteroclada Zhang et Xia.

PubMed

Martinez, Joval N; Padilla, Philip Ian P

2016-08-01

Gracilariopsis heteroclada Zhang et Xia (Gracilariaceae, Rhodophyta) is one of the most studied marine seaweeds due to its economic importance. This has been cultivated extensively on commercial scale in the Philippines and other Asian countries. However, sustainable production of G. heteroclada in the Philippines could not be maximized due to the occurrence of rotten thallus disease. Thus, isolation and characterization of agar-digesting bacteria from the rotten thalli of G. heteroclada was conducted. A total of seven representative bacterial isolates were randomly selected based on their ability to digest agar as evidenced by the formation of depressions around the bacterial colonies on nutrient agar plates supplemented with 1.5% NaCl and liquefaction of agar. Gram-staining and biochemical characterization revealed that isolates tested were gram-negative rods and taxonomically identified as Vibrio parahaemolyticus (86-99.5%) and Vibrio alginolyticus (94.2-97.7%), respectively. It is yet to be confirmed whether these agar-digesting vibrios are involved in the induction and development of rotten thallus disease in G. heteroclada in concomitance with other opportunistic bacterial pathogens coupled with adverse environmental conditions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Phytochemical Screening and Antimicrobial Activity of Some Medicinal Plants Against Multi-drug Resistant Bacteria from Clinical Isolates

PubMed Central

Dahiya, Praveen; Purkayastha, Sharmishtha

2012-01-01

The in vitro antibacterial activity of various solvents and water extracts of aloe vera, neem, bryophyllum, lemongrass, tulsi, oregano, rosemary and thyme was assessed on 10 multi-drug resistant clinical isolates from both Gram-positive and Gram-negative bacteria and two standard strains including Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922. The zone of inhibition as determined by agar well diffusion method varied with the plant extract, the solvent used for extraction, and the organism tested. Klebsiella pneumoniae 2, Escherichia coli 3 and Staphylococcus aureus 3 were resistant to the plant extracts tested. Moreover, water extracts did not restrain the growth of any tested bacteria. Ethanol and methanol extracts were found to be more potent being capable of exerting significant inhibitory activities against majority of the bacteria investigated. Staphylococcus aureus 1 was the most inhibited bacterial isolate with 24 extracts (60%) inhibiting its growth whereas Escherichia coli 2 exhibited strong resistance being inhibited by only 11 extracts (28%). The results obtained in the agar diffusion plates were in fair correlation with that obtained in the minimum inhibitory concentration tests. The minimum inhibitory concentration of tulsi, oregano, rosemary and aloe vera extracts was found in the range of 1.56-6.25 mg/ml for the multi-drug resistant Staphylococcus aureus isolates tested whereas higher values (6.25-25 mg/ml) were obtained against the multi-drug resistant isolates Klebsiella pneumoniae 1 and Escherichia coli 1 and 2. Qualitative phytochemical analysis demonstrated the presence of tannins and saponins in all plants tested. Thin layer chromatography and bioautography agar overlay assay of ethanol extracts of neem, tulsi and aloe vera indicated flavonoids and tannins as major active compounds against methicillin-resistant Staphylococcus aureus. PMID:23716873

ISOLATION AND GENOTYPING OF CLOSTRIDIUM PERFRINGENS FROM FREE-LIVING SOUTH AMERICAN COATI (NASUA NASUA).

PubMed

Silva, Rodrigo O S; Almeida, Lara R; Oliveira Junior, Carlos A; Lima, Paula C S; Soares, Danielle F M; Pereira, Pedro L L; Silva, Israel J; Lobato, Francisco C F

2016-03-01

The importance of Clostridium perfringens for most wild animal species remains unclear. This study aimed to isolate and genotype C. perfringens in stool samples from free-living South American coati (Nasua nasua) in Brazil. Forty-six free-living N. nasua were trapped and stool samples were collected. Two different protocols for C. perfringens isolation were tested: direct plating onto selective agar and pre-enrichment in broth followed by plating in selective agar. Clostridium perfringens type A was isolated from 15 (32.6%) animals by direct plating and 36 (78.3%) animals by broth PE, and the rate of isolation was significantly different between these two methods (P < 0.01). Twelve of the 36 (33.3%) isolated strains by the PE protocol were positive for the β-2 toxin-encoding gene (cpb2) whereas the enterotoxin-encoding gene (cpe) and necrotic enteritis like-B toxin gene (netb) were not found. These results suggest that C. perfringens is commonly part of the microbiota of free-living coatis. Additionally, the use of a PE protocol appears to be essential for studies on C. perfringens in this species.

Effect of selected solutes on growth and recovery of a radiation-resistant Moraxella

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bruns, M.A.; Maxcy, R.B.

1978-01-01

A highly radiation-resistant Moraxella sp. from beef was more resistant to gamma radiation in frozen beef than Clostridium botulinum 33A spores. Even though the Moraxella sp. was extremely radiation-resistant, its recovery after irradiation was markedly influenced by the plating medium. Fewer colony-forming units were recovered in Tryptic Soy Agar (TSA) than in Plate Count Agar (PCA), and differences in recovery became more pronounced with increasing radiation dose. Growth studies of the nonirradiated Moraxella sp. suggested the presence of dialyzable inhibitory factor(s) in Trypticase Soy Broth (TSB) and TSA. The low (0.5 percent) concentration of NaCl in TSA was shown tomore » be mainly responsible for the slow growth and reduced recovery after irradiation. Reduced recovery was also obtained by plating the Moraxella sp. in PCA plus 0.5 percent NaCl or PCA plus 6 percent glucose after irradiation. It was noted that 2 other highly radiation-resistant isolates identified as Moraxella sp. gave similar results. Sensitivity to low solute concentrations, therefore, appeared to be a general phenomenon for this group.« less

Spatial Control of Bacteria Using Screen Printing

PubMed Central

Moon, Soonhee; Fritz, Ian L.; Singer, Zakary S.

2016-01-01

Abstract Synthetic biology has led to advances in both our understanding and engineering of genetic circuits that affect spatial and temporal behaviors in living cells. A growing array of native and synthetic circuits such as oscillators, pattern generators, and cell–cell communication systems has been studied, which exhibit spatiotemporal properties. To better understand the design principles of these genetic circuits, there is a need for versatile and precise methods for patterning cell populations in various configurations. In this study, we develop a screen printing methodology to pattern bacteria on agar, glass, and paper surfaces. Initially, we tested three biocompatible resuspension media with appropriate rheological properties for screen printing. Using microscopy, we characterized the resolution and bleed of bacteria screen prints on agar and glass surfaces, obtaining resolutions as low as 188 μm. Next, we engineered bacterial strains producing visible chromoproteins analogous to the cyan, magenta, and yellow subtractive color system for the creation of multicolored bacteria images. Using this system, we printed distinct populations in overlapping or interlocking designs on both paper and agar substrates. These proof-of-principle experiments demonstrated how the screen printing method could be used to study microbial community interactions and pattern formation of biofilms at submillimeter length scales. Overall, our approach allows for rapid and precise prototyping of patterned bacteria species that will be useful in the understanding and engineering of spatiotemporal behaviors in microbial communities. PMID:29577061

A highly efficient nonchemical method for isolating live nematodes (Caenorhabditis elegans) from soil during toxicity assays.

PubMed

Kim, Shin Woong; Moon, Jongmin; An, Youn-Joo

2015-01-01

The success of soil toxicity tests using Caenorhabditis elegans may depend in large part on recovering the organisms from the soil. However, it can be difficult to learn the International Organization for Standardization/ASTM International recovery process that uses the colloidal silica flotation method. The present study determined that a soil-agar isolation method provides a highly efficient and less technically demanding alternative to the colloidal silica flotation method. Test soil containing C. elegans was arranged on an agar plate in a donut shape, a linear shape, or a C curve; and microbial food was placed outside the soil to encourage the nematodes to leave the soil. The effects of ventilation and the presence of food on nematode recovery were tested to determine the optimal conditions for recovery. A linear arrangement of soil on an agar plate that was sprinkled with microbial food produced nearly 83% and 90% recovery of live nematodes over a 3-h and a 24-h period, respectively, without subjecting the nematodes to chemical stress. The method was tested using copper (II) chloride dihydrate, and the resulting recovery rate was comparable to that obtained using colloidal silica flotation. The soil-agar isolation method portrayed in the present study enables live nematodes to be isolated with minimal additional physicochemical stress, making it a valuable option for use in subsequent sublethal tests where live nematodes are required. © 2014 SETAC.

Aseptic Laboratory Techniques: Plating Methods

PubMed Central

Sanders, Erin R.

2012-01-01

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) as well as Material Safety Data Sheets (MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection (ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to: ● Perform plating procedures without contaminating media. ● Isolate single bacterial colonies by the streak-plating method. ● Use pour-plating and spread-plating methods to determine the concentration of bacteria. ● Perform soft agar overlays when working with phage. ● Transfer bacterial cells from one plate to another using the replica-plating procedure. ● Given an experimental task, select the appropriate plating method. PMID:22617405

l-Asparaginase from Proteus vulgaris1

PubMed Central

Tosa, Tetsuya; Sano, Ryujiro; Yamamoto, Kozo; Nakamura, Masatoshi; Ando, Katsuko; Chibata, Ichiro

1971-01-01

To produce an immunologically and enzymologically new type of l-asparaginase, 108 strains of bacteria were screened for enzyme production. As a result, 13 bacteria belonging to the genera Alcaligenes, Bacterium, and Proteus were found to produce l-asparaginases in high levels. Among these l-asparaginases, partially purified l-asparaginases from B. cadaveris and P. vulgaris showed antitumor activity. A partially purified l-asparaginase preparation of P. vulgaris did not react with the antibody of Escherichia colil-asparaginase on the Ouchterlony agar plate. Culture conditions for the production of l-asparaginase by P. vulgaris were investigated in detail. The enzyme was produced in high yields when cells were grown aerobically in a medium containing sodium fumarate and corn steep liquor. The addition of glucose or ammonium ion to the medium, however, resulted in depressed production of l-asparaginase. Under the optimum conditions, 3,700 international units of l-asparaginase was obtained from 1 liter of culture medium. Images PMID:5000866

Decolorization of RBBR by plant cells and correlation with the transformation of PCBs.

PubMed

Chroma, Ludmila; Macek, Tomas; Demnerova, Katerina; Macková, Martina

2002-11-01

An extracellular H2O2-requiring Remazol Brilliant Blue R (RBBR) decolorizing enzyme activity was detected after cultivation of cells of various plant species both in liquid medium and when growing on agar plates containing RBBR. Level of the enzyme activity was compared with the ability to metabolize polychlorinated biphenyls (PCBs). The ability to decolorize RBBR was tested in the presence and absence of PCBs. The cultures with high PCB-transforming activity proved to exhibit RBBR oxidase much more resistant towards the influence of PCBs. In addition low activities of lignin peroxidase (LiP) and manganese dependent peroxidase (MnP) were detected in medium and in plant cells. No correlation of MnP and LiP activities with PCB degradation could be found. The RBBR decolorization could be used as a rough screening method for plant cultures able to metabolize PCBs.

Molecular identification of Aspergillus and Eurotium species isolated from rice and their toxin-producing ability.

PubMed

Yazdani, D; Zainal Abidin, M A; Tan, Y H; Kamaruzaman, S

2011-01-01

Thirty milled rice samples were collected from retailers in 4 provinces of Malaysia. These samples were evaluated for Aspergillus spp. infection by direct plating on malt extract salt agar (MESA). All Aspergillus holomorphs were isolated and identified using nucleotide sequences of ITS 1 and ITS 2 of rDNA. Five anamorphs (Aspergillus flavus, A. oryzae, A. tamarii, A. fumigatus and A. niger) and 5 teleomorphs (Eurotium rubrum, E. amstelodami, E. chevalieri, E. cristatum and E. tonophilum) were identified. The PCR-sequencing based technique for sequences of ITS 1 and ITS 2 is a fast technique for identification of Aspergillus and Eurotium species, although it doesn't work flawlessly for differentiation of Eurotium species. All Aspergillus and Eurotium isolates were screened for their ability to produce aflatoxin and ochratoxin A (OTA) by HPLC and TLC techniques. Only A. flavus isolate UPM 89 was able to produce aflatoxins B1 and B2.

Validation of FMTV Modular VHP/mVHP System and Fumigation Decontamination Process in a C-141B Starlifter Aircraft

DTIC Science & Technology

2007-08-01

each location was aseptically transferred to 5 mL Tryptic Soy Broth (TSB) and incubated at 55 ’C. Coupons were observed the following day. If...Samples were then serially diluted in buffered peptone water and pour plated (1 mL per plate) using Tryptic Soy Agar (TSA). Plates were gently swirled in...1260 area. aft surface inside of hydraulic oil box 19 39 Starboard - 750. 59 On V2 79 On platform, 99 Aft, overhead in on oxygen box overhead, forward

Performance of a New Chromogenic Medium, BBL CHROMagar MRSA II (BD), for Detection of Methicillin-Resistant Staphylococcus aureus in Screening Samples ▿

PubMed Central

Van Vaerenbergh, Kristien; Cartuyvels, Reinoud; Coppens, Guy; Frans, Johan; Van den Abeele, Anne-Marie; De Beenhouwer, Hans

2010-01-01

Two chromogenic media for the detection of MRSA were compared: BBL CHROMagar MRSA II (BD) and MRSA ID agar (bioMérieux). Following overnight nonselective enrichment, 1,919 screening samples were inoculated on both chromogenic agars. After 24 h, the sensitivities of both media were high and comparable. Both media showed an important decrease in specificity after 48 h of incubation (decreases of 8% for MRSA II and 10% for MRSA ID), but MRSA II was significantly more specific at both time points. PMID:20181915

Development of a colony lift immunoassay to facilitate rapid detection and quantification of Escherichia coli O157:H7 from agar plates and filter monitor membranes.

PubMed

Ingram, D T; Lamichhane, C M; Rollins, D M; Carr, L E; Mallinson, E T; Joseph, S W

1998-07-01

E. coli O157:H7 is a food-borne adulterant that can cause hemorrhagic ulcerative colitis and hemolytic uremic syndrome. Faced with an increasing risk of foods contaminated with E. coli O157:H7, food safety officials are seeking improved methods to detect and isolate E. coli O157:H7 in hazard analysis and critical control point systems in meat- and poultry-processing plants. A colony lift immunoassay was developed to facilitate the positive identification and quantification of E. coli O157:H7 by incorporating a simple colony lift enzyme-linked immunosorbent assay with filter monitors and traditional culture methods. Polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, Mass.) were prewet with methanol and were used to make replicates of every bacterial colony on agar plates or filter monitor membranes that were then reincubated for 15 to 18 h at 36 +/- 1 degree C, during which the colonies not only remained viable but were reestablished. The membranes were dried, blocked with blocking buffer (Kirkegaard and Perry Laboratories [KPL], Gaithersburg, Md.), and exposed for 7 min to an affinity-purified horseradish peroxidase-labeled goat anti-E. coli O157 antibody (KPL). The membranes were washed, exposed to a 3,3',5,5'-tetramethylbenzidine membrane substrate (TMB; KPL) or aminoethyl carbazole (AEC; Sigma Chemical Co., St. Louis, Mo.), rinsed in deionized water, and air dried. Colonies of E. coli O157:H7 were identified by either a blue (via TMB) or a red (via AEC) color reaction. The colored spots on the PVDF lift membrane were then matched to their respective parent colonies on the agar plates or filter monitor membranes. The colony lift immunoassay was tested with a wide range of genera in the family Enterobacteriaceae as well as different serotypes within the E. coli genus. The colony lift immunoassay provided a simple, rapid, and accurate method for confirming the presence of E. coli O157:H7 colonies isolated on filter monitors or spread plates by traditional culture methods. An advantage of using the colony lift immunoassay is the ability to test every colony serologically on an agar plate or filter monitor membrane simultaneously for the presence of the E. coli O157 antigen. This colony lift immunoassay has recently been successfully incorporated into a rapid-detection, isolation, and quantification system for E. coli O157:H7, developed in our laboratories for retail meat sampling.

Development of a Colony Lift Immunoassay To Facilitate Rapid Detection and Quantification of Escherichia coli O157:H7 from Agar Plates and Filter Monitor Membranes

PubMed Central

Ingram, David T.; Lamichhane, Chinta M.; Rollins, David M.; Carr, Lewis E.; Mallinson, Edward T.; Joseph, Sam W.

1998-01-01

E. coli O157:H7 is a food-borne adulterant that can cause hemorrhagic ulcerative colitis and hemolytic uremic syndrome. Faced with an increasing risk of foods contaminated with E. coli O157:H7, food safety officials are seeking improved methods to detect and isolate E. coli O157:H7 in hazard analysis and critical control point systems in meat- and poultry-processing plants. A colony lift immunoassay was developed to facilitate the positive identification and quantification of E. coli O157:H7 by incorporating a simple colony lift enzyme-linked immunosorbent assay with filter monitors and traditional culture methods. Polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, Mass.) were prewet with methanol and were used to make replicates of every bacterial colony on agar plates or filter monitor membranes that were then reincubated for 15 to 18 h at 36 ± 1°C, during which the colonies not only remained viable but were reestablished. The membranes were dried, blocked with blocking buffer (Kirkegaard and Perry Laboratories [KPL], Gaithersburg, Md.), and exposed for 7 min to an affinity-purified horseradish peroxidase-labeled goat anti-E. coli O157 antibody (KPL). The membranes were washed, exposed to a 3,3′,5,5′-tetramethylbenzidine membrane substrate (TMB; KPL) or aminoethyl carbazole (AEC; Sigma Chemical Co., St. Louis, Mo.), rinsed in deionized water, and air dried. Colonies of E. coli O157:H7 were identified by either a blue (via TMB) or a red (via AEC) color reaction. The colored spots on the PVDF lift membrane were then matched to their respective parent colonies on the agar plates or filter monitor membranes. The colony lift immunoassay was tested with a wide range of genera in the family Enterobacteriaceae as well as different serotypes within the E. coli genus. The colony lift immunoassay provided a simple, rapid, and accurate method for confirming the presence of E. coli O157:H7 colonies isolated on filter monitors or spread plates by traditional culture methods. An advantage of using the colony lift immunoassay is the ability to test every colony serologically on an agar plate or filter monitor membrane simultaneously for the presence of the E. coli O157 antigen. This colony lift immunoassay has recently been successfully incorporated into a rapid-detection, isolation, and quantification system for E. coli O157:H7, developed in our laboratories for retail meat sampling. PMID:9665968

Uropathogenic Escherichia coli Metabolite-Dependent Quiescence and Persistence May Explain Antibiotic Tolerance during Urinary Tract Infection

PubMed Central

Leatham-Jensen, Mary P.; Mokszycki, Matthew E.; Rowley, David C.; Deering, Robert; Camberg, Jodi L.; Sokurenko, Evgeni V.; Tchesnokova, Veronika L.; Frimodt-Møller, Jakob; Leth Nielsen, Karen; Sun, Gongqin

2016-01-01

ABSTRACT In the present study, it is shown that although Escherichia coli CFT073, a human uropathogenic (UPEC) strain, grows in liquid glucose M9 minimal medium, it fails to grow on glucose M9 minimal medium agar plates seeded with ≤106 CFU. The cells on glucose plates appear to be in a “quiescent” state that can be prevented by various combinations of lysine, methionine, and tyrosine. Moreover, the quiescent state is characteristic of ~80% of E. coli phylogenetic group B2 multilocus sequence type 73 strains, as well as 22.5% of randomly selected UPEC strains isolated from community-acquired urinary tract infections in Denmark. In addition, E. coli CFT073 quiescence is not limited to glucose but occurs on agar plates containing a number of other sugars and acetate as sole carbon sources. It is also shown that a number of E. coli CFT073 mini-Tn5 metabolic mutants (gnd, gdhA, pykF, sdhA, and zwf) are nonquiescent on glucose M9 minimal agar plates and that quiescence requires a complete oxidative tricarboxylic acid (TCA) cycle. In addition, evidence is presented that, although E. coli CFT073 quiescence and persistence in the presence of ampicillin are alike in that both require a complete oxidative TCA cycle and each can be prevented by amino acids, E. coli CFT073 quiescence occurs in the presence or absence of a functional rpoS gene, whereas maximal persistence requires a nonfunctional rpoS. Our results suggest that interventions targeting specific central metabolic pathways may mitigate UPEC infections by interfering with quiescence and persistence. IMPORTANCE Recurrent urinary tract infections (UTIs) affect 10 to 40% of women. In up to 77% of those cases, the recurrent infections are caused by the same uropathogenic E. coli (UPEC) strain that caused the initial infection. Upon infection of urothelial transitional cells in the bladder, UPEC appear to enter a nongrowing quiescent intracellular state that is thought to serve as a reservoir responsible for recurrent UTIs. Here, we report that many UPEC strains enter a quiescent state when ≤106 CFU are seeded on glucose M9 minimal medium agar plates and show that mutations in several genes involved in central carbon metabolism prevent quiescence, as well as persistence, possibly identifying metabolic pathways involved in UPEC quiescence and persistence in vivo. PMID:27303698

Evaluation of anti-plaque microbial activity of Azadirachta indica (neem oil) in vitro: A pilot study.

PubMed

Elavarasu, Sugumari; Abinaya, P; Elanchezhiyan, S; Thangakumaran; Vennila, K; Naziya, K B

2012-08-01

Probably microbial plaque is the main etiology for periodontal tissue inflammation. Various chemical agents have been evaluated over the years with respect to their antimicrobial effects in the oral cavity. However, all are associated with side effects that prohibit regular long-term use. Therefore, the effectiveness of Azadirachta indica (neem) against plaque formation is considered to be vital, with lesser side effects. The aim of the present study is to evaluate and prove the antimicrobial activity of neem using plaque samples. Culture was prepared using brain heart infusion broth reagent. Dental plaque samples were used for that. Kirby-Bauer antimicrobial susceptibility test procedure was carried away with the sample. Neem oil was kept in the agar plate with culture and the diameter of inhibition zones was calculated. Results showed inhibition zones on the agar plate around neem oil. Study shows definite antiplaque activity of neem oil.

Infection Assay of Cyst Nematodes on Arabidopsis Roots.

PubMed

Bohlmann, Holger; Wieczorek, Krzysztof

2015-09-20

Plant parasitic nematodes are devastating pests on many crops. Juveniles (J2) of cyst nematodes invade the roots to induce a syncytium. This feeding site is their only source of nutrients. Male nematodes leave the roots after the fourth molt to mate with females. The females stay attached to their syncytia throughout their life and produce hundreds of eggs, which are contained in their bodies. When the females die their bodies form the cysts, which protect the eggs. Cysts can survive for many years in the soil until favorable conditions induce hatching of the juveniles. The beet cyst nematode Heterodera schachtii ( H. schachtii )is a pathogen of sugar beet ( Beta vulgaris ) but can also complete its life cycle on Arabidopsis roots growing on agar plates under sterile conditions. We present here protocols for a stock culture of H. schachtii and an infection assay on agar plates.

Isolation of New Aureobasidium Strains That Produce High-Molecular-Weight Pullulan with Reduced Pigmentation

PubMed Central

Pollock, Thomas J.; Thorne, Linda; Armentrout, Richard W.

1992-01-01

New isolates of Aureobasidium pullulans were obtained from plant leaf surfaces gathered in San Diego County. The new fungal isolates were identified as A. pullulans on the basis of the appearance of polymorphic colonies formed on agar plates, the electrophoretic profiles of repeated genomic DNA sequences, and the production of pullulan in shake flask cultures. The isolates showed different degrees of pigmentation. One of the natural isolates was nonpigmented under mock production conditions in liquid culture, but was still able to synthesize a reduced amount of pigment on agar plates at late times. A mutagenic treatment with ethidium bromide produced derivatives of normally pigmented natural isolates that exhibited an increased tendency toward yeastlike growth and reduced pigmentation. Additionally, some of the new isolates and mutant derivatives accumulated pullulan of relatively high molecular weight in the culture broths. Images PMID:16348676

Comparison of respiratory activity and culturability during monochloramine disinfection of binary population biofilms.

PubMed Central

Stewart, P S; Griebe, T; Srinivasan, R; Chen, C I; Yu, F P; deBeer, D; McFeters, G A

1994-01-01

Biofilm bacteria challenged with monochloramine retained significant respiratory activity, even though they could not be cultured on agar plates. Microbial colony counts on agar media declined by approximately 99.9% after 1 h of disinfection, whereas the number of bacteria stained by a fluorescent redox dye experienced a 93% reduction. Integrated measures of biofilm respiratory activity, including net oxygen and glucose utilization rates, showed only a 10 to 15% reduction. In this biofilm system, measures of microbial respiratory activity and culturability yielded widely differing estimates of biocide efficacy. PMID:8017950

The Shiga and Shiga-Like Cytotoxins: Gene Regulation and Functional Analysis of the Binding Subunits

DTIC Science & Technology

1989-05-05

SLT-I and SLT-II operons, designated slt-I and slt-II respectively, have been cloned from toxin-converting coliphage (Newland et al. 1985; Willshaw...The plasmid bands were removed through the sides of the tubes with a 20-gauge needle, the EtBr was extracted with water -saturated butanol, and the...pBluescript vectors were spread on LB agar plates with 50 ~1 Bluo-gal (BRL; 2% in dimethyl formamide) and 25 ~1 IPTG (BRL; lOOmM in water ) on LB agar

Practical direct plaque assay for coliphages in 100-ml samples of drinking water.

PubMed Central

Grabow, W O; Coubrough, P

1986-01-01

A practical single-agar-layer plaque assay for the direct detection of coliphages in 100-ml samples of water was designed and evaluated. With this assay a 100-ml sample of water, an agar medium containing divalent cations, and the host Escherichia coli C (ATCC 13706) were mixed in a single container, and the mixture was plated on 10 14-cm-diameter petri dishes. It was more sensitive, reliable, and accurate than various other methods and proved rapid, simple, and economic. PMID:3532952

Molecular cloning, overexpression, and enzymatic characterization of glycosyl hydrolase family 16 β-Agarase from marine bacterium Saccharophagus sp. AG21 in Escherichia coli.

PubMed

Lee, Youngdeuk; Oh, Chulhong; De Zoysa, Mahanama; Kim, Hyowon; Wickramaarachchi, Wickramaarachchige Don Niroshana; Whang, Ilson; Kang, Do-Hyung; Lee, Jehee

2013-01-01

An agar-degrading bacterium was isolated from red seaweed (Gelidium amansii) on a natural seawater agar plate, and identified as Saccharophagus sp. AG21. The β-agarase gene from Saccharophagus sp. AG21 (agy1) was screened by long and accurate (LA)-PCR. The predicted sequence has a 1,908 bp open reading frame encoding 636 amino acids (aa), and includes a glycosyl hydrolase family 16 (GH16) β-agarase module and two carbohydrate binding modules of family 6 (CBM6). The deduced aa sequence showed 93.7% and 84.9% similarity to β-agarase of Saccharophagus degradans and Microbulbifer agarilyticus, respectively. The mature agy1 was cloned and overexpressed as a His-tagged recombinant β-agarase (rAgy1) in Escherichia coli, and had a predicted molecular mass of 69 kDa and an isoelectric point of 4.5. rAgy1 showed optimum activity at 55oC and pH 7.6, and had a specific activity of 85 U/mg. The rAgy1 activity was enhanced by FeSO4 (40%), KCl (34%), and NaCl (34%), compared with the control. The newly identified rAgy1 is a β-agarase, which acts to degrade agarose to neoagarotetraose (NA4) and neoagarohexaose (NA6) and may be useful for applications in the cosmetics, food, bioethanol, and reagent industries.

Candida krusei form mycelia along agar surfaces towards each other and other Candida species.

PubMed

Fleischmann, Jacob; Broeckling, Corey D; Lyons, Sarah

2017-03-11

Candida krusei has been known to exhibit communal interactions such as pellicle formation and crawling out of nutritional broth. We noticed another possible interaction on agar surfaces, where C. krusei yeast cells formed mycelia along agar surfaces toward each other. We report here the results of experiments to study this interaction. When C.krusei yeast cells are plated in parallel streaks, they form mycelia along agar surfaces toward other yeasts. They also detect the presence of Candida albicans and Candida glabrata across agar surfaces, while the latter two react neither to their own kind, nor to C. krusei. Secreted molecule(s) are likely involved as C.krusei does not react to heat killed C. krusei. Timing and rate of mycelia formation across distances suggests that mycelia start forming when a secreted molecule(s) on agar surface reaches a certain concentration. We detected farnesol, tyrosol and tryptophol molecules that may be involved with mycelial formation, on the agar surfaces between yeast streaks. Unexpectedly the amounts detected between streaks were significantly higher than would have expected from additive amounts of two streaks. All three Candida species secreted these molecules. When tested on agar surface however, none of these molecules individually or combined induced mycelia formation by C. krusei. Our data confirms another communal interaction by C. krusei, manifested by formation of mycelia by yeast cells toward their own kind and other yeasts on agar surfaces. We detected secretion of farnesol, tyrosol and tryptophol by C. krusei but none of these molecules induced this activity on agar surface making it unlikely that they are the ones utilized by this yeast for this activity.

Screening of antagonistic bacteria for biological control of nursery wilt of black pepper (Piper nigrum).

PubMed

Anith, K N; Radhakrishnan, N V; Manomohandas, T P

2003-01-01

Bacterial antagonists of Phytophthora capsici were isolated from underground shoot portions of rooted cuttings of black pepper. Initially isolates were screened by dual culture on potato dextrose agar and carrot agar. Further, a screening was done on black pepper shoots for supression of lesion caused by the pathogen. Most of the antagonists showed varying levels of antagonism in the dual culture and the shoot assay. Isolate PN-026, showing the highest suppression of lesion development in the shoot assay was found to be the most efficient antagonist in reducing Phytophthora capsici induced nursery wilt of black pepper. This screening involving the host, pathogen, and the antagonist, performed on black pepper shoot (the planting material for this vegetatively propagated crop), could be used as a rapid and reliable method for the isolation of efficient bacterial antagonists of P. capsici.

Aerosol, a health hazard during ultrasonic scaling: A clinico-microbiological study.

PubMed

Singh, Akanksha; Shiva Manjunath, R G; Singla, Deepak; Bhattacharya, Hirak S; Sarkar, Arijit; Chandra, Neeraj

2016-01-01

Ultrasonic scaling is a routinely used treatment to remove plaque and calculus from tooth surfaces. These scalers use water as a coolant which is splattered during the vibration of the tip. The splatter when mixed with saliva and plaque of the patients causes the aerosol highly infectious and acts as a major risk factor for transmission of the disease. In spite of necessary protection, sometimes, the operator might get infected because of the infectious nature of the splatter. To evaluate the aerosol contamination produced during ultrasonic scaling by the help of microbiological analysis. This clinico-microbiological study consisted of twenty patients. Two agar plates were used for each patient; the first was kept at the center of the operatory room 20 min before the treatment while the second agar plate was kept 40 cm away from the patient's chest during the treatment. Both the agar plates were sent for microbiological analysis. The statistical analysis was done with the help of STATA 11.0 (StataCorp. 2013. Stata Statistical Software, Release 13. College Station, TX: StataCorp LP, 4905 Lakeway Drive College Station, Texas, USA). Statistical software was used for data analysis and the P < 0.001 was considered to be statistically significant. The results for bacterial count were highly significant when compared before and during the treatment. The Gram staining showed the presence of Staphylococcus and Streptococcus species in high numbers. The aerosols and splatters produced during dental procedures have the potential to spread infection to dental personnel. Therefore, proper precautions should be taken to minimize the risk of infection to the operator.

Study of the lactic acid bacteria throughout the manufacture of dry-cured lacón (a Spanish traditional meat product). Effect of some additives.

PubMed

Lorenzo, José M; García Fontán, María C; Cachaldora, Aida; Franco, Inmaculada; Carballo, Javier

2010-04-01

Total aerobic mesophilic microflora (on SPC agar), lactic acid bacteria (on MRS agar) and lactobacilli (on Rogosa agar) were enumerated in samples from the surface and the interior of the pieces throughout the manufacture of six batches of lacón. Three of the batches were made without additives and three with additives (glucose (2 g/kg), sodium nitrite (E(250)) (125 mg/kg), sodium nitrate (E(251)) (175 mg/kg), sodium ascorbate (E(301)) (500 mg/kg), and sodium citrate (E(331)) (100 mg/kg)). The counts decreased throughout the manufacturing process, particularly after the salting stage. The use of additives did not affect the counts or the evolution of the microbial groups, except for the lactobacilli, which were present in higher numbers in the batches with additives. In four batches (two without and two with additives), from MRS agar and from Rogosa agar plates, 10 colonies were randomly taken from each sampling point of each batch (five from the surface sample and five from the interior sample) and from each culture medium; a total of 224 strains from MRS agar, and 176 strains from Rogosa agar that were identified by classical methods. The MRS agar displayed moderate selectivity for the isolation of lactic acid bacteria, and only 59% of the isolated strains belonged to this microbial group. Homofermentative and facultative heterofermentative lactobacilli (particularly Lactobacillus curvatus and Lactobacillus sakei) were the most abundant species isolated on this medium. The selectivity of the Rogosa agar for lactobacilli was extremely high. The species of lactobacilli isolated on this medium at different stages of manufacture of the four batches of lacón were consistent with those isolated from MRS agar. The use of additives in the lacón did not appreciably affect the kinds and proportions of species isolated on either MRS agar or Rogosa agar.

Occurrence of Extended Spectrum β-Lactamases, KPC-Type, and MCR-1.2-Producing Enterobacteriaceae from Wells, River Water, and Wastewater Treatment Plants in Oltrepò Pavese Area, Northern Italy

PubMed Central

Caltagirone, Mariasofia; Nucleo, Elisabetta; Spalla, Melissa; Zara, Francesca; Novazzi, Federica; Marchetti, Vittoria M.; Piazza, Aurora; Bitar, Ibrahim; De Cicco, Marica; Paolucci, Stefania; Pilla, Giorgio; Migliavacca, Roberta; Pagani, Laura

2017-01-01

To evaluate the water compartment antibiotic-resistance contamination rates, 11 wells, five streams, and four treatment plants located in the Oltrepò Pavese area were screened for the presence of third generation cephalosporins resistant Gram-negative bacteria. Enterobacteriaceae were also characterized for the Extended-Spectrum-β-Lactamases (ESBLs), carbapenemases, and mcr-1 genes presence. From December 2014 to November 2015, 246 water samples were filtered, plated on Plate Count Agar, MacConkey Agar, and MacConkey Agar with cefotaxime. Isolates were species identified using AutoSCAN-4-System and ESBLs, carbapenemases, and colistin resistance determinants were characterized by PCR, sequencing, and microarray. Plasmid conjugative transfer experiments, PCR-based Replicon typing, Pulsed-Field Gel Electrophoresis, Multi-Locus-Sequence-Typing, and in-silico plasmid characterization were performed. A total of 132 enterobacteria isolates grew on MacConkey agar with cefotaxime: 82 (62.1%) were obtained from streams, 41 (31.1%) from treatment plants, and 9 (6.8%) from wells. Thirty out of 132 (22.7%) isolates, mainly belonging to Escherichia coli (n = 15) species, showed a synergic effect with piperacillin-tazobactam. A single ESBL gene of blaCTX−M-type was identified in 19/30 isolates. In further two E. coli strains, a blaCTX−M−1 gene co-existed with a blaSHV-type ESBL determinant. A blaSHV−12 gene was detected in two isolates of E. coli (n = 1) and Klebsiella oxytoca (n = 1), while any ESBL determinant was ascertained in seven Yersinia enterocolitica strains. A blaDHA-type gene was detected in a cefoxitin resistant Y. enterocolitica from a stream. Interestingly, two Klebsiella pneumoniae strains of ST307 and ST258, collected from a well and a wastewater treatment plant, resulted KPC-2, and KPC-3 producers, respectively. Moreover, we report the first detection of mcr-1.2 ST10 E. coli on a conjugative IncX4 plasmid (33.303 bp in size) from a stream of Oltrepò Pavese (Northern Italy). Both ESBLs E. coli and ESBLs/carbapenemase-producing K. pneumoniae strains showed clonal heterogeneity by Pulsed-Field Gel Electrophoresis and Multi-Locus-Sequence-Typing. During one-year study and taking in account the whole Gram-negative bacterial population, an average percentage of cefotaxime resistance of 69, 32, and 10.3% has been obtained for the wastewater treatment plants, streams, and wells, respectively. These results, of concern for public health, highlight the need to improve hygienic measures to reduce the load of discharged bacteria with emerging resistance mechanisms. PMID:29176971

Isolation of Shiga toxin-producing Escherichia coli from fresh produce using STEC heart infusion washed blood agar with mitomycin-C.

PubMed

Lin, Andrew; Nguyen, Lam; Clotilde, Laurie M; Kase, Julie A; Son, Insook; Lauzon, Carol R

2012-11-01

The ability to detect and isolate Shiga toxin-producing Escherichia coli (STEC) remains a major challenge for food microbiologists. Although methods based on nucleic acids and antibodies have improved detection of STECs in foods, isolation of these bacteria remains arduous. STEC isolation is necessary for matching food, environmental, and clinical isolates during outbreak investigations and for distinguishing between pathogenic and nonpathogenic organisms. STEC heart infusion washed blood agar with mitomycin-C (SHIBAM) is a modification of washed sheep blood agar prepared by adding mitomycin-C and optimizing both the washed blood and base agar to better isolate STECs. Most STEC isolates produce a zone of hemolysis on SHIBAM plates and are easily distinguishable from background microbiota. Here, we present data supporting the use of SHIBAM to isolate STECs from fresh produce. SHIBAM was tested for accuracy in identifying STECs (365 of 410 STEC strains were hemolytic, and 63 of 73 E. coli strains that did not produce Shiga toxin were not hemolytic) and for recovery from artificially inoculated fresh produce (11 of 24 romaine lettuce samples and 6 of 24 tomato samples). STEC recovery with SHIBAM agar was greatly improved when compared with recovery on Levine's eosin-methylene blue agar as a reference method.

Polymer Film-Based Screening and Isolation of Polylactic Acid (PLA)-Degrading Microorganisms.

PubMed

Kim, Mi Yeon; Kim, Changman; Moon, Jungheun; Heo, Jinhee; Jung, Sokhee P; Kim, Jung Rae

2017-02-28

Polylactic acid (PLA) has been highlighted as an alternative renewable polymer for the replacement of petroleum-based plastic materials, and is considered to be biodegradable. On the other hand, the biodegradation of PLA by terminal degraders, such as microorganisms, requires a lengthy period in the natural environment, and its mechanism is not completely understood. PLA biodegradation studies have been conducted using mainly undefined mixed cultures, but only a few bacterial strains have been isolated and examined. For further characterization of PLA biodegradation, in this study, the PLA-degrading bacteria from digester sludge were isolated and identified using a polymer film-based screening method. The enrichment of sludge on PLA granules was conducted with the serial transference of a subculture into fresh media for 40 days, and the attached biofilm was inoculated on a PLA film on an agar plate. 3D optical microscopy showed that the isolates physically degraded the PLA film due to bacterial degradation. 16S rRNA gene sequencing identified the microbial colonies to be Pseudomonas sp. MYK1 and Bacillus sp. MYK2. The two isolates exhibited significantly higher specific gas production rates from PLA biodegradation compared with that of the initial sludge inoculum.

Biocidal and inhibitory activity screening of de novo synthesized surfactants against two eukaryotic and two prokaryotic microbial species.

PubMed

Tiecco, Matteo; Cardinali, Gianluigi; Roscini, Luca; Germani, Raimondo; Corte, Laura

2013-11-01

Thirty-six quaternary ammonium salts, of which 28 structurally different non-commercially available surfactants, were tested to screen their biocidal and inhibitory antimicrobial activity. Their activity was compared to commercially available amphiphiles as well as to non-amphiphilic quaternary ammonium salts. As target of these compounds four microbial species were employed of which two (Saccharomyces cerevisiae and Candida albicans) were important yeast in the food and clinical environment and the other two (Escherichia coli and Listeria innocua) represented the Gram negative and positive bacteria, respectively. The surfactants showed the ability to kill the microbial cells in water solution and to variably hamper their growth onto agar medium. The non-amphiphilic compounds (which represent analogues of some surfactants used in this study, since they have the same head group but no hydrophobic portion) had little effect in solution and no effect against the microbial growth on plate. Amphoteric and non-amphoteric zwitterionic surfactants showed reduced biocidal activity. The most active antimicrobial agent was N-tetradecyltropinium bromide (23S) surfactant. The presence of cells did not significantly affect the ability to form micelles, as demonstrated by comparative conductometric measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

On the cultivation of free-living marine and estuarine nematodes

NASA Astrophysics Data System (ADS)

Moens, T.; Vincx, M.

1998-06-01

Although a large body of literature exists on the systematics and ecology of free-living marine and brackish-water nematodes, key questions on the nature and magnitude of interactions between nematodes and other organisms in the benthos remain unanswered. Relatively few authors have investigated live nematodes in food web studies or in experiments dealing with the nematodes’ response to a varying environment. It is mainly for the latter purpose that attempts have been made to maintain, rear and cultivate selected species. This paper describes the methodology used for the maintenance, rearing, and eventual permanent agnotobiotic cultivation of a variety of estuarine nematodes. Spot plates, where small samples of sediment or macrophyte material are inoculated on a sloppy agar layer, have been used for the purpose of maintenance and initial cultivation. Those species that reproduce on spot plates are then selected for monospecific cultivation on agar layers with different nutrient enrichments and with micro-organisms cotransferred from the spot plates as food. Mixtures of bacto and nutrient agar prepared in artificial seawater were specifically suitable for the xenic cultivation of nine bacterivorous and, when supplied with Erdschreiber nutrients, two algivorous/bacterivorous nematode species. Up to three generations of five other nematode species have been reared under laboratory conditions, and several more were kept alive and active for variable periods of time on agar. Generation times observed on spot plates for Adoncholaimus fuscus and Oncholaimus oxyuris were substantially shorter than previously published estimates and suggest a correspondingly higher predatory and scavenging potency for these and related enoplids. A procedure for the long-term storage of nematodes at -80°C with glycerol as a cryoprotectant was successfully used for Diplolaimella dievengatensis, Panagrolaimus sp. 1, and Pellioditis marina, but not for Diplolaimelloides meyli. The authors have also summarized the existing literature on the cultivation of marine and brackish-water nematodes. Continuous cultivation appears to have been successful mainly for Aufwuchs and epiphytic nematodes; only few sediment-dwellers have been established in permanent culture. Of only just over 30 species that have ever been cultivated, more than half belong to one family (Monhysteridae) and three are Rhabditida, an order poorly represented in the marine environment. Four species have been grown in monoxenic and one in axenic culture, the latter though with limited success. It is concluded that our understanding of the basic nutritional requirements of marine nematodes is as yet insufficient, and that the culture techniques which have so far mainly deployed agar or liquid substrates, while being suitable for the cultivation of Aufwuchs and epiphytic nematodes, do not accurately enough mimic gradients specific of the natural habitat of many sediment-dwellers.

Evaluation of microbiological accumulation capability of the commercial sponge Spongia officinalis var. adriatica (Schmidt) (Porifera, Demospongiae).

PubMed

Stabili, Loredana; Licciano, Margherita; Longo, Caterina; Corriero, Giuseppe; Mercurio, Maria

2008-05-01

This study was carried out to evaluate the microbiological accumulation capability of the demosponge Spongia officinalis var. adriatica. Six microbiological parameters were researched in two sampling periods in the water and in reared sponge samples coming from sites with different degrees of microbial contamination: an off-shore fish farm displaced off the Apulian coast (Southern Adriatic Sea) and a no-impacted area displaced into the Marine Protected Area of Porto Cesareo (Apulian coast-Ionian Sea). We detected the density of culturable heterotrophic bacteria by spread plate on marine agar, total culturable bacteria at 37 degrees C on Plate Count Agar and vibrios on thiosulphate-citrate-bile-sucrose-salt (TCBS) agar. Total and fecal coliforms as well as fecal streptococci concentrations were detected by the MPN method. Bacterial densities were always higher in the sponge homogenates compared with the corresponding seawater in the sampling points and in both sampling periods. As regard vibrios, total culturable bacteria at 37 degrees C and fecal streptococci concentrations, the highest values were observed in the sponge samples coming from the off-shore fish farm during the summer period. The ability of Spongia officinalis var. adriatica to accumulate the microbial pollution indicators suggests that this species can be employed as a bioindicator for monitoring water quality.

Biodegradative potential of fungal isolates from sacral ambient: In vitro study as risk assessment implication for the conservation of wall paintings

PubMed Central

Dimkić, Ivica; Stupar, Miloš; Stanković, Slaviša; Vukojević, Jelena; Ljaljević Grbić, Milica

2018-01-01

The principal purpose of the study was to evaluate in vitro the potential ability of fungal isolates obtained from the painted layer of frescoes and surrounding air to induce symptoms of fresco deterioration, associated with their growth and metabolism, so that the risk of such deterioration can be precisely assessed and appropriate conservation treatments formulated. Biodegradative properties of the tested microfungi were qualitatively characterized through the use of a set of special agar plates: CaCO3 glucose agar (calcite dissolution), casein nutrient agar (casein hydrolysis), Czapek-Dox minimal medium (pigment secretion); and Czapek-Dox minimal broth (acid and alkali production). Most of the tested isolates (71.05%) demonstrated at least one of the degradative properties, with Penicillium bilaiae as the most potent, since it tested positive in all four. The remaining isolates (28.95%) showed no deterioration capabilities and were hence considered unlikely to partake in the complex process of fungal deterioration of murals via the tested mechanisms. The obtained results clearly indicate that utilization of fast and simple plate assays can provide insight into the biodegradative potential of deteriogenic fungi and allow for their separation from allochthonous transients, a prerequisite for precise assessment of the amount of risk posed by a thriving mycobiota to mural paintings. PMID:29309432

High-throughput screening of coenzyme preference change of thermophilic 6-phosphogluconate dehydrogenase from NADP + to NAD +

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Rui; Chen, Hui; Zhong, Chao

Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP + to NAD +. Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfatemore » (PMS), NAD +, and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP + to NAD +. Furthermore, this screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT.« less

High-throughput screening of coenzyme preference change of thermophilic 6-phosphogluconate dehydrogenase from NADP + to NAD +

DOE PAGES

Huang, Rui; Chen, Hui; Zhong, Chao; ...

2016-09-02

Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP + to NAD +. Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfatemore » (PMS), NAD +, and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP + to NAD +. Furthermore, this screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT.« less

Isolation and Characterization of Isogenic Pairs of Domed Hemolytic and Flat Nonhemolytic Colony Types of Bordetella pertussis

PubMed Central

Peppler, Mark S.

1982-01-01

Four different serotype strains of Bordetella pertussis, 3779BL2S4, Tohama I, 353/Z, and 2753, were plated on Bordet-Gengou agar, where they grew as domed, hemolytic (D+H+) wild-type colonies. Cloned D+H+ colony types of all four strains were passed onto modified Stainer-Scholte medium solidified with 1% Noble Agar. Colonies were selected from Stainer-Scholte agar, and these subsequently grew as flat, nonhemolytic (D−H−) colonies when transferred back onto Bordet-Gengou agar. The frequency of D−H− organisms within a population of cloned D+H+ was determined to be between 5 × 10−5 and 5 × 10−6. The D−H− colony types maintained their flat, nonhemolytic characteristics for over 80 single-colony passages on Bordet-Gengou agar. The isogenic pairs of D+H+ and D−H− colony types from the four strains were compared for hemagglutination titer, lymphocytosis-promoting activity, adenylate cyclase activity, and presence of agglutinogens by agglutination. In all cases the D−H− colony types showed reduced activities or amounts of antigen compared with their D+H+ parents. Freely diffusible antigens were markedly different between the two phenotypes as noted by double diffusion of antisera added to plates on which colonies of the variants were growing. Antigens solubilized from the two colony types by Triton X-100 were also markedly different as judged by radial immunodiffusion with antifimbrial hemagglutinin, antilymphocytosis-promoting factor, and anti-353/Z adsorbed with autoclaved 353/Z. In addition, autoradiographs of 125I-surface-labeled whole cells separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed unique banding patterns for each colony type. Since all organisms, regardless of colony type, were grown on Bordet-Gengou agar, the differences reported could not be due to medium composition. Differences between phenotypes were also independent of passage number on Bordet-Gengou agar. By analogy to previous studies, the D−H− organisms appear to fulfill the criteria for phase III or phase IV in the system of Leslie and Gardner (P. H. Leslie and A. D. Gardner, J. Hyg. 31:423-434, 1931) or phase III in the system of Kasuga et al. (T. Kasuga, Y. Nakase, K. Ukishima, and K. Takatsu, Kitasato Arch. Exp. Med. 26:121-134, 1954). Images PMID:6279517

40 CFR 141.21 - Coliform sampling.

Code of Federal Regulations, 2014 CFR

2014-07-01

... water) with the intent that if the method they choose has an unacceptable false-positive or negative...), supplemented with 100 µg/mL of MUG. If the 18th edition is used, incubate the agar plate at 35 °C for 4 hours...

40 CFR 141.21 - Coliform sampling.

Code of Federal Regulations, 2012 CFR

2012-07-01

... water) with the intent that if the method they choose has an unacceptable false-positive or negative...), supplemented with 100 µg/mL of MUG. If the 18th edition is used, incubate the agar plate at 35 °C for 4 hours...

40 CFR 141.21 - Coliform sampling.

Code of Federal Regulations, 2013 CFR

2013-07-01

... water) with the intent that if the method they choose has an unacceptable false-positive or negative...), supplemented with 100 µg/mL of MUG. If the 18th edition is used, incubate the agar plate at 35 °C for 4 hours...

Reflected scatterometry for noninvasive interrogation of bacterial colonies

USDA-ARS?s Scientific Manuscript database

A phenotyping of bacterial colonies on agar plates using forward-scattering diffraction-pattern analysis provided promising classification of several different bacteria such as Salmonella, Vibrio, Listeria, and E. coli. Since the technique is based on forward-scattering phenomena, light transmittanc...

Adaptation response of Pseudomonas fragi on refrigerated solid matrix to a moderate electric field.

PubMed

Chen, Wenbo; Hu, Honghai; Zhang, Chunjiang; Huang, Feng; Zhang, Dequan; Zhang, Hong

2017-02-10

Moderate electric field (MEF) technology is a promising food preservation strategy since it relies on physical properties-rather than chemical additives-to preserve solid cellular foods during storage. However, the effectiveness of long-term MEF exposure on the psychrotrophic microorganisms responsible for the food spoilage at cool temperatures remains unclear. The spoilage-associated psychrotroph Pseudomonas fragi MC16 was obtained from pork samples stored at 7 °C. Continuous MEF treatment attenuated growth and resulted in subsequent adaptation of M16 cultured on nutrient agar plates at 7 °C, compared to the control cultures, as determined by biomass analysis and plating procedures. Moreover, intracellular dehydrogenase activity and ATP levels also indicated an initial effect of MEF treatment followed by cellular recovery, and extracellular β-galactosidase activity assays indicated no obvious changes in cell membrane permeability. Furthermore, microscopic observations using scanning and transmission electron microscopy revealed that MEF induced sublethal cellular injury during early treatment stages, but no notable changes in morphology or cytology on subsequent days. Our study provides direct evidence that psychrotrophic P. fragi MC16 cultured on nutrient agar plates at 7 °C are capable of adapting to MEF treatment.

Addition of Carbon to the Culture Medium Improves the Detection Efficiency of Aflatoxin Synthetic Fungi

PubMed Central

Suzuki, Tadahiro; Iwahashi, Yumiko

2016-01-01

Aflatoxin (AF) is a harmful secondary metabolite that is synthesized by the Aspergillus species. Although AF detection techniques have been developed, techniques for detection of AF synthetic fungi are still required. Techniques such as plate culture methods are continually being modified for this purpose. However, plate culture methods require refinement because they suffer from several issues. In this study, activated charcoal powder (carbon) was added to a culture medium containing cyclodextrin (CD) to enhance the contrast of fluorescence and improve the detection efficiency for AF synthetic fungi. Two culture media, potato dextrose agar and yeast extract sucrose agar, were investigated using both plate and liquid cultures. The final concentrations of CD and carbon in the media were 3 mg/mL and 0.3 mg/mL, respectively. Addition of carbon improved the visibility of fluorescence by attenuating approximately 30% of light scattering. Several fungi that could not be detected with only CD in the medium were detected with carbon addition. The carbon also facilitated fungal growth in the potato dextrose liquid medium. The results suggest that addition of carbon to media can enhance the observation of AF-derived fluorescence. PMID:27854283

Growth and mycotoxin production by Chaetomium globosum.

PubMed

Fogle, Matthew R; Douglas, David R; Jumper, Cynthia A; Straus, David C

2007-07-01

Chaetomium globosum, the most common species within this genus, produces chaetoglobosins A and C when cultured on building material. Relatively low levels of these compounds have been shown to be lethal to various tissue culture cell lines. This study had two major objectives: (1) to determine the frequency at which Chaetomium species are isolated in water-damaged buildings and (2) to examine the production of chaetoglobosins A and C in isolates of C. globosum obtained from different buildings. Out of 794 water-damaged buildings, Chaetomium species were isolated in 49% of these structures. C. globosum ATCC 16021 was grown on four different media: oatmeal agar (OA), potato dextrose agar (PDA), corn meal agar (CMA), and malt extract agar (MEA). After 4 weeks, fungal growth was evaluated based on colony diameter and the quantity of spores produced on agar plates. In addition, production of chaetoglobosin A and C was monitored using high performance liquid chromatography. Colony diameter, spore production, and mycotoxin production by C. globosum were the highest on OA. Out of 30 C. globosum isolates cultured on OA for 4 weeks, 16 produced detectable amounts of chaetoglobosin A and every isolate produced chaetoglobosin C.

Comparison of four chromogenic media and Hektoen agar for detection and presumptive identification of Salmonella strains in human stools.

PubMed

Perez, J M; Cavalli, P; Roure, C; Renac, R; Gille, Y; Freydiere, A M

2003-03-01

Several chromogenic media have been developed to enhance the specificity of Salmonella detection. We compared the performance of four commercial chromogenic media-namely, ABC medium (Lab M. Ltd., Bury, United Kingdom), COMPASS Salmonella agar (Biokar Diagnostics, Beauvais, France), CHROMagar Salmonella agar (CHROMagar Company, Paris, France), and SM ID agar (bioMerieux, Marcy l'Etoile, France)-with conventional Hektoen medium. Nine hundred sixteen stool samples from inpatients at three hospitals were cultured, in parallel, on the five media, both by direct inoculation and after selective enrichment in selenite broth. Sixty-four Salmonella strains with 12 serotypes were isolated on at least one medium. After 48 h of incubation, sensitivity before and after enrichment was 62.5 and 89.1% with ABC medium, 77.1 and 93.8% with COMPASS agar, 66.7 and 89.1% with CHROMagar, 68.8 and 85.9% with SM ID agar, and 85.4 and 98.4% with Hektoen agar, respectively. Broth enrichment and prolonged incubation (48 versus 24 h) increased the sensitivity of all five media. Only one strain was not isolated on Hektoen agar. The number of false-positive isolates was higher with all five media after enrichment in selenite broth and after incubation for 48 h compared to 24 h. The specificity of the four chromogenic media was better than 91% after incubation for 24 h (77.7% with Hektoen agar) and better than 84% after incubation for 48 h (74.8% with Hektoen agar). This higher specificity reduces the need for confirmatory tests, thereby cutting technical time and reagent requirements. Both COMPASS agar and CHROMagar Salmonella, which after simple additional tests showed close efficiencies (96 and 97%, respectively), can be recommended as single-plate media of choice for the detection and presumptive identification of salmonellae in stools.

Dial-A-Decon Solution Chemistry GAP Testing

DTIC Science & Technology

2012-04-01

34 The tubes were serially diluted using Buttcrfield’s buffer solution and plated in triplicate on Tryptic Soy Agar. Plates were enumerated the...of 200 uL HD to 10 mL of the surfactant solution. The energy to create the oil in water (O/W) emulsions was provided by magnetic stirring. Solutions...emulsify a mixture of water and oil such as HD, one or more emulsifiers are required. Each surfactant system can be characterized by an HLB value

R-plasmid transfer in a wastewater treatment plant.

PubMed Central

Mach, P A; Grimes, D J

1982-01-01

Enteric bacteria have been examined for their ability to transfer antibiotic resistance in a wastewater treatment plant. Resistant Salmonella enteritidis, Proteus mirabilis, and Escherichia coli were isolated from clinical specimens and primary sewage effluent. Resistance to ampicillin, chloramphenicol, streptomycin, sulfadiazine, and tetracycline was demonstrated by spread plate and tube dilution techniques. Plasmid mediation of resistance was shown by ethidium bromide curing, agarose gel electrophoresis, and direct cell transfer. Each donor was mated with susceptible E. coli and Shigella sonnei. Mating pairs (and recipient controls) were suspended in unchlorinated primary effluent that had been filtered and autoclaved. Suspensions were added to membrane diffusion chambers which were then placed in the primary and secondary setting tanks of the wastewater treatment plant. Resistant recombinants were detected by replica plating nutrient agar master plates onto xylose lysine desoxycholate agar plates that contained per milliliter of medium 10 micrograms of ampicillin, 30 micrograms of chloramphenicol, 10 micrograms of streptomycin, 100 micrograms of sulfadiazine, or 30 micrograms of tetracycline. Mean transfer frequencies for laboratory matings were 2.1 X 10(-3). In situ matings for primary and secondary settling resulted in frequencies of 4.9 X 10(-5) and 7.5 X 10(-5), respectively. These values suggest that a significant level of resistance transfer occurs in wastewater treatment plants in the absence of antibiotics as selective agents. Images PMID:6760813

Improved vectors for transcriptional/translational signal screening in corynebacteria using the melC operon from Streptomyces glaucescens as reporter.

PubMed

Adham, Sirin A I; Rodríguez, Sonia; Ramos, Angelina; Santamaría, Ramón I; Gil, José A

2003-07-01

The tyrosinase operon ( melC) from Streptomyces glaucescens was cloned and functionally expressed in Brevibacterium lactofermentum and Corynebacterium glutamicum under the control of the promoter of the kan gene from Tn 5. Recombinant corynebacterial cells containing the tyrosinase operon produced melanin on agar plates and in liquid culture when supplemented with copper and tyrosine. A conjugative bifunctional replacement vector for transcriptional/translational signal screening (pEMel-1) was constructed using expression of the melC operon from S. glaucescens, which can be used for cloning promoter sequences as EcoRI- NdeI fragments. When the DNA fragments with promoter activity such as cspBp or trpp were inserted into pEMel-1, B. lactofermentum harboring the chimeric plasmids produced melanin at different stages of growth, allowing temporal detection of promoter activity. The vector was also used to detect the activity of a Streptomyces promoter ( xysAp), which was inactive in B. lactofermentum, after PCR mutagenesis. The melC operon can be used for the visual, inexpensive (compared to the high price of starch azure for amylase detection), and non-selective (in contrast to the kan or cat genes) screening of several thousand clones at high colony density without killing of the transformants due to the presence of iodine (as in the case of amylase assay).

Miniaturizing 3D assay for high-throughput drug and genetic screens for small patient-derived tumor samples (Conference Presentation)

NASA Astrophysics Data System (ADS)

Rotem, Asaf; Garraway, Levi; Su, Mei-Ju; Basu, Anindita; Regev, Aviv; Struhl, Kevin

2017-02-01

Three-dimensional growth conditions reflect the natural environment of cancer cells and are crucial to be performed at drug screens. We developed a 3D assay for cellular transformation that involves growth in low attachment (GILA) conditions and is strongly correlated with the 50-year old benchmark assay-soft agar. Using GILA, we performed high-throughput screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. This phenotypic approach is complementary to our genetic approach that utilizes single-cell RNA-sequencing of a patient sample to identify putative oncogenes that confer sensitivity to drugs designed to specifically inhibit the identified oncoprotein. Currently, we are dealing with a big challenge in our field- the limited number of cells that might be extracted from a biopsy. Small patient-derived samples are hard to test in the traditional multiwell plate and it will be helpful to minimize the culture area and the experimental system. We managed to design a suitable microfluidic device for limited number of cells and perform the assay using image analysis. We aim to test drugs on tumor cells, outside of the patient body- and recommend on the ideal treatment that is tailored to the individual. This device will help to minimize biopsy-sampling volumes and minimize interventions in the patient's tumor.

Three-dimensional characterization of bacterial microcolonies on solid agar-based culture media.

PubMed

Drazek, Laurent; Tournoud, Maud; Derepas, Frédéric; Guicherd, Maryse; Mahé, Pierre; Pinston, Frédéric; Veyrieras, Jean-Baptiste; Chatellier, Sonia

2015-02-01

For the last century, in vitro diagnostic process in microbiology has mainly relied on the growth of bacteria on the surface of a solid agar medium. Nevertheless, few studies focused in the past on the dynamics of microcolonies growth on agar surface before 8 to 10h of incubation. In this article, chromatic confocal microscopy has been applied to characterize the early development of a bacterial colony. This technology relies on a differential focusing depth of the white light. It allows one to fully measure the tridimensional shape of microcolonies more quickly than classical confocal microscopy but with the same spatial resolution. Placing the device in an incubator, the method was able to individually track colonies growing on an agar plate, and to follow the evolution of their surface or volume. Using an appropriate statistical modeling framework, for a given microorganism, the doubling time has been estimated for each individual colony, as well as its variability between colonies, both within and between agar plates. A proof of concept led on four bacterial strains of four distinct species demonstrated the feasibility and the interest of the approach. It showed in particular that doubling times derived from early tri-dimensional measurements on microcolonies differed from classical measurements in micro-dilutions based on optical diffusion. Such a precise characterization of the tri-dimensional shape of microcolonies in their late-lag to early-exponential phase could be beneficial in terms of in vitro diagnostics. Indeed, real-time monitoring of the biomass available in a colony could allow to run well established microbial identification workflows like, for instance, MALDI-TOF mass-spectrometry, as soon as a sufficient quantity of material is available, thereby reducing the time needed to provide a diagnostic. Moreover, as done for pre-identification of macro-colonies, morphological indicators such as three-dimensional growth profiles derived from microcolonies could be used to perform a first pre-identification step, but in a shorten time. Copyright © 2014 Elsevier B.V. All rights reserved.

Spray method for recovery of heat-injured Salmonella Typhimurium and Listeria monocytogenes.

PubMed

Back, Kyeong-Hwan; Kim, Sang-Oh; Park, Ki-Hwan; Chung, Myung-Sub; Kang, Dong-Hyun

2012-10-01

Selective agar is inadequate for supporting recovery of injured cells. During risk assessment of certain foods, both injured and noninjured cells must be enumerated. In this study, a new method (agar spray method) for recovering sublethally heat-injured microorganisms was developed and used for recovery of heat-injured Salmonella Typhimurium and Listeria monocytogenes. Molten selective agar was applied as an overlay to presolidified nonselective tryptic soy agar (TSA) by spray application. Heat-injured cells (55°C for 10 min in 0.1% peptone water or 55°C for 15 min in sterilized skim milk) were inoculated directly onto solidified TSA. After a 2-h incubation period for cell repair, selective agar was applied to the TSA surface with a sprayer, and the plates were incubated. The recovery rate for heat-injured Salmonella Typhimurium and L. monocytogenes with the spray method was compared with the corresponding rates associated with TSA alone, selective media alone, and the conventional overlay method (selective agar poured on top of resuscitated cells grown on TSA and incubated for 2 h). No significant differences (P > 0.05) were found in pathogen recovery obtained with TSA, the overlay method, and the spray method. However, a lower recovery rate (P < 0.05) was obtained for isolation of injured cells on selective media. Overall, these results indicate that the agar spray method is an acceptable alternative to the conventional overlay method and is a simpler and more convenient approach to recovery and detection of injured cells.

How do microorganisms influence trace element uptake by plants? Screening in an agar model rhizosphere.

NASA Astrophysics Data System (ADS)

Marchetti, M.; Robinson, B. H.; Evangelou, M. W. H.; Vachey, A.; Schwitzguebel, J. P.; Bernier-Latmani, R.; Schulin, R.

2009-04-01

Trace elements (TE) are essential for humans and plants, but they may be toxic if their concentration is too high. For this reason, the management of TE in soils is very important. In some cases it may be necessary to increase the uptake of nutrients or TE by plants, for example in a biofortification perspective. Conversely, in some other cases TE uptake by plants should be decreased, for instance to avoid heavy metals entering the food chain via edible crops. Microorganisms living in the rhizosphere affect trace element (TE) uptake by plants. However, due to the complexity of this space and the variety of microorganisms that occur there, it is difficult to isolate the effect of any particular strain. To overcome this hurdle, we developed a system in which we grew plants under sterile conditions in agar and inoculated their rhizosphere with a single, well-defined microbial strain. For many years, agar has been used as a growth substrate for microorganisms and plant tissues. It is cheap, easy to use, and can be autoclaved to ensure its sterility. Because of its widespread use, an experiment conducted using this substrate can be reproduced under the same conditions in any laboratory. In contrast to soil, there is little interaction between the trace elements and the agar matrix. There are many studies investigating the influence of microorganisms on TE uptake by plants. However, so far only a small variety of microorganisms has been tested on few plant species. Therefore, the first objective of our research was to develop a method to rapidly screen a large variety of microorganisms on various plant species. Once this goal was achieved, we sought to study the effect of single, well-defined microbial strains on TE uptake by sunflower and wheat. The substrate for plants growth was a 10% agar solution prepared with modified Hoagland's solution and a TE solution containing 1 mg/kg Pb and molar equivalents of Cu, Ni and Zn. The agar solution was autoclaved and poured into sterile, transparent plastic boxes, whose lid was equipped with a filter allowing gas exchanges without contamination by external microorganisms. The seed surface was sterilised and the plants grew one week in agar before their rhizosphere was inoculated with LB broth containing a pure bacterial strain or agar plugs colonized by fungal hyphae. We tested 14 strains, with 5 replicates per treatment and a control where the system was inoculated with sterile LB broth. The plants grew for 2 weeks in a climate chamber and their shoots were analysed for their TEs by ICP-OES. Samples of agar and roots were collected to confirm microbial colonization of the rhizosphere, respectively sterile conditions in the control treatments. Concerning the method development, the plants grew without visible toxicity in all the boxes, and the analysis of root and agar samples indicated that the controls were sterile and the strains inoculated were growing along the roots. More than 90% of the TE and nutrients added to the system were in the liquid fraction of the agar medium, thus available for root uptake. The screening showed that the microorganisms in general decreased TE uptake by wheat and sunflower, although some of them had an opposite effect on the plants. However, with the same plant species, the microorganisms had a consistent effect on all TE tested, i.e. a given single strain caused the same effect (increase or decrease of TE uptake) on all TE tested. In sunflower, 3 microorganisms (Paenibacillus polymyxa, Pythium ultimum and Rhizoctonia solani) decreased Cu and Zn uptake by 50% compared to the control treatment. These three species are common soil microorganisms. All three are known to exude auxin, a phytohormone. This hormone can modify root morphology and physiology and thus may affect TE uptake by plants. R. solani and P. ultimum are root pathogens. Their effect was opposite to what we expected. If roots are damaged, TE should have flooded into the plant and accumulate in the tissues, but this was not the case. One explanation could be the biosorption of TE by these microorganisms, reducing the uptake by plant. Conversely to sunflower, none of the microorganisms tested showed a significant effect on TE uptake by wheat. With our research, we created an agar system allowing the screening of several microbial strains for their effect on plant TE uptake. Future work will involve screening of several other strains in a wide range of conditions in agar. A method validation with a pot experiment is also needed, as some interactions in this artificial rhizosphere may be different from those that would take place in soil. We will also pursue the investigation of two interesting mechanisms revealed by the screening: the effect of pathogens and phytohormone-exuding microorganisms on TE uptake by plants.

40 CFR 180.1027 - Nuclear polyhedrosis virus of Heliothis zea; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2010 CFR

2010-07-01

... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1027 Nuclear polyhedrosis virus of... should not exceed 107 colonies per gram as determined by an aerobic plate on trypticase soy agar. (2...

40 CFR 180.1027 - Nuclear polyhedrosis virus of Heliothis zea; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2011 CFR

2011-07-01

... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1027 Nuclear polyhedrosis virus of... should not exceed 107 colonies per gram as determined by an aerobic plate on trypticase soy agar. (2...

Detection of Campylobacter Colonies using Hyperspectral Imaging

USDA-ARS?s Scientific Manuscript database

Isolation and detection of Campylobacter in foods via direct plating involves lengthy laboratory procedures including enrichments and microaerobic incubations, which take several days to a week. The incubation time for growing Campylobacter colonies in agar media is typically 24 hours to 48 hours. F...

MICROBIAL VOLATILE ORGANIC COMPOUND EMISSION RATES AND EXPOSURE MODEL

EPA Science Inventory

This paper presents the results from a study that examined microbial volatile organic compound (MVOC) emissions from six fungi and one bacterial species (Streptomyces spp.) commonly found in indoor environments. Data are presented on peak emission rates from inoculated agar plate...

The demosponge Halichondria (Halichondria) panicea (Pallas, 1766) as a novel source of biosurfactant-producing bacteria.

PubMed

Rizzo, Carmen; Syldatk, Christoph; Hausmann, Rudolf; Gerçe, Berna; Longo, Caterina; Papale, Maria; Conte, Antonella; De Domenico, Emilio; Michaud, Luigi; Lo Giudice, Angelina

2018-06-01

The Mediterranean sponge Halichondria (Halichondria) panicea was explored as a novel matrix for the isolation of biosurfactant-producing bacteria. A total of 38 (out of 56) isolates gave a good response to the employed screening tests (e.g., stable emulsion detection, surface tension measurement, hemolytic activity, and blue agar plate assay) and were selected for further analyses. The thin layer chromatography revealed a possible glucidic composition of biosurfactants. Most promising strains, i.e., those able to produce stable emulsion with percentage higher than 30% and yellow spots on TLC plates, were affiliated to the genera Pseudovibrio, Acinetobacter, and Bacillus. The biosurfactant production by two isolates (i.e., Acinetobacter sp. SpN134 and Pseudovibrio sp. SpE85) was evaluated under different culture conditions, in terms of temperature, NaCl concentration, and pH. Surface tension reduction ability was more stable than the emulsification, and resulted differently influenced by salinity, temperature, and pH. Acinetobacter sp. SpN134 resulted particularly efficient and competitive if compared with other well-known biosurfactant producers. Data suggest that sponges may represent a promising matrix for the isolation of biosurfactant-producing bacteria, reinforcing the growing interest towards filter-feeding organisms as underexplored sources of specialized bacteria. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Screening complex hazardous wastes for mutagenic activity using a modified version of the TLC/Salmonella assay.

PubMed

Houk, V S; Claxton, L D

1986-03-01

10 complex hazardous wastes were tested for mutagenic activity using a modified version of the TLC/Salmonella assay developed by Bjørseth et al. (1982). This fractionation/bioassay scheme couples thin-layer chromatography (TLC) with the Salmonella/mammalian-microsome (Ames) assay for the detection of mutagenic constituents in complex mixtures. Crude (unadulterated) hazardous wastes and selected hazardous waste extracts were fractionated on commercially available cellulose TLC plates. Mutagenicity testing was performed in situ by applying a single overlay of minimal growth agar, tester strain TA98 or TA100, and the optional metabolic activation system directly onto the developed chromatogram. A mutagenic effect was indicated either by the appearance of localized clusters of revertant colonies or by an increase in total revertant growth vis-à-vis control plates. 7 of 10 hazardous wastes (including tars, emulsions, sludges, and spent acids and caustics) demonstrated mutagenic activity when tested by this method. To assess the sensitivity of the modified TLC/Salmonella assay, 14 Salmonella mutagens from a wide range of chemical classes and polarities were tested. Selected compounds included heterocyclics, aromatic amines, alkylating agents, antitumor agents, a nitrosamine and a nitroaromatic. 11 of the 14 mutagens were positive in this test system. The 3 compounds refractory to analysis included a polycyclic aromatic hydrocarbon and two volatiles.

Effect of Soybean Casein Digest Agar Lot on Number of Bacillus stearothermophilus Spores Recovered †

PubMed Central

Pflug, I. J.; Smith, Geraldine M.; Christensen, Ronald

1981-01-01

In recent years it has become increasingly apparent that Bacillus stearothermophilus spores are affected by various environmental factors that influence the performance of the spores as biological indicators. One environmental factor is the recovery medium. The effect of different lots of commercial soybean casein digest agar on the number of colony-forming units per plate was examined in two series of experiments: (i) several lots of medium from two manufacturers were compared in single experiments, and (ii) paired media experiments with four lots of medium were carried out and yielded three-point survivor curves. The results demonstrate that commercial soybean casein digest agar is variable on a lot-to-lot basis. The variation was lowest when recovering unheated or minimally heated spores and increased greatly with the severity of heating. PMID:16345822

Roles of curli, cellulose and BapA in Salmonella biofilm morphology studied by atomic force microscopy.

PubMed

Jonas, Kristina; Tomenius, Henrik; Kader, Abdul; Normark, Staffan; Römling, Ute; Belova, Lyubov M; Melefors, Ojar

2007-07-24

Curli, cellulose and the cell surface protein BapA are matrix components in Salmonella biofilms. In this study we have investigated the roles of these components for the morphology of bacteria grown as colonies on agar plates and within a biofilm on submerged mica surfaces by applying atomic force microscopy (AFM) and light microscopy. AFM imaging was performed on colonies of Salmonella Typhimurium grown on agar plates for 24 h and on biofilms grown for 4, 8, 16 or 24 h on mica slides submerged in standing cultures. Our data show that in the wild type curli were visible as extracellular material on and between the cells and as fimbrial structures at the edges of biofilms grown for 16 h and 24 h. In contrast to the wild type, which formed a three-dimensional biofilm within 24 h, a curli mutant and a strain mutated in the global regulator CsgD were severely impaired in biofilm formation. A mutant in cellulose production retained some capability to form cell aggregates, but not a confluent biofilm. Extracellular matrix was observed in this mutant to almost the same extent as in the wild type. Overexpression of CsgD led to a much thicker and a more rapidly growing biofilm. Disruption of BapA altered neither colony and biofilm morphology nor the ability to form a biofilm within 24 h on the submerged surfaces. Besides curli, the expression of flagella and pili as well as changes in cell shape and cell size could be monitored in the growing biofilms. Our work demonstrates that atomic force microscopy can efficiently be used as a tool to monitor the morphology of bacteria grown as colonies on agar plates or within biofilms formed in a liquid at high resolution.

Development of novel agar media for isolating guaiacol producing Alicyclobacillus spp.

PubMed

Chang, S S; Park, S H; Kang, D H

2013-06-03

The purpose of this study is to develop a selective and differential medium (SK2 agar) for isolating guaiacol producing Alicyclobacillus. Forty-one selected dyes and vanillic acid were incorporated in SK agar for screening selective and differential agents. Two guaiacol producing (1016, 1101) and two non-guaiacol producing (19220, C-GD 1-1) Alicyclobacillus isolates were streaked onto media and color differentiation of the isolates was assessed. Among 41 tested dyes, Chrome Azurol S (CAS) allowed color differentiation of the two types of Alicyclobacillus. Colonies of guaiacol producing Alicyclobacillus isolates appeared as dark purple to royal blue color with yellow background, whereas non-guaiacol producing Alicyclobacillus isolates produced cream colored colonies with yellow background. Vanillic acid not only served as a precursor for guaiacol formation but also inhibited non-guaiacol producing Alicyclobacillus. Non-guaiacol producing isolates did not grow on SK agar containing more than 70 ppm vanillic acid, whereas the recovery of guaiacol producing isolates was unaffected. When compared with other Alicyclobacillus isolation media, not only was SK2 agar capable of selectively recovering guaiacol-producing Alicyclobacillus, the degree of growth was also approximately equal if not better than orange serum agar, potato dextrose agar, and K agar. The development of SK2 agar provides the fruit juice industry with an inexpensive, simple to use alternative for the detection of guaiacol producing Alicyclobacillus. Copyright © 2013 Elsevier B.V. All rights reserved.

Salmonella spp. contamination in commercial layer hen farms using different types of samples and detection methods.

PubMed

Soria, M C; Soria, M A; Bueno, D J; Godano, E I; Gómez, S C; ViaButron, I A; Padin, V M; Rogé, A D

2017-08-01

The performance of detection methods (culture methods and polymerase chain reaction assay) and plating media used in the same type of samples were determined as well as the specificity of PCR primers to detected Salmonella spp. contamination in layer hen farms. Also, the association of farm characteristics with Salmonella presence was evaluated. Environmental samples (feces, feed, drinking water, air, boot-swabs) and eggs were taken from 40 layer hen houses. Salmonella spp. was most detected in boot-swabs taken around the houses (30% and 35% by isolation and PCR, respectively) follow by fecal samples (15.2% and 13.6% by isolation and PCR, respectively). Eggs, drinking water, and air samples were negative for Salmonella detection. Salmonella Schwarzengrund and S. Enteritidis were the most isolated serotypes. For plating media, relative specificity was 1, and the relative sensitivity was greater for EF-18 agar than XLDT agar in feed and fecal samples. However, relative sensitivity was greater in XLDT agar than EF-18 agar for boot-swab samples. Agreement was between fair to good depending on the sample, and it was good between isolation and PCR (feces and boot-swabs), without agreement for feed samples. Salmonella spp. PCR was positive for all strains, while S. Typhimurium PCR was negative. Salmonella Enteritidis PCR used was not specific. Based in the multiple logistic regression analyses, categorization by counties was significant for Salmonella spp. presence (P-value = 0.010). This study shows the importance of considering different types of samples, plating media and detection methods during a Salmonella spp. monitoring study. In addition, it is important to incorporate the sampling of floors around the layer hen houses to learn if biosecurity measures should be strengthened to minimize the entry and spread of Salmonella in the houses. Also, the performance of some PCR methods and S. Enteritidis PCR should be improved, and biosecurity measures in hen farms must be reinforced in the region of more concentrated layer hen houses to reduce the probability of Salmonella spp. presence. © 2017 Poultry Science Association Inc.

Evaluation of two commercially available chromogenic media for confirmation of methicillin-resistant Staphylococcus aureus from human, animal, and food samples.

PubMed

Ariza-Miguel, Jaime; Oniciuc, Elena-Alexandra; Sanz, Iván; Fernández-Natal, Isabel; Hernández, Marta; Rodríguez-Lázaro, David

2015-09-16

We compared the diagnostic performance of two chromogenic media, Brilliance MRSA 2 agar (Thermo Fisher Scientific) and ChromID MRSA agar (bioMérieux), for MRSA confirmation of 239 Staphylococcus aureus isolates from clinical, animal and food samples. Statistically significant differences were not observed between MRSA confirmation by mecA/mecC PCR, and by culture in both chromogenic media. However, a statistically significant difference was observed between the results obtained by both chromogenic media (p = 0.003). Segregated analysis of the results depending on the origin of the isolates (clinical, animal, and food) revealed a significant lower performance in the MRSA confirmation of food-derived isolates by using Brilliance MRSA 2 agar in comparison to PCR confirmation (p = 0.003) or ChromID MRSA agar (p<0.001). Both chromogenic media provided a good diagnostic performance for detection of MRSA isolates of human and animal origin. In conclusion, the use of chromogenic agar plates for MRSA confirmation of S. aureus isolates can provide a good diagnostic performance (sensitivity >92% and specificity >89%) regardless of the type of chromogenic media used or the origin of the S. aureus isolates. However, our results revealed a lower diagnostic performance for MRSA confirmation of S. aureus isolates from food samples by using Brilliance MRSA 2 agar. Copyright © 2015 Elsevier B.V. All rights reserved.

Responses of Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus to Simulated Food Processing Treatments, Determined Using Fluorescence-Activated Cell Sorting and Plate Counting▿

PubMed Central

Kennedy, Deirdre; Cronin, Ultan P.; Wilkinson, Martin G.

2011-01-01

Three common food pathogenic microorganisms were exposed to treatments simulating those used in food processing. Treated cell suspensions were then analyzed for reduction in growth by plate counting. Flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) were carried out on treated cells stained for membrane integrity (Syto 9/propidium iodide) or the presence of membrane potential [DiOC2(3)]. For each microbial species, representative cells from various subpopulations detected by FCM were sorted onto selective and nonselective agar and evaluated for growth and recovery rates. In general, treatments giving rise to the highest reductions in counts also had the greatest effects on cell membrane integrity and membrane potential. Overall, treatments that impacted cell membrane permeability did not necessarily have a comparable effect on membrane potential. In addition, some bacterial species with extensively damaged membranes, as detected by FCM, appeared to be able to replicate and grow after sorting. Growth of sorted cells from various subpopulations was not always reflected in plate counts, and in some cases the staining protocol may have rendered cells unculturable. Optimized FCM protocols generated a greater insight into the extent of the heterogeneous bacterial population responses to food control measures than did plate counts. This study underlined the requirement to use FACS to relate various cytometric profiles generated by various staining protocols with the ability of cells to grow on microbial agar plates. Such information is a prerequisite for more-widespread adoption of FCM as a routine microbiological analytical technique. PMID:21602370

Screening the thermophilic and hyperthermophilic bacterial population of three Iranian hot-springs to detect the thermostable α-amylase producing strain

PubMed Central

Fooladi, J; Sajjadian, A

2010-01-01

Background Screening is a routine procedure for isolation of microorganisms which are able to produce special metabolites. Purified thermostable α-amylase from bacterial sources is widely used in different industries. In this study we analyzed samples collected from three different hot springs in Iran to detect any strains capable of producing thermostable α-amylase. Materials and Methods Hot water samples from Larijan (67°C, pH 6.5), Mahallat (46°C, pH 7), and Meshkinshahr (82°C, pH 6), were cultivated in screening starch agar plates and incubated at 65°C for 24 hours. Thereafter, the plates were stained with Gram's iodine solution. Results and Discussion The bacterial colonies from the Meshkinshahr hot-spring produced the largest haloforming zone. Based on the phenotypic tests, the strain was identified as Bacillus sp. The culture condition was optimized for biosynthesis of α-amylase. The enzyme was produced at maximum level when it was incubated at 70°C in the presence of soluble starch (1%) at pH 6. The addition of calcium (10 mM) and peptone (1%) to the mineral medium, shortened the lag period and improved the growth and α-amylase synthesis. The addition of glucose (1%) to the culture greatly diminished the syntheses of α -amylase. Importantly, the enzyme extract retained 100% activity when incubated for 45 minutes at 100°C. Conclusion The Meshkinshahr hot-spring is rich in the Bacillus spp thermostable α-amylase producing strain of the thermophilic bacterial population. Iranian hot-springs like Meshkinshahr, have large microbial storages and can be used as sources of different biological products like enzymes. The enzyme which was produced with Bacillus sp. could hydrolyse polymers like starch and was used at laboratory scale successfully. PMID:22347550

Cocultivation of Legionella pneumophila and free-living amoebae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tyndall, R.L.; Domingue, E.L.

1982-10-01

Studies of the interaction of Legionella pneumophila with free-living amoebae showed that Naegleria lovaniensis and Acanthamoeba royreba could use L. pneumophia as a sole food source. However, growth of the amoebae on nonnutrient agar plates seeded with L. pneumophila was slower than growth on nonnutrient agar plates seeded with Escherichia coli. On inoculation of L. pneumophila into axenic cultures of N. lovaniensis and A. roryba, 99.9% of the L. pneumophila was destroyed within 24 h. After several weeks, however, some amoeba cultures became chronically infected and supported the growth of L. pneumophila. Amoebae exposed to L. pneumophila and containing adheredmore » L. pneumophila, L. pneumophila antigens, or both, showed no increased pathogenic potential on intranasal inoculation of weanling mice. Similarly, L. pneumophila propagated in chronically infected amoeba cultures showed no increase in virulence on intraperitoneal inoculation of guinea pigs relative to L. pneumophila grown in yeast extract broth. 20 references, 1 figure, 4 tables.« less

Evaluation of anti-plaque microbial activity of Azadirachta indica (neem oil) in vitro: A pilot study

PubMed Central

Elavarasu, Sugumari; Abinaya, P.; Elanchezhiyan, S.; Thangakumaran; Vennila, K.; Naziya, K. B.

2012-01-01

Background: Probably microbial plaque is the main etiology for periodontal tissue inflammation. Various chemical agents have been evaluated over the years with respect to their antimicrobial effects in the oral cavity. However, all are associated with side effects that prohibit regular long-term use. Therefore, the effectiveness of Azadirachta indica (neem) against plaque formation is considered to be vital, with lesser side effects. The aim of the present study is to evaluate and prove the antimicrobial activity of neem using plaque samples. Materials and Methods: Culture was prepared using brain heart infusion broth reagent. Dental plaque samples were used for that. Kirby–Bauer antimicrobial susceptibility test procedure was carried away with the sample. Neem oil was kept in the agar plate with culture and the diameter of inhibition zones was calculated. Results: Results showed inhibition zones on the agar plate around neem oil. Conclusion: Study shows definite antiplaque activity of neem oil. PMID:23066297

Detecting Staphylococcus aureus in milk from dairy cows using sniffer dogs.

PubMed

Fischer-Tenhagen, C; Theby, V; Krömker, V; Heuwieser, W

2018-05-01

Fast and accurate identification of disease-causing pathogens is essential for specific antimicrobial therapy in human and veterinary medicine. In these experiments, dogs were trained to identify Staphylococcus aureus and differentiate it from other common mastitis-causing pathogens by smell. Headspaces from agar plates, inoculated raw milk samples, or field samples collected from cows with Staphylococcus aureus and other mastitis-causing pathogens were used for training and testing. The ability to learn the specific odor of Staphylococcus aureus in milk depended on the concentration of the pathogens in the training samples. Sensitivity and specificity for identifying Staphylococcus aureus were 91.3 and 97.9%, respectively, for pathogens grown on agar plates; 83.8 and 98.0% for pathogens inoculated in raw milk; and 59.0 and 93.2% for milk samples from mastitic cows. The results of these experiments underline the potential of odor detection as a diagnostic tool for pathogen diagnosis. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Rapid identification of staphylococci by Raman spectroscopy.

PubMed

Rebrošová, Katarína; Šiler, Martin; Samek, Ota; Růžička, Filip; Bernatová, Silvie; Holá, Veronika; Ježek, Jan; Zemánek, Pavel; Sokolová, Jana; Petráš, Petr

2017-11-01

Clinical treatment of the infections caused by various staphylococcal species differ depending on the actual cause of infection. Therefore, it is necessary to develop a fast and reliable method for identification of staphylococci. Raman spectroscopy is an optical method used in multiple scientific fields. Recent studies showed that the method has a potential for use in microbiological research, too. Our work here shows a possibility to identify staphylococci by Raman spectroscopy. We present a method that enables almost 100% successful identification of 16 of the clinically most important staphylococcal species directly from bacterial colonies grown on a Mueller-Hinton agar plate. We obtained characteristic Raman spectra of 277 staphylococcal strains belonging to 16 species from a 24-hour culture of each strain grown on the Mueller-Hinton agar plate using the Raman instrument. The results show that it is possible to distinguish among the tested species using Raman spectroscopy and therefore it has a great potential for use in routine clinical diagnostics.

Mycoflora and mycotoxin-producing fungi of air-dust particles from Egypt.

PubMed

Abdel-Hafez, S I; Shoreit, A A; Abdel-Hafez, A I; el Maghraby, O M

1986-01-01

Using the dilution-plate method, 27 genera and 64 species were collected from 20 air-dust samples on glucose - (24 genera and 57 species) and cellulose - (21 genera and 45 species) Czapek's agar at 28 degrees C. There are basic similarities between the mycoflora of air-dust on the two media and the most prevalent species were Aspergillus niger, A. flavus, A. ochraceus, A. terreus, A. versicolor, Penicillium chrysogenum, P. funiculosum, Alternaria alternata, Cladosporium herbarum, Fusarium oxysporum, Rhizopus stolonifer and Trichoderma viride. Chaetomium globosum, Stachybotrys chartarum, Humicola grisea and Arthrobotrys oligospora were common only on cellulose agar plates. Extracts of mycelium from 25 isolates were tested with brine schrimp (Artemia salina); of these 23 displayed varying degrees of toxicity. Thin layer chromatographic analysis of 12 isolates of Aspergillus flavus revealed that 4 strains were producing detectable aflatoxin. Zearalenone production was noted for 3 out of 5 strains of Fusarium oxysporum and 2 out of 5 strains of F. solani.

Presence and changes in populations of yeasts on raw and processed poultry products stored at refrigeration temperature.

PubMed

Ismail, S A; Deak, T; El-Rahman, H A; Yassien, M A; Beuchat, L R

2000-12-05

A study was undertaken to determine populations and profiles of yeast species on fresh and processed poultry products upon purchase from retail supermarkets and after storage at 5 degrees C until shelf life expiration, and to assess the potential role of these yeasts in product spoilage. Fifty samples representing 15 commercial raw, marinated, smoked, or roasted chicken and turkey products were analyzed. Yeast populations were determined by plating on dichloran rose bengal chloramphenicol (DRBC) agar and tryptone glucose yeast extract (TGY) agar. Proteolytic activity was determined using caseinate and gelatin agars and lipolytic activity was determined on plate count agar supplemented with tributyrin. Populations of aerobic microorganisms were also determined. Initial populations of yeasts (log10 cfu/g) ranged from less than 1 (detection limit) to 2.89, and increased by the expiration date to 0.37-5.06, indicating the presence of psychrotrophic species. Highest initial populations were detected in raw chicken breast, wings, and ground chicken, as well as in turkey necks and legs, whereas roasted chicken and turkey products contained less than 1 log10 cfu/g. During storage, yeast populations increased significantly (P < or = 0.05) in whole chicken, ground chicken, liver, heart and gizzard, and in ground turkey and turkey sausage. Isolates (152 strains) of yeasts from poultry products consisted of 12 species. Yarrowia lipolytica and Candida zeylanoides were predominant, making up 39 and 26% of the isolates, respectively. Six different species of basidiomycetous yeasts representing 24% of the isolates were identified. Most Y. lipolytica strains showed strong proteolytic and lipolytic activities, whereas C. zeylanoides was weakly lipolytic. Results suggest that yeasts, particularly Y. lipolytica, may play a more prominent role than previously recognized in the spoilage of fresh and processed poultry stored at 5 degrees C.

Characterization of tetracycline-resistant bacteria in an urbanizing subtropical watershed.

PubMed

Sullivan, B A; Gentry, T; Karthikeyan, R

2013-09-01

The objective of this study was to determine whether varying levels of urbanization influence the dominant bacterial species of mildly resistant (0·03 mmol l(-1) tetracycline) and highly resistant (0·06 mmol l(-1) tetracycline) bacteria in sediment and water. Also, the level of urbanization was further evaluated to determine whether the diversity of tetracycline resistance genes present in the isolates and the capability of transferring their resistance were influenced. Sediment and water samples collected from five sampling sites were plated in triplicate on nutrient agar plates with a mild dose (0·03 mmol l(-1) ) and a high dose (0·06 mmol l(-1) ) of tetracycline. Five colonies from each plate plus an additional five from each triplicate group were randomly selected and isolated on nutrient agar containing 0·03 mmol l(-1) tetracycline (400 isolates). The isolates were identified by 16S rRNA gene sequencing and comparison to GenBank using blast. The isolates were also screened for 15 tetracycline resistance genes using a multiplex PCR assay and their ability to transfer resistance through conjugation experiments using a kanamycin-resistant Escherichia. coli K-12 strain labelled with a green fluorescent protein gene. Results from this study indicate that the dominant resistant organisms in this watershed are Acinetobacter spp., Chryseobacterium spp., Serratia spp., Pseudomonas spp., Aeromonas spp. and E. coli. All of these organisms are Gram negative and are closely related to pathogenic species. A majority of the isolates (66%) were capable of transferring their resistance, and there was a greater incidence of tet resistance transfer with increasing urbanization. Also, it was determined that the dominant resistance genes in the watershed are tet(W) and tet(A). Urbanization significantly affected dominant tetracycline-resistant bacteria species, but did not affect dominant resistance genes. There was correlation between increased urbanization with an increase in the ability to transfer tetracycline resistance. This indicates that urban areas may select for bacterial species that are capable of transferring resistance. These results indicate that urbanization influences the occurrence of tetracycline-resistant bacteria and the potential for transfer of resistance genes. © 2013 The Society for Applied Microbiology.

Performance of CHROMAGAR candida and BIGGY agar for identification of yeast species.

PubMed

Yücesoy, Mine; Marol, Serhat

2003-10-29

The importance of identifying the pathogenic fungi rapidly has encouraged the development of differential media for the presumptive identification of yeasts. In this study two differential media, CHROMagar Candida and bismuth sulphite glucose glycine yeast agar, were evaluated for the presumptive identification of yeast species. A total number of 270 yeast strains including 169 Candida albicans, 33 C. tropicalis, 24 C. glabrata, 18 C. parapsilosis, 12 C. krusei, 5 Trichosporon spp., 4 C. kefyr, 2 C. lusitaniae, 1 Saccharomyces cerevisiae and 1 Geotrichum candidum were included. The strains were first identified by germ tube test, morphological characteristics on cornmeal tween 80 agar and Vitek 32 and API 20 C AUX systems. In parallel, they were also streaked onto CHROMagar Candida and bismuth sulphite glucose glycine yeast agar plates. The results were read according to the color, morphology of the colonies and the existance of halo around them after 48 hours of incubation at 37 degrees C. The sensitivity and specificity values for C. albicans strains were found to be 99.4, 100% for CHROMagar Candida and 87.0, 75.2% for BiGGY agar, respectively. The sensitivity of CHROMagar Candida to identify C. tropicalis, C. glabrata and C. krusei ranged between 90.9 and 100% while the specificity was 100%. The sensitivity rates for BiGGY agar were 66.6 and 100% while the specificity values were found to be 95.4 and 100% for C. tropicalis and C. krusei, respectively. It can be concluded that the use of CHROMagar Candida is an easy and reliable method for the presumptive identification of most commonly isolated Candida species especially C. albicans, C. tropicalis and C. krusei. The lower sensitivity and specificity of BiGGY agar to identify commonly isolated Candida species potentially limits the clinical usefulness of this agar.

Phylogenetic diversity of Flavobacteria isolated from the North Sea on solid media.

PubMed

Hahnke, Richard L; Harder, Jens

2013-10-01

Flavobacteria are abundant in the North Sea, an epeiric sea on the continental shelf of Europe. However, this abundance has so far not been reflected by the number of strains in culture collections. In this study, Flavobacteria were isolated from pelagic and benthic samples, such as seawater, phytoplankton, sediment and its porewater, and from surfaces of animals and seaweeds on agar plates with a variety of carbon sources. Dilution cultivation with a new medium, incubation at low temperatures and with long incubation times, and colony screening by a Flavobacteria-Cytophagia-specific PCR detecting 16S rRNA gene sequences led to a collection of phylogenetically diverse strains. Two strains affiliated with Flammeovirgaceae and seven strains affiliated with Cyclobacteriaceae, whereas within the Flavobacteriaceae 20 isolated strains presumably represented seven novel candidate genera and 355 strains affiliated with 26 of 80 validly described marine Flavobacteriaceae genera, based on a genus boundary of 95.0% 16S rRNA gene sequence identity. The majority of strains (276) affiliated with 37 known species in 16 genera (based on a boundary of 98.7% 16S rRNA gene sequence identity), whereas 79 strains likely represented 42 novel species in 22 established Flavobacteriaceae genera. Pigmentation, iridescence, gliding motility, agar lysis, and flexirubin as a chemical marker supported the taxonomy at the species level. This study demonstrated the culturability on solid medium of phylogenetically diverse Flavobacteria originating from the North Sea. Copyright © 2013 Elsevier GmbH. All rights reserved.

Extracellular deoxyribonuclease production by periodontal bacteria.

PubMed

Palmer, L J; Chapple, I L C; Wright, H J; Roberts, A; Cooper, P R

2012-08-01

Whilst certain bacteria have long been known to secrete extracellular deoxyribonuclease (DNase), the purpose in microbial physiology was unclear. Recently, however, this enzyme has been demonstrated to confer enhanced virulence, enabling bacteria to evade the host's immune defence of extruded DNA/chromatin filaments, termed neutrophil extracellular traps (NETs). As NETs have recently been identified in infected periodontal tissue, the aim of this study was to screen periodontal bacteria for extracellular DNase activity. To determine whether DNase activity was membrane bound or secreted, 34 periodontal bacteria were cultured in broth and on agar plates. Pelleted bacteria and supernatants from broth cultures were analysed for their ability to degrade DNA, with relative activity levels determined using an agarose gel electrophoresis assay. Following culture on DNA-supplemented agar, expression was determined by the presence of a zone of hydrolysis and DNase activity related to colony size. Twenty-seven bacteria, including red and orange complex members Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, Parvimonas micra, Prevotella intermedia, Streptococcus constellatus, Campylobacter rectus and Prevotella nigrescens, were observed to express extracellular DNase activity. Differences in DNase activity were noted, however, when bacteria were assayed in different culture states. Analysis of the activity of secreted DNase from bacterial broth cultures confirmed their ability to degrade NETs. The present study demonstrates, for the first time, that DNase activity is a relatively common property of bacteria associated with advanced periodontal disease. Further work is required to determine the importance of this bacterial DNase activity in the pathogenesis of periodontitis. © 2011 John Wiley & Sons A/S.

Use of sucrose-agar globule with root exudates for mass production of vesicular arbuscular mycorrhizal fungi.

PubMed

Selvaraj, Thangaswamy; Kim, Hoon

2004-03-01

A sucrose-agar globule (SAG) was newly introduced to increase production of the vesicular arbuscular mycorrhizal (VAM) fungal spores, Gigaspora gigantea and Glomus fasciculatum. An SAG inoculum and a sucrose-agar globule with root exudates (SAGE) inoculum were prepared, and their spore productions were compared with a soil inoculum. When the SAGE was used as the inoculum on sucrose-agar medium plates the number of spores was increased (35% more than the soil inoculum). After the soil inoculum and SAGE were inoculated on an experimental plant, Zingiber officinale, the percentage root colonization, number of VAM spores, and dry matter content were analyzed. It was observed that the SAGE showed a higher percentage of root colonization (about 10% more), and increases in the number of spores (about 26%) and dry matter (more than 13%) for the two VAM fungal spores than the soil inoculum. The results of this study suggested that the SAGE inoculum may be useful for the mass production of VAM fungi and also for the large scale production of VAM fungal fertilizer.

Comparative evaluation of antimicrobial effect of herbal root canal irrigants (Morinda citrifolia, Azadirachta indica, Aloe vera) with sodium hypochlorite: An in vitro study.

PubMed

Babaji, Prashant; Jagtap, Kiran; Lau, Himani; Bansal, Nandita; Thajuraj, S; Sondhi, Priti

2016-01-01

Successful root canal treatment involves the complete elimination of microorganism from the root canal and the three-dimensional obturation of the canal space. Enterococcus faecalis is the most commonly found bacteria in failed root canal. Chemical irrigation of canals along with biomechanical preparation helps in the elimination of microorganisms. The present study was aimed to evaluate the antimicrobial effect of herbal root canal irrigants (Morinda citrifolia, Azadirachta indica extract, Aloe vera) with sodium hypochlorite (NaOCl). The bacterial E. faecalis (ATCC) culture was grown overnight in brain heart infusion (BHI) broth and inoculated in Mueller-Hinton agar plates. Antibacterial inhibition was assessed using agar well diffusion method. All five study irrigants were added to respective wells in agar plates and incubated at 37°C for 24 h. Bacterial inhibition zone around each well was recorded. Results were tabulated and statistically analyzed using Statistical Package for the Social Sciences software for Windows, version 19.0. (IBM Corp., Armonk, NY. Highest inhibitory zone against E. faecalis was seen in NaOCl fallowed by M. citrifolia and A. indica extract, and the least by A. vera extract. Tested herbal medicine (A. indica extract, M. citrifolia, A. vera) showed inhibitory zone against E. faecalis. Hence, these irrigants can be used as root canal irrigating solutions.

A 96-well screen filter plate for high-throughput biological sample preparation and LC-MS/MS analysis.

PubMed

Peng, Sean X; Cousineau, Martin; Juzwin, Stephen J; Ritchie, David M

2006-01-01

A novel 96-well screen filter plate (patent pending) has been invented to eliminate a time-consuming and labor-intensive step in preparation of in vivo study samples--to remove blood or plasma clots. These clots plug the pipet tips during a manual or automated sample-transfer step causing inaccurate pipetting or total pipetting failure. Traditionally, these blood and plasma clots are removed by picking them out manually one by one from each sample tube before any sample transfer can be made. This has significantly slowed the sample preparation process and has become a bottleneck for automated high-throughput sample preparation using robotic liquid handlers. Our novel screen filter plate was developed to solve this problem. The 96-well screen filter plate consists of 96 stainless steel wire-mesh screen tubes connected to the 96 openings of a top plate so that the screen filter plate can be readily inserted into a 96-well sample storage plate. Upon insertion, the blood and plasma clots are excluded from entering the screen tube while clear sample solutions flow freely into it. In this way, sample transfer can be easily completed by either manual or automated pipetting methods. In this report, three structurally diverse compounds were selected to evaluate and validate the use of the screen filter plate. The plasma samples of these compounds were transferred and processed in the presence and absence of the screen filter plate and then analyzed by LC-MS/MS methods. Our results showed a good agreement between the samples prepared with and without the screen filter plate, demonstrating the utility and efficiency of this novel device for preparation of blood and plasma samples. The device is simple, easy to use, and reusable. It can be employed for sample preparation of other biological fluids that contain floating particulates or aggregates.

Isolation of Escherichia coli mutants defective in enzymes of membrane lipid synthesis.

PubMed Central

Raetz, C R

1975-01-01

A new method has been developed which permits the rapid screening of E. coli colonies for mutants with defective enzymes of phospholipid metabolism. In this procedure, a disc of filter paper is pressed down on an agar plate containing several hundred colonies of mutagen-treated cells, after which the paper is lifted off. In the process the colonies are transferred to the paper, giving rise to a replica print of the master plate. The few cells from each colony left on the master keep growing in the original pattern. The pattern of colonies is also retained on the filter paper, even after the cells are rendered permeable with lysozyme and EDTA. Colonies treated in this manner remain absorbed to the paper, where they can convert sn-(U-14-C)glycero-3-P to phosphatidyl(U-14-C)glycerophosphate, dependent on added CDP-diglyceride. Unrelated reactions of sn-(U-14-C)glycero-3-P that may obscure the synthesis of phosphatidyl-glycerophosphate are inhibited by the addition of reagents poisoning energy generation. The radioactive phospholipid that forms around each colony on the paper is precipitated in situ with trichloroacetic acid, and unreacted sn-(U-14-C)glycero-3-P is washed away. After autoradiography, the colonies on the filter paper are stained with Coomassie blue. When the autoradiogram is superimposed on the strained paper, mutants are identified as blue colonies lacking a black halo. With this method, 20,000 colonies were screened in several days. Four mutants were identified with low levels of CDP-diglyceride:snglycero-3-P phosphatidyl transferase (EC 2.7.8.5, GLYCEROL-PHOSPHATE PHOSPHATIDYLTRANSFERASE, PHOSPHATIDYLGLYCEROPHOSPHATE SYNTHETASE) IN EXTRACTS. With a similar assay, 10,000 additional colonies were screened for mutants with altered CDP-diglyceride:L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthetase), and four strains were found in which the enzyme is thermolabile. The screening technique described here is termed replica printing and should be applicable not only to studies of phospholipid metabolism but also to nucleic acid and protein synthesis. Images PMID:49056

Diversity of bacteria and archaea from a landfill in Chandigarh, India as revealed by culture-dependent and culture-independent molecular approaches.

PubMed

Krishnamurthi, S; Chakrabarti, T

2013-02-01

The bacterial community structure of a municipal landfill in Chandigarh, India was analysed by culture-dependent as well as culture-independent molecular approaches, and archaeal structure by the latter method. Samples were collected in two phases from the surface and a depth of 0.91 m in June, 2004 and from 0.91 m, 1.52 m and 1.68 m in May, 2005. After serial dilutions, samples were plated onto tryptic soy agar (TSA), plate count agar (PCA), tryptic soy broth agar (TSBA) and TSBA100 (TSBA diluted 100 times and solidified with agarose), and incubated aerobically at 30°C. The number of bacteria (CFU) on different media ranged between 9.4×10⁵g⁻¹ (on PCA) and 1.9×10⁷g⁻¹ (on TSA) (wet weight). The numbers of bacteria enumerated from plates incubated anaerobically (anaerobic agar and reinforced clostridial agar) were 2.1×10⁷and 1.7×10⁶g⁻¹, respectively. Of the 468 isolated and purified bacteria (183 in the first phase and 285 in the second phase), 135 were characterised using phenotypic characteristics as well as 16S rRNA gene sequence analysis. It was found that members of the phylum Firmicutes were overwhelmingly predominant (86.6%) in the landfill, followed by Actinobacteria (9.6%) and Proteobacteria (3.7%). Among the Firmicutes, at least 17 species from the single genus Bacillus were the most abundant inhabitants of the landfill. Detailed polyphasic characterisation of many of these isolates led to the discovery of a novel genus Paenisporosarcina (and the species P. quisquiliarum), a novel species of Microbacterium, M. immunditiarum, and reclassification of Sporosarcina macmurdoensis, Pelagibacillus goriensis, Bacillus silvestris, Bacillus insolitus, Bacillus psychrotolerans and Bacillus psychrodurans. Culture-independent analysis of two 16S rRNA gene libraries also revealed that the phylum Firmicutes was the predominant group in this community. The diversity of Archaea was found to be limited mainly to members of two orders: Methanosarcinales and Methanomicrobiales of the phylum Euryarchaeota. When these results were compared to those reported earlier on similar studies, it was found that irrespective of differences in composition of municipal solid waste (especially compostable organic matter and paper) and climate, the members of bacterial and archaeal communities in landfills of many countries remained broadly similar. Copyright © 2012 Elsevier GmbH. All rights reserved.

Plate-based diversity subset screening generation 2: an improved paradigm for high-throughput screening of large compound files.

PubMed

Bell, Andrew S; Bradley, Joseph; Everett, Jeremy R; Loesel, Jens; McLoughlin, David; Mills, James; Peakman, Marie-Claire; Sharp, Robert E; Williams, Christine; Zhu, Hongyao

2016-11-01

High-throughput screening (HTS) is an effective method for lead and probe discovery that is widely used in industry and academia to identify novel chemical matter and to initiate the drug discovery process. However, HTS can be time consuming and costly and the use of subsets as an efficient alternative to screening entire compound collections has been investigated. Subsets may be selected on the basis of chemical diversity, molecular properties, biological activity diversity or biological target focus. Previously, we described a novel form of subset screening: plate-based diversity subset (PBDS) screening, in which the screening subset is constructed by plate selection (rather than individual compound cherry-picking), using algorithms that select for compound quality and chemical diversity on a plate basis. In this paper, we describe a second-generation approach to the construction of an updated subset: PBDS2, using both plate and individual compound selection, that has an improved coverage of the chemical space of the screening file, whilst only selecting the same number of plates for screening. We describe the validation of PBDS2 and its successful use in hit and lead discovery. PBDS2 screening became the default mode of singleton (one compound per well) HTS for lead discovery in Pfizer.

Determining Enzyme Activity by Radial Diffusion

ERIC Educational Resources Information Center

Davis, Bill D.

1977-01-01

Discusses advantages of radial diffusion assay in determining presence of enzyme and/or rough approximation of amount of enzyme activities. Procedures are included for the preparation of starch-agar plates, and the application and determination of enzyme. Techniques using plant materials (homogenates, tissues, ungerminated embryos, and seedlings)…

Rathayibacter iranicus isolated from asymptomatic wheat seeds in Turkey

USDA-ARS?s Scientific Manuscript database

Asymptomatic wheat seeds collected from 799 farmers in six central provinces of Turkey were checked for the presence of Rathayibacter species by plating 100 µl of the diluted and undiluted seed wash suspension onto modified 523 agar. Of the 25 isolated strains presumptively identified as Rathayibac...

Antifungal Effects of Silver Nanoparticles (AgNPs) against Various Plant Pathogenic Fungi.

PubMed

Kim, Sang Woo; Jung, Jin Hee; Lamsal, Kabir; Kim, Yun Seok; Min, Ji Seon; Lee, Youn Su

2012-03-01

This research is concerned with the fungicidal properties of nano-size silver colloidal solution used as an agent for antifungal treatment of various plant pathogens. We used WA-CV-WA13B, WA-AT-WB13R, and WA-PR-WB13R silver nanoparticles (AgNPs) at concentrations of 10, 25, 50, and 100 ppm. Eighteen different plant pathogenic fungi were treated with these AgNPs on potato dextrose agar (PDA), malt extract agar, and corn meal agar plates. We calculated fungal inhibition in order to evaluate the antifungal efficacy of silver nanoparticles against pathogens. The results indicated that AgNPs possess antifungal properties against these plant pathogens at various levels. Treatment with WA-CV-WB13R AgNPs resulted in maximum inhibition of most fungi. Results also showed that the most significant inhibition of plant pathogenic fungi was observed on PDA and 100 ppm of AgNPs.

Recognition of anaerobic bacterial isolates in vitro using electronic nose technology.

PubMed

Pavlou, A; Turner, A P F; Magan, N

2002-01-01

Use of an electronic nose (e.nose) system to differentiation between anaerobic bacteria grown in vitro on agar media. Cultures of Clostridium spp. (14 strains) and Bacteroides fragilis (12 strains) were grown on blood agar plates and incubated in sampling bags for 30 min before head space analysis of the volatiles. Qualitative analyses of the volatile production patterns was carried out using an e.nose system with 14 conducting polymer sensors. Using data analysis techniques such as principal components analysis (PCA), genetic algorithms and neural networks it was possible to differentiate between agar blanks and individual species which accounted for all the data. A total of eight unknowns were correctly discriminated into the bacterial groups. This is the first report of in vitro complex volatile pattern recognition and differentiation of anaerobic pathogens. These results suggest the potential for application of e.nose technology in early diagnosis of microbial pathogens of medical importance.

Plate-based diversity subset screening: an efficient paradigm for high throughput screening of a large screening file.

PubMed

Bell, Andrew S; Bradley, Joseph; Everett, Jeremy R; Knight, Michelle; Loesel, Jens; Mathias, John; McLoughlin, David; Mills, James; Sharp, Robert E; Williams, Christine; Wood, Terence P

2013-05-01

The screening files of many large companies, including Pfizer, have grown considerably due to internal chemistry efforts, company mergers and acquisitions, external contracted synthesis, or compound purchase schemes. In order to screen the targets of interest in a cost-effective fashion, we devised an easy-to-assemble, plate-based diversity subset (PBDS) that represents almost the entire computed chemical space of the screening file whilst comprising only a fraction of the plates in the collection. In order to create this file, we developed new design principles for the quality assessment of screening plates: the Rule of 40 (Ro40) and a plate selection process that insured excellent coverage of both library chemistry and legacy chemistry space. This paper describes the rationale, design, construction, and performance of the PBDS, that has evolved into the standard paradigm for singleton (one compound per well) high-throughput screening in Pfizer since its introduction in 2006.

Environmental bacteria produce abundant and diverse antibiofilm compounds.

PubMed

Farmer, J T; Shimkevitch, A V; Reilly, P S; Mlynek, K D; Jensen, K S; Callahan, M T; Bushaw-Newton, K L; Kaplan, J B

2014-12-01

The aim of this study was to isolate novel antibiofilm compounds produced by environmental bacteria. Cell-free extracts were prepared from lawns of bacteria cultured on agar. A total of 126 bacteria isolated from soil, cave and river habitats were employed. Extracts were tested for their ability to inhibit Staphylococcus aureus biofilm in a 96-well microtitre plate assay. A total of 55/126 extracts (44%) significantly inhibited Staph. aureus biofilm. Seven extracts were selected for further analysis. The antibiofilm activities in all seven extracts exhibited unique patterns of molecular mass, chemical polarity, heat stability and spectrum of activity against Staph. aureus, Staphylococcus epidermidis and Pseudomonas fluorescens, suggesting that these seven antibiofilm activities were mediated by unique chemical compounds with different mechanisms of action. Environmental bacteria produce abundant and diverse antibiofilm compounds. Screening cell-free extracts is a useful method for identifying secreted compounds that regulate biofilm formation. Such compounds may represent a novel source of antibiofilm agents for technological development. © 2014 The Society for Applied Microbiology.

Improved acid tolerance of Lactobacillus pentosus by error-prone whole genome amplification.

PubMed

Ye, Lidan; Zhao, Hua; Li, Zhi; Wu, Jin Chuan

2013-05-01

Acid tolerance of Lactobacillus pentosus ATCC 8041 was improved by error-prone amplification of its genomic DNA using random primers and Taq DNA polymerase. The resulting amplification products were transferred into wild-type L. pentosus by electroporation and the transformants were screened for growth on low-pH agar plates. After only one round of mutation, one mutant (MT3) was identified that was able to completely consume 20 g/L of glucose to produce lactic acid at a yield of 95% in 1L MRS medium at pH 3.8 within 36 h, whereas no growth or lactic acid production was observed for the wild-type strain under the same conditions. The acid tolerance of mutant MT3 remained genetically stable for at least 25 subcultures. Therefore, the error-prone whole genome amplification technique is a very powerful tool for improving phenotypes of this lactic acid bacterium and may also be applicable for other microorganisms. Copyright © 2012 Elsevier Ltd. All rights reserved.

Genetic Stability of Streptomyces Lividans pIJ702 in Response to Spaceflight

NASA Astrophysics Data System (ADS)

Lim, K. S.; Goins, T. L.; Voeikova, T. A.; Pyle, B. H.

2008-06-01

Streptomyces lividans carrying plasmid pIJ702 encoding genes for thiostrepton resistance (tsr-) and melanin production (mel+) was plated on agar and flown on the Russian satellite Foton-M3 for 16 days. The percentage loss of plasmid expression in flight samples was lower than that in ground samples when both samples were grown in enriched (ISP) media. Differences in media content also affect plasmid expression rate; ISP media have a higher loss of plasmid expression than samples in minimum media when both were grown on ground conditions. Results suggest that stress resulted in the increased expression of plasmid pIJ702 by S. lividans. Screening of thiostrepton resistant white (tsr+ mel-) mutants showed similar proportions of variants in ground samples and flight samples. To determine if there are mutations in the mel gene, DNA extracted from flight and control white mutants was amplified and gel electrophoresis of amplified products show no major mutation in the products. Sequencing of amplified products is required to identify mutations resulting in loss of pigmentation.

Enhanced production of xylanase from locally isolated fungal strain using agro-industrial residues under solid-state fermentation.

PubMed

Abdullah, Roheena; Nisar, Kinza; Aslam, Aafia; Iqtedar, Mehwish; Naz, Shagufta

2015-01-01

This study is related to the isolation of fungal strain for xylanase production using agro-industrial residues. Forty fungal strains with xylanolytic potential were isolated by using xylan agar plates and quantitatively screened in solid-state fermentation. Of all the tested isolates, the strain showing highest ability to produce xylanase was assigned the code Aspergillus niger LCBT-14. For the enhanced production of the enzyme, five different fermentation media were evaluated. Out of all media, M4 containing wheat bran gave maximum enzyme production. Effect of different variables including incubation time, temperature, pH, carbon and nitrogen sources has been investigated. The optimum enzyme production was obtained after 72 h at 30°C and pH 4. Glucose as a carbon source while ammonium sulphate and yeast extract as nitrogen sources gave maximum xylanase production (946 U/mL/min). This study was successful in producing xylanase by A. niger LCBT-14 economically by utilising cheap indigenous substrate.

Agarolytic culturable bacteria associated with three antarctic subtidal macroalgae.

PubMed

Sánchez Hinojosa, Verónica; Asenjo, Joel; Leiva, Sergio

2018-05-21

Bacterial communities of Antarctic marine macroalgae remain largely underexplored in terms of diversity and biotechnological applications. In this study, three Antarctic subtidal macroalgae (Himantothallus grandifolius, Pantoneura plocamioides and Plocamium cartilagineum), two of them endemic of Antarctica, were investigated as a source for isolation of agar-degrading bacteria. A total of 21 epiphytic isolates showed agarolytic activity at low temperature on agar plates containing agar as the sole carbon source. 16S rRNA identification showed that the agar-degrading bacteria belonged to the genera Cellulophaga, Colwellia, Lacinutrix, Olleya, Paraglaciecola, Pseudoalteromonas and Winogradskyella. The agarase enzyme from a potential new species of the genus Olleya was selected for further purification. The enzyme was purified from the culture supernatant of Olleya sp. HG G5.3 by ammonium sulfate precipitation and ion-exchange chromatography. Molecular weight of the agarase was estimated to be 38 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme exhibited activity at 4 °C, retaining > 50% of its maximum activity at this temperature. This is the first study reporting the phylogeny of agar-degrading bacteria isolated from Antarctic subtidal macroalgae and the results suggest the huge potential of Antarctic algae-associated bacteria as a source of cold-active hydrolytic enzymes of biotechnological interest.

Comparison of direct-plating and broth-enrichment culture methods for detection of potential bacterial pathogens in respiratory secretions.

PubMed

Kaur, Ravinder; Wischmeyer, Jareth; Morris, Matthew; Pichichero, Michael E

2017-11-01

We compared the recovery of potential respiratory bacterial pathogens and normal flora from nasopharyngeal specimens collected from children during health and at the onset of acute otitis media (AOM) by selective direct-plating and overnight broth-enrichment. Overall, 3442 nasal wash (NW) samples collected from young children were analysed from a 10-year prospective study. NWs were cultured by (1) direct-plating to TSAII/5 % sheep blood agar and chocolate agar plates and (2) overnight broth-enrichment in BacT/ALERT SA-broth followed by plating. Standard microbiology techniques were applied to identify three dominant respiratory bacterial pathogens: Streptococcus pneumoniae (Spn), Haemophilus influenzae (Hflu) and Moraxella catarrhalis (Mcat) as well as two common nasal flora, Staphylococcus aureus (SA) and alpha-haemolytic Streptococci (AHS).Results/Key findings. Direct-plating of NW resulted in isolation of Spn from 37.8 %, Hflu from 13.6 % and Mcat from 33.2 % of samples. In comparison, overnight broth-enrichment isolated fewer Spn (30.1 %), Hflu (6.2 %) and Mcat (16.2 %) (P<0.001-0.0001). Broth-enrichment resulted in significant increased isolation of SA (6.0 %) and AHS (30.1 %) (P<0.0001). Competition between bacterial species in broth when both species were detected by direct-plating was assessed, and it was found that SA and AHS out-competed other species during broth-enrichment when samples were collected from healthy children but not during AOM. In middle ear fluids (MEF) at the onset of AOM, broth-enrichment resulted in higher recovery of Spn (+10.4 %, P<0.001), Hflu (+4.4 %, P=0.39) and Mcat (+13.5 %, <0.001). Broth-enrichment significantly reduces the accurate detection of bacterial respiratory pathogens and increases identification of SA and AHS in NW. Broth-enrichment improves detection of bacterial respiratory pathogens in MEF samples.

In Vitro Lethal Activity of the Nematophagous Fungus Clonostachys rosea (Ascomycota: Hypocreales) against Nematodes of Five Different Taxa.

PubMed

Rodríguez-Martínez, Rosalia; Mendoza-de-Gives, Pedro; Aguilar-Marcelino, Liliana; López-Arellano, María Eugenia; Gamboa-Angulo, Marcela; Hanako Rosas-Saito, Greta; Reyes-Estébanez, Manuela; Guadalupe García-Rubio, Virginia

2018-01-01

This study was aimed to evaluate the in vitro lethal activity of the nematophagous fungi Clonostachys rosea against 5 nematodes species belonging to different taxa. Two groups of 35 Petri dishes (PD) each were divided into 5 series of 7 (PD). Group 1 (series 1, 2, 3, 4, and 5) contained only water agar; meanwhile group 2 plates (series 6, 7, 8, 9, and 10) contained C. rosea cultures growth on water agar. Every plate from the two groups was added with 500 nematodes corresponding to the following genera/specie: Haemonchus contortus , Caenorhabditis elegans, Rhabditis sp., Panagrellus redivivus , and Butlerius sp. After 5-day incubation at room temperature, free (nontrapped) larvae were recovered from plates using the Baermann funnel technique. Recovered nematodes were counted and compared with their proper controls. Results shown an important reduction percentage of the nematode population attributed to the fungal lethal activity as follows: H. contortus (L 3 ) 87.7%; C. elegans 94.7%; Rhabditis sp. 71.9%; P. redivivus 92.7%; and Butlerius sp. 100% ( p ≤ 0.05). The activity showed by C. rosea against the H. contortus can be crucial for further studies focused to the biological control of sheep haemonchosis, although the environmental impact against beneficial nematodes should be evaluated.

Comparison of the Antimicrobial Efficacy of Two Antibiotics Sparfloxacin and Augmentin as Experimental Root Canal Irrigating Solutions against Enterococcus faecalis - An Invitro Study

PubMed Central

Venigalla, Bhuvan Shome; Surakanti, Jayaprada Reddy; Thumu, Jayaprakash; Chennamaneni, Krishna Chaitanya; Kalluru, Rama S.

2016-01-01

Introduction One of the main goals of endodontic treatment is root canal disinfection and to prevent subsequent chances of reinfection. Adjuvant to instrumentation, root canal irrigants are required to eliminate the bacteria found on the root canal walls and lateral canals within the dentinal tubules. Aim To measure and compare the antibacterial efficacy of two antibiotics as experimental root canal irrigating solutions against Enterococcus faecalis (E. faecalis). Materials and Methods Fifteen Brain Heart Infusion agar plates were inoculated with Enterococcus faecalis-American Type Culture Collection (ATCC) 29212. 5 micrograms (mcg) Sparfloxacin discs, 30mcg Augmentin discs, and sterile paper test discs saturated with 2% Chlorhexidine (CHX), 3% Sodium Hypochlorite (NaOCl) and 5% NaOCl solutions were placed on agar plates. Sodium Chloride 0.9% (NaCl) paper discs were used as controls. Fifteen plates were incubated aerobically at 37°C. Results were expressed as per the terms of the diameter of the inhibition zone. Results Results suggested a statistically significant difference in the zones of inhibition between five irrigating solutions (p < 0.001). Conclusion Although, zones of inhibition were found in all the groups, 5mcg Sparfloxacin and 30mcg Augmentin showed maximum antimicrobial activity against E.faecalis. PMID:27135003

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

PubMed Central

Morales-Soto, Nydia; Anyan, Morgen E.; Mattingly, Anne E.; Madukoma, Chinedu S.; Harvey, Cameron W.; Alber, Mark; Déziel, Eric; Kearns, Daniel B.; Shrout, Joshua D.

2015-01-01

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more “temperate swarmers” that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. “Wettability”, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment. PMID:25938934

Evaluation of a New Selective Medium, BD BBL CHROMagar MRSA II, for Detection of Methicillin-Resistant Staphylococcus aureus in Stool Specimens ▿

PubMed Central

Havill, Nancy L.; Boyce, John M.

2010-01-01

We compared the recovery of methicillin-resistant Staphylococcus aureus (MRSA) on a new selective chromogenic agar, BD BBL CHROMagar MRSA II (CMRSAII), to that on traditional culture media with 293 stool specimens. The recovery of MRSA was greater on the CMRSAII agar. Screening of stool samples can identify patients who were previously unknown carriers of MRSA. PMID:20392908

Immobilization of pectin depolymerising polygalacturonase using different polymers.

PubMed

Ur Rehman, Haneef; Aman, Afsheen; Nawaz, Muhammad Asif; Karim, Asad; Ghani, Maria; Baloch, Abdul Hameed; Ul Qader, Shah Ali

2016-01-01

Polygalacturonase catalyses the hydrolysis of pectin substances and widely has been used in food and textile industries. In current study, different polymers such as calcium alginate beads, polyacrylamide gel and agar-agar matrix were screened for the immobilization of polygalacturonase through entrapment technique. Polyacrylamide gel was found to be most promising one and gave maximum (89%) immobilization yield as compared to agar-agar (80%) and calcium alginate beads (46%). The polymers increased the reaction time of polygalacturonase and polymers entrapped polygalacturonases showed maximum pectinolytic activity after 10 min of reaction as compared to free polygalacturonase which performed maximum activity after 5.0 min of reaction time. The temperature of polygalacturonase for maximum enzymatic activity was increased from 45°C to 50°C and 55°C when it was immobilized within agar-agar and calcium alginate beads, respectively. The optimum pH (pH 10) of polygalacturonase was remained same when it was immobilized within polyacrylamide gel and calcium alginate beads, but changed from pH 10 to pH 9.0 after entrapment within agar-agar. Thermal stability of polygalacturonase was improved after immobilization and immobilized polygalacturonases showed higher tolerance against different temperatures as compared to free enzyme. Polymers entrapped polygalacturonases showed good reusability and retained more than 80% of their initial activity during 2nd cycles. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of Sodium Fluorescein and Plating Medium on Recovery of Irradiated Escherichia coli and Serratia marcescens from Aerosols

PubMed Central

Dorsey, Emerson L.; Berendt, Richard F.; Neff, Everett L.

1970-01-01

Irradiation of aerosols of either Escherichia coli or Serratia marcescens with simulated solar (xenon) radiation caused a significant decrease in viability. When sodium fluorescein was employed to determine the physical loss of organisms from the aerosol, an additional adverse effect upon survival was noted. The decay curves indicated that at least two mechanisms of inactivation were operative, one due to aerosolization, the other to irradiation. After collection from aerosols, both species of microorganisms grew better on blood agar base than on Casitone agar, but this finding did not appear to be related to the effect of irradiation. PMID:4922085

Usefulness of Chromogenic CromoCen® AGN agar medium for the identification of the genus Aeromonas: Assessment of faecal samples.

PubMed

Aguilera-Arreola, M G; Portillo-Muñoz, M I; Rodríguez-Martínez, C; Castro-Escarpulli, G

2012-08-01

Selective screening media for the detection and identification of Aeromonas strains are needed to guide primary isolation procedures in the clinical laboratory. This study compared the selective CromoCen® AGN chromogenic agar medium for the detection and identification of Aeromonas strains that were isolated from various samples against the conventional selective agar media that are commonly used for the isolation of this organism in food, environmental and clinical samples. The Miles and Misra and ecometric methods were used to evaluate the microbiological performance of CromoCen® AGN chromogenic agar medium, which was shown to be satisfactory. A total of 14 reference Aeromonas strains, 44 wild strains and 106 clinical stool specimens were examined using both non-chromogenic selective agars that are commonly used for Aeromonas isolation and CromoCen® AGN agar. The latter exhibited 94.73% sensitivity and 100% specificity for the various samples. On CromoCen® AGN agar medium, Aeromonas formed colonies with light green, greenish and salmon pigments with or without a surrounding wide transparent zone (halo) of 2-3mm in diameter around the entire border. This medium is recommended for the isolation and potential identification of the Aeromonas genus. Copyright © 2012 Elsevier B.V. All rights reserved.

First time identification of Acanthamoeba genotypes in the cornea samples of wild birds; Is Acanthamoeba keratitis making the predatory birds a target?

PubMed

Karakavuk, Muhammet; Aykur, Mehmet; Şahar, Esra Atalay; Karakuş, Mehmet; Aldemir, Duygu; Döndüren, Ömer; Özdemir, Hüseyin Gökhan; Can, Hüseyin; Gürüz, Adnan Yüksel; Dağcı, Hande; Döşkaya, Mert

2017-12-01

Acanthamoeba is a free-living amoeba which can be isolated from environment and among others well known as an opportunist protozoan parasite causing infections in humans and animals. Eyes are extremely important for the wild birds and losing sight ability due to Acanthamoeba can be dangerous. The studies on Acanthamoeba infection in wild birds is very few in world and Turkey therefore we aimed to screen deceased wild birds found in İzmir and Manisa provinces located in western Turkey using PCR and non-nutrition agar (NNA) plate method. Cornea samples were obtained from 18 deceased wild birds. During the external examination, signs of keratitis were observed in two Eurasian sparrowhawks (Accipiter nisus). All of the corneal samples were analyzed by two PCR methods and NNA plate. According to results, the Acanthamoeba positivity in corneal samples was 16.6% and 5.5% by PCR and plate method, respectively. According to sequencing data, two of isolates belonged to genotype T5 and one was genotype T4. In conclusion, Acanthamoeba infection was detected in wild bird cornea samples with/without keratitis for the first time in the world. The result of this study also show that Acanthamoeba can be a cause of keratitis in wild birds of Turkey and thus these predator birds can be a target of other wild animals due to loss of sight ability. In terms of public health, these results show the importance of wild birds as a source of Acanthamoeba infection in nature. Copyright © 2017 Elsevier Inc. All rights reserved.

Protein Chips for Detection of Salmonella spp. from Enrichment Culture

PubMed Central

Poltronieri, Palmiro; Cimaglia, Fabio; De Lorenzis, Enrico; Chiesa, Maurizio; Mezzolla, Valeria; Reca, Ida Barbara

2016-01-01

Food pathogens are the cause of foodborne epidemics, therefore there is a need to detect the pathogens in food productions rapidly. A pre-enrichment culture followed by selective agar plating are standard detection methods. Molecular methods such as qPCR have provided a first rapid protocol for detection of pathogens within 24 h of enrichment culture. Biosensors also may provide a rapid tool to individuate a source of Salmonella contamination at early times of pre-enrichment culture. Forty mL of Salmonella spp. enrichment culture were processed by immunoseparation using the Pathatrix, as in AFNOR validated qPCR protocols. The Salmonella biosensor combined with immunoseparation showed a limit of detection of 100 bacteria/40 mL, with a 400 fold increase to previous results. qPCR analysis requires processing of bead-bound bacteria with lysis buffer and DNA clean up, with a limit of detection of 2 cfu/50 μL. Finally, a protein chip was developed and tested in screening and identification of 5 common pathogen species, Salmonella spp., E. coli, S. aureus, Campylobacter spp. and Listeria spp. The protein chip, with high specificity in species identification, is proposed to be integrated into a Lab-on-Chip system, for rapid and reproducible screening of Salmonella spp. and other pathogen species contaminating food productions. PMID:27110786

Asymptomatic bacteriuria, bacteremia, and other infections due to NSU corynebacteria.

PubMed

Furness, G; Kaminski, Z

1975-11-01

By means of the new medium, nonspecific urethritis (NSU) chocolate agar, NSU corymebacteria were isolated from patients with asymptomatic bacteriuria, bacteremia, cervicitis, conjuctivitis, and pericarditis, and also with bone marrow, wound, and cul-de-sac infections. The NSU corynebacteria were considered the etiologic agents. On the basis of biochemical reactions, antibiotic sensitivity, and complement fixation tests some isolates were the same microorganisms. Both patients with conjunctivitis were infected with the same NSU corynebacteria. A second isolate was cultured from patients with osteomyelitis and cervicitis, while a third was recovered from an infected leg wound and from a patient with pericarditis. Seven of the isolates, when injected into rabbits hypersensitive to four NSU corynebacteria isolated from the inflamed epididymis of patients with epididymitis, elicited delayed hypersensitivity reactions, which indicated that they also were related antigenically. It is suggested that nonspecific urethritis and eididymitis may represent an infection with NSU corynebacteria, or may be an extension of bacteriuria due to these microorganisms, with a delayed hypersensitivity reaction as a possible additional complication. Colony counts on NSU chocolate agar of the bacteria in urines from male and female patients were higher than those obtained on conventional agar media. NSU chocolate agar is superior to other agar media for the isolation of pathogenic and saprophytic bacteria not only from the urogenital tract but also from other foci of infection. It is easily prepared from commercial blood agar plates and its use should be considered when a selective medium is not required.

An evidential example of airborne bacteria in a crowded, underground public concourse in Tokyo

NASA Astrophysics Data System (ADS)

Seino, Kaoruko; Takano, Takehito; Nakamura, Keiko; Watanabe, Masafumi

2005-01-01

We examined airborne bacteria in an underground concourse in Tokyo and investigated conditions that influenced bacterial counts. Airborne bacteria were collected by using an impactor sampler. Colonies on plate count agar (PCA) and Columbia colistin-nalidixic acid agar with 5% sheep blood (CNA agar) were enumerated. The range, geometric mean, and 95% CI of the bacterial counts (CFU m-3) on PCA and CNA agar were 150-1380, 456, 382-550 and 50-990, 237, 182-309, respectively. Bacterial counts on PCA significantly correlated with number of the pedestrians (r=0.89), relative humidity (r=0.70) and airborne dust (PM5.0) (r=0.73). Results of a multiple regression indicated independent positive association between the number of pedestrians and bacterial counts on PCA (p<0.01) after excluding the influence of relative humidity and airborne dust. Similar results were obtained with the statistical analysis for the counts of bacteria on CNA agar. Gram-positive cocci were dominant on PCA and CNA agar. Staphylococcus epidermidis and Micrococcus spp. were dominant among the 11 genera and 19 species identified in the present study. Considering the pattern of identified species and the significant independent association between number of pedestrians and bacterial counts, airborne bacteria in a crowded underground concourse were mostly originated from the pedestrians who were walking in the underground concourse. This study gave an evidential example of bacterial conditions in the air of an underground crowded public space in Tokyo.

Establishment of HPC(R2A) for regrowth control in non-chlorinated distribution systems.

PubMed

Uhl, Wolfgang; Schaule, Gabriela

2004-05-01

Drinking water distributed without disinfection and without regrowth problems for many years may show bacterial regrowth when the residence time and/or temperature in the distribution system increases or when substrate and/or bacterial concentration in the treated water increases. An example of a regrowth event in a major German city is discussed. Regrowth of HPC bacteria occurred unexpectedly at the end of a very hot summer. No pathogenic or potentially pathogenic bacteria were identified. Increased residence times in the distribution system and temperatures up to 25 degrees C were identified as most probable causes and the regrowth event was successfully overcome by changing flow regimes and decreasing residence times. Standard plate counts of HPC bacteria using the spread plate technique on nutrient rich agar according to German Drinking Water Regulations (GDWR) had proven to be a very good indicator of hygienically safe drinking water and to demonstrate the effectiveness of water treatment. However, the method proved insensitive for early regrowth detection. Regrowth experiments in the lab and sampling of the distribution system during two summers showed that spread plate counts on nutrient-poor R2A agar after 7-day incubation yielded 100 to 200 times higher counts. Counts on R2A after 3-day incubation were three times less than after 7 days. As the precision of plate count methods is very poor for counts less than 10 cfu/plate, a method yielding higher counts is better suited to detect upcoming regrowth than a method yielding low counts. It is shown that for the identification of regrowth events HPC(R2A) gives a further margin of about 2 weeks for reaction before HPC(GDWR). Copyright 2003 Elsevier B.V.

Preparation and screening of an arrayed human genomic library generated with the P1 cloning system.

PubMed Central

Shepherd, N S; Pfrogner, B D; Coulby, J N; Ackerman, S L; Vaidyanathan, G; Sauer, R H; Balkenhol, T C; Sternberg, N

1994-01-01

We describe here the construction and initial characterization of a 3-fold coverage genomic library of the human haploid genome that was prepared using the bacteriophage P1 cloning system. The cloned DNA inserts were produced by size fractionation of a Sau3AI partial digest of high molecular weight genomic DNA isolated from primary cells of human foreskin fibroblasts. The inserts were cloned into the pAd10sacBII vector and packaged in vitro into P1 phage. These were used to generate recombinant bacterial clones, each of which was picked robotically from an agar plate into a well of a 96-well microtiter dish, grown overnight, and stored at -70 degrees C. The resulting library, designated DMPC-HFF#1 series A, consists of approximately 130,000-140,000 recombinant clones that were stored in 1500 microtiter dishes. To screen the library, clones were combined in a pooling strategy and specific loci were identified by PCR analysis. On average, the library contains two or three different clones for each locus screened. To date we have identified a total of 17 clones containing the hypoxanthine-guanine phosphoribosyltransferase, human serum albumin-human alpha-fetoprotein, p53, cyclooxygenase I, human apurinic endonuclease, beta-polymerase, and DNA ligase I genes. The cloned inserts average 80 kb in size and range from 70 to 95 kb, with one 49-kb insert and one 62-kb insert. Images PMID:8146166

Microbiological analysis of debris from STS-42 IML-1 by direct plating of rinse waters

NASA Technical Reports Server (NTRS)

Smithers, G. A.

1992-01-01

Microbial analysis of air filter debris from the Spacelab International Microgravity Laboratory-1 (IML-1) mission was performed via direct plating of rinse waters on a battery of selective and nonselective nutrient agars. Microbial isolates were identified using Minitek and Biolog technologies. Twenty-four types of bacteria were recovered and classified; a similar number of fungal types was observed, but these were not identified. This procedure can provide information about the proportions of organism types present at the time of debris collection.

Identification of Tumor Rejection Antigens for Breast Cancer Using a Mouse Tumor Rejection Model

DTIC Science & Technology

2009-05-01

plaques were randomly picked and PCR with T3 and T7 primer was done to validate the cDNA insert. The inserts ranged from 500 to 3,500 bp. A...modifications. Briefly, 5 103 phage clones were plated with XL-Blue on NZY agar plates. After 4 hours of incubation at 37jC, isopropyl-L-thio-h-D...using XLOLR cells and ExAssist helper phage (Stratagene). Plasmid DNA was prepared using a FastPlasmid kit (Eppendorf, Hamburg, Germany). The nucleotide

[Effects of combined application of culture supernatant of human umbilical cord mesenchymal stem cells and ciprofloxacin on Staphylococcus aureus in vitro].

PubMed

Zhou, B; Tu, H L; Ba, T; Wang, L F; Wang, S J; Nie, S Y

2017-06-20

Objective: To explore the effects of combined application of culture supernatant of human umbilical cord mesenchymal stem cells (hUCMSCs) and ciprofloxacin on Staphylococcus aureus (SA) in vitro. Methods: hUCMSCs were isolated from umbilical cord tissue of full-term healthy fetus after cesarean section and cultured. Cells in the third passage were used in the experiments after identification. SA strains isolated from wounds of burn patients in our burn wards were used in the experiments. Cells were divided into 0, 10, 100, and 1 000 ng/mL lipopolysaccharide (LPS) groups according to the random number table (the same dividing method below). Cells were cultured with culture medium of mesenchymal stem cells (MSCs) after being treated with medium containing the corresponding mass concentrations of LPS for 12 h. At post culture hour (PCH) 6, 12, and 24, 6 wells of culture supernatant of cells in each group were obtained to measure the content of LL-37 with enzyme-linked immunosorbent assay. Ninety blood agar plates were divided into ciprofloxacin control group (CC), ciprofloxacin+ supernatant group (CS), and ciprofloxacin+ supernatant+ LL-37 antibody group (CSL), with 30 blood agar plates in each group. Blood agar plates in group CC were coated with 1.5×10(8) colony forming unit (CFU)/mL bacteria solution prepared with normal saline. Blood agar plates in group CS were coated with 1.5×10(8) CFU/mL bacteria solution prepared with normal saline and culture supernatant of hUCMSCs (cultured by culture medium of MSCs, the same below) in double volume of normal saline. Blood agar plates in group CSL were coated with 1.5×10(8) CFU/mL bacteria solution prepared with normal saline, culture supernatant of hUCMSCs in double volume of normal saline, and 2.6 μL LL-37 antibody in the concentration of 2 μg/mL. At PCH 12, 24, and 48, 10 blood agar plates of each group were harvested to observe the distribution of SA colony on blood agar plate and to measure the diameter of bacterial inhibition ring of ciprofloxacin. The minimum inhibitory concentration (MIC) of ciprofloxacin against SA of each group was recorded. Fractional inhibitory concentration (FIC) indexes of ciprofloxacin in groups CS and CSL at PCH 12, 24, and 48 were calculated, and the effect of synergy was evaluated. Data were processed with analysis of variance of factorial design, one-way analysis of variance, LSD- t test, Kruskal-Wallis test, and Mann-Whitney U test. Results: (1) At each PCH, the content of LL-37 in culture supernatant of cells in 10, 100, and 1 000 ng/mL LPS groups was higher than that in 0 ng/mL LPS group (with t values from 11.22 to 33.36, P values below 0.01); the content of LL-37 in culture supernatant of cells in 100 and 1 000 ng/mL LPS groups was higher than that in 10 ng/mL LPS group (with t values from 2.24 to 18.73, P <0.05 or P <0.01); the content of LL-37 in culture supernatant of cells in 1 000 ng/mL LPS group was higher than that in 100 ng/mL LPS group (with t values from 12.46 to 14.70, P values below 0.01). (2) At PCH 12, 24, and 48, the bacterial colonies in groups CC, CS, and CSL began to integrate over time. At PCH 12, 24, and 48, the diameters of bacterial inhibition ring of ciprofloxacin in group CC were 26, 24, and 23 mm, respectively, with no obvious change. At PCH 12, 24, and 48, the diameters of bacterial inhibition ring of ciprofloxacin in groups CS and CSL were 82, 71, 68 mm, and 74, 59, 56 mm, respectively, significantly longer than those of group CC. (3) At each PCH, the MIC of ciprofloxacin against SA was significantly higher in group CC than in groups CS and CSL (with Z values from 6.22 to 6.71, P values below 0.01); the MIC of ciprofloxacin against SA was significantly higher in group CSL than in group CS (with Z values all equal to 6.72, P values below 0.01). (4) FIC indexes of ciprofloxacin in groups CS and CSL at PCH 12, 24, and 48 were 0.011, 0.032, 0.032, and 0.122, 0.350, 0.350, respectively. The results indicated that culture supernatant of hUCMSCs had synergistically antibacterial effect on ciprofloxacin. Conclusions: hUCMSCs can secrete LL-37, and the secretion level is increased with increase of LPS concentration. Combination of culture supernatant of hUCMSCs and ciprofloxacin can decrease the dosage of ciprofloxacin in resisting SA. Once LL-37 is neutralized, the synergistically antibacterial effect of culture supernatant of hUCMSCs is decreased.

Plant-based culture media: Efficiently support culturing rhizobacteria and correctly mirror their in-situ diversity.

PubMed

Youssef, Hanan H; Hamza, Mervat A; Fayez, Mohamed; Mourad, Elhussein F; Saleh, Mohamed Y; Sarhan, Mohamed S; Suker, Ragab M; Eltahlawy, Asmaa A; Nemr, Rahma A; El-Tahan, Mahmod; Ruppel, Silke; Hegazi, Nabil A

2016-03-01

Our previous publications and the data presented here provide evidences on the ability of plant-based culture media to optimize the cultivability of rhizobacteria and to support their recovery from plant-soil environments. Compared to the tested chemically-synthetic culture media (e.g. nutrient agar and N-deficient combined-carbon sources media), slurry homogenates, crude saps, juices and powders of cactus (Opuntia ficus-indica) and succulent plants (Aloe vera and Aloe arborescens) were rich enough to support growth of rhizobacteria. Representative isolates of Enterobacter spp., Klebsiella spp., Bacillus spp. and Azospirillum spp. exhibited good growth on agar plates of such plant-based culture media. Cell growth and biomass production in liquid batch cultures were comparable to those reported with the synthetic culture media. In addition, the tested plant-based culture media efficiently recovered populations of rhizobacteria associated to plant roots. Culturable populations of >10(6)-10(8) cfu g(-1) were recovered from the ecto- and endo-rhizospheres of tested host plants. More than 100 endophytic culture-dependent isolates were secured and subjected to morphophysiological identification. Factor and cluster analyses indicated the unique community structure, on species, genera, class and phyla levels, of the culturable population recovered with plant-based culture media, being distinct from that obtained with the chemically-synthetic culture media. Proteobacteria were the dominant (78.8%) on plant-based agar culture medium compared to only 31% on nutrient agar, while Firmicutes prevailed on nutrient agar (69%) compared to the plant-based agar culture media (18.2%). Bacteroidetes, represented by Chryseobacterium indologenes, was only reported (3%) among the culturable rhizobacteria community of the plant-based agar culture medium.

Plant-based culture media: Efficiently support culturing rhizobacteria and correctly mirror their in-situ diversity

PubMed Central

Youssef, Hanan H.; Hamza, Mervat A.; Fayez, Mohamed; Mourad, Elhussein F.; Saleh, Mohamed Y.; Sarhan, Mohamed S.; Suker, Ragab M.; Eltahlawy, Asmaa A.; Nemr, Rahma A.; El-Tahan, Mahmod; Ruppel, Silke; Hegazi, Nabil A.

2015-01-01

Our previous publications and the data presented here provide evidences on the ability of plant-based culture media to optimize the cultivability of rhizobacteria and to support their recovery from plant-soil environments. Compared to the tested chemically-synthetic culture media (e.g. nutrient agar and N-deficient combined-carbon sources media), slurry homogenates, crude saps, juices and powders of cactus (Opuntia ficus-indica) and succulent plants (Aloe vera and Aloe arborescens) were rich enough to support growth of rhizobacteria. Representative isolates of Enterobacter spp., Klebsiella spp., Bacillus spp. and Azospirillum spp. exhibited good growth on agar plates of such plant-based culture media. Cell growth and biomass production in liquid batch cultures were comparable to those reported with the synthetic culture media. In addition, the tested plant-based culture media efficiently recovered populations of rhizobacteria associated to plant roots. Culturable populations of >106–108 cfu g−1 were recovered from the ecto- and endo-rhizospheres of tested host plants. More than 100 endophytic culture-dependent isolates were secured and subjected to morphophysiological identification. Factor and cluster analyses indicated the unique community structure, on species, genera, class and phyla levels, of the culturable population recovered with plant-based culture media, being distinct from that obtained with the chemically-synthetic culture media. Proteobacteria were the dominant (78.8%) on plant-based agar culture medium compared to only 31% on nutrient agar, while Firmicutes prevailed on nutrient agar (69%) compared to the plant-based agar culture media (18.2%). Bacteroidetes, represented by Chryseobacterium indologenes, was only reported (3%) among the culturable rhizobacteria community of the plant-based agar culture medium. PMID:26966571

A comparative study of biofilm formation by Shiga toxigenic Escherichia coli using epifluorescence microscopy on stainless steel and a microtitre plate method.

PubMed

Rivas, Lucia; Dykes, Gary A; Fegan, Narelle

2007-04-01

Attachment of Shiga toxigenic Escherichia coli (STEC) to surfaces and the formation of biofilms may enhance persistence in a food processing environment and present a risk of contaminating products. Seven strains of STEC and three non-STEC strains were selected to compare two biofilm quantification methods; epifluorescence microscopy on stainless steel (SS) and a microtitre plate assay. The influence of prior growth in planktonic (nutrient broth) and sessile (nutrient agar) culture on biofilm production, as well as expression of surface structures and the possession of antigen 43 (encoded by agn43) on biofilm formation were also investigated. Biofilms were produced in diluted nutrient broth at 25 degrees C for 24 and 48 h. Curli expression was determined using congo red indicator agar, while the presence of agn43 was determined using polymerase chain reaction. No correlation was found between counts for epifluorescence microscopy on SS and the absorbance values obtained with the microtitre plate method for planktonic and sessile grown cultures. Different abilities of individual STEC strains to attach to SS and microtitre plates were found with some strains attaching better to each surface following growth in either planktonic or sessile culture. All O157 STEC strains had low biofilm counts on SS for planktonic and sessile grown cultures; however, one STEC O157:H- strain (EC516) had significantly greater (p<0.05) biofilm production on microtitre plates compared to the other O157 STEC strains. EC516 and other STEC (O174:H21 and O91:H21) strains expressing curli fimbriae were found to produce significantly greater (p<0.05) biofilms on microtitre plates compared to the non-curli expressing strains. No relationship was found between the production of type-I fimbriae, motility, agn43 and bacterial physicochemical properties (previously determined) and biofilm formation on SS or microtitre plates. Variations between the two biofilm determination methods may suggest that the biofilm production on microtitre plates may not be appropriate to represent other surfaces such as SS and that caution should be taken when selecting a method to quantify biofilm production on a surface.

Species Identification and Antibiotic Susceptibilities of Coagulase- Negative Staphylococci Isolated from Urinary Tract Infection Specimens.

PubMed

Hashmi, Asra; Abdullah, Farhan Essa; Abdullah, Nihal Essa; Kazmi, Shahana Urooj

2016-07-01

To determine the incidence of Coagulase- negative S. aureusin urinary tract infections and sensitivities of these isolates to antimicrobial agents. Cohort study. Dr. Essa Laboratory and Immunology and Infectious Disease Research Laboratory (IIDRL), Microbiology Department, University of Karachi, from January 2009 to January 2010. Urine specimens, suggestive of urinary tract infection (UTI), were identified. Speciation of isolates was done using API-20 Staph.system. Screening of extracellular products was done using SDS-PAGE electrophoresis and Hemolysin on blood-agar plates. Minimum inhibitory concentration (MICs) of antibiotics was estimated by microtiter well plate method. Frequency and percentages were determined and chi-square test was used for comparing proportions with significance at p < 0.05. Coagulase - negative S. aureus(CONS) were the cause of urinary tract infection in 56 out of 1866 outpatient (3%) and 164 of 1261 inpatient (13%), urinary tract infections (p < 0.001). Two hundred and twenty CONS isolates were identified. The most common CONS identified was S. saprophyticus (31%, 68 strains). The relative frequency of Coagulase - negative S. aureuswas 6% (13 strains). All isolates were sensitive to Vancomycin and Linezolid. Resistance was 69% to Ampicillin, 53% to Methicillin, and 37.5% to Ciprofloxacin. CONS are a potential uropathogens, with capability of slime production and resistance to common empirical prescriptions. This also warrants formulation of an appropriate antibiotic policy that covers CONS.

21 CFR 610.30 - Test for Mycoplasma.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Test for Mycoplasma. 610.30 Section 610.30 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS... inoculated in evenly distributed amounts over the surface of no less than 10 plates of at least two agar...

21 CFR 610.30 - Test for Mycoplasma.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Test for Mycoplasma. 610.30 Section 610.30 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS... inoculated in evenly distributed amounts over the surface of no less than 10 plates of at least two agar...

Anthrax

DTIC Science & Technology

2009-01-01

antimicrobials listed below) Initial therapye (intravenous dosing) Oral dosing Initial therapy (intravenous) IV treatment initially Switch to oral...high index of suspicion for anthrax, initiation of therapy should not be delayed for results of these confirmatory tests. 1. MICROBIOLOGICAL TESTS...Sputum specimens from inhalational anthrax patients should be plated on chocolate agar, SBA, and MAC. Cultures will show isolated B. anthracis

Fluorogenic membrane overlays to enumerate total coliforms, Escherichia coli, and total Vibrionaceae in shellfish and seawater

USDA-ARS?s Scientific Manuscript database

Three assays were developed to enumerate total coliforms, Escherichia coli, and total Vibrionaceae in shellfish and other foods and in seawater and other environmental samples. Assays involve membrane overlays of overnight colonies on non-selective agar plates to detect ß-glucuronidase and lysyl am...

Directional movement of entomopathogenic nematodes in response to electrical fields: Effects of species, magnitude of voltage, and infective juvenile age

USDA-ARS?s Scientific Manuscript database

Entomopathogenic nematodes respond to a variety of stimuli when foraging. Previously, we reported a directional response to electrical fields for two entomopathogenic nematode species; specifically, when electrical fields were generated on agar plates Steinernema glaseri (a nematode that utilizes a...

Automatic counting and classification of bacterial colonies using hyperspectral imaging

USDA-ARS?s Scientific Manuscript database

Detection and counting of bacterial colonies on agar plates is a routine microbiology practice to get a rough estimate of the number of viable cells in a sample. There have been a variety of different automatic colony counting systems and software algorithms mainly based on color or gray-scale pictu...

Three Strains of Pseudomonas fluorescens Exhibit Differential Toxicity Against Drosophila melanogaster

USDA-ARS?s Scientific Manuscript database

Three strains of Pseudomonas fluorescens were tested for toxicity to Drosophila melanogaster in an insect feeding assay. Insect eggs were placed on the surface of a non-nutritive agar plate supplemented with a food source that was non-inoculated or inoculated with P. fluorescens Pf0-1, SBW25, or Pf-...

Phytophthora species associated with tanoak stem cankers in southwestern Oregon

Treesearch

Paul Reeser; Wendy Sutton; Everett Hansen

2009-01-01

From 2001 through 2006 stem cankers on tanoak (Lithocarpus densiflorus) were sampled during surveys to detect and eradicate Phytophthora ramorum from forests in southwestern Oregon. Pieces of bark from stem canker margins were plated on cornmeal agar amended with 10 ppm natamycin, 200 ppm Na-ampicillin, and 10 ppm rifampicin....

DETECTION OF BACTERIAL CYTOTOXIC ACTIVITIES FROM WATER-DAMAGED CEILING TILE MATERIAL FOLLOWING INCUBATION ON BLOOD AGAR

EPA Science Inventory

Samples of ceiling tiles with high levels of bacteria exhibited cytotoxic activities on a HEP-2 tissue culture assay. Ceiling tiles containing low levels of bacterial colonization did not show cytotoxic activities on the HEP-2 tissue culture assay. Using a spread plate procedure ...

Seed mycoflora of Ephedra aphylla and amino acid profile of seed-borne Aspergillus flavus.

PubMed

Al-Qarawi, Abdulaziz A; Hashem, Abeer; Abd-Allah, Elsayed F

2012-09-01

Twenty-seven seed samples of Ephedra aphylla were collected from different rangelands in Riyadh region, Saudi Arabia during seed production season of 2010. They were assessed to determine the incidence of seedborne fungal flora using both agar plate and blotter paper methods. The investigation of the seeds yielded thirty four fungal species belonging to twelve genera, which are new record to seed-brone mycoflora of E. aphylla in Saudi Arabia. The agar plate method was found superior over blotter methods. The genus Aspergillus was the most prevalent one followed by Fusarium, Penicillium, Alternaria, and Chaetomium. Only eighteen isolates of A. flavus (∼ 28.6% of total isolates) were able to produce aflatoxins. Mycelial amino acids profile of selected aflatoxigenic isolates of A. flavus was investigated and five amino acids, namely cystein, lysine, praline, tryptophan and valine were common in mycelia and all of them were aflatoxins producers. Based on the dissimilarity coefficient between the isolates and their amino acids patterns, high diversity among the population of A. flavus has been recorded.

Growth inhibition of heat-injured Enterococcus faecium by oligophosphates in a cured meat model.

PubMed

Houben, J H; Tjeerdsma-van Bokhoven, J L M

2004-12-01

Cells of two heat-resistant strains of Enterococcus faecium were heated and incubated in meat suspensions containing curing ingredients. The concentrations of the curing ingredients were those frequently used for pasteurized ham-type products, except that the concentrations of the oligophosphates (triphosphate and diphosphate) varied. Heating tests at 69 degrees C were performed with inoculated meat suspensions in heat-sealed plastic pouches. Numbers of bacteria were counted immediately after heating and in parallel series of heated pouches incubated at 37 degrees C. Plating was performed in Tryptone Dextrose Yeast Meat Peptonised Milk Agar (TDYMP); in TDYMP Agar to which the curing ingredients were added; and in TDYMP Agar to which the curing ingredients except oligophosphates were added. The inclusion of oligophosphates in the heating medium increased the heat-injury sustained by the E. faecium cells, and in combination with rather severe heat treatment even completely blocked the growth of surviving organisms in the meat suspension incubated at 37 degrees C. The presence of oligophosphates in the culture medium TDYMP Agar severely reduced the counts of freshly heated cells; however, this effect disappeared after repair and growth of the surviving organisms in the meat suspension.

High-throughput measurements of biochemical responses using the plate::vision multimode 96 minilens array reader.

PubMed

Huang, Kuo-Sen; Mark, David; Gandenberger, Frank Ulrich

2006-01-01

The plate::vision is a high-throughput multimode reader capable of reading absorbance, fluorescence, fluorescence polarization, time-resolved fluorescence, and luminescence. Its performance has been shown to be quite comparable with other readers. When the reader is integrated into the plate::explorer, an ultrahigh-throughput screening system with event-driven software and parallel plate-handling devices, it becomes possible to run complicated assays with kinetic readouts in high-density microtiter plate formats for high-throughput screening. For the past 5 years, we have used the plate::vision and the plate::explorer to run screens and have generated more than 30 million data points. Their throughput, performance, and robustness have speeded up our drug discovery process greatly.

Improved quantitative recovery of Listeria monocytogenes from stainless steel surfaces using a one-ply composite tissue.

PubMed

Vorst, Keith L; Todd, Ewen C D; Rysert, Elliot T

2004-10-01

Four sampling devices, a sterile environmental sponge (ES), a sterile cotton-tipped swab (CS), a sterile calcium alginate fiber-tipped swab (CAS), and a one-ply composite tissue (CT), were evaluated for quantitative recovery of Listeria monocytogenes from a food-grade stainless steel surface. Sterile 304-grade stainless steel plates (6 by 6 cm) were inoculated with approximately 106 CFU/cm2 L. monocytogenes strain Scott A and dried for 1 h. The ES and CT sampling devices were rehydrated in phosphate buffer solution. After plate swabbing, ES and CT were placed in 40 ml of phosphate buffer solution, stomached for 1 min and hand massaged for 30 s. Each CS and CAS device was rehydrated in 0.1% peptone before swabbing. After swabbing, CS and CAS were vortexed in 0.1% peptone for 1 min. Samples were spiral plated on modified Oxford agar with modified Oxford agar Rodac Contact plates used to recover any remaining cells from the stainless steel surface. Potential inhibition from CT was examined in both phosphate buffer solution and in a modified disc-diffusion assay. Recovery was 2.70, 1.34, and 0.62 log greater using CT compared with ES, CS, and CAS, respectively, with these differences statistically significant (P < 0.001) for ES and CT and for CAS, CS, and CT (P < 0.05). Rodac plates were typically overgrown following ES, positive after CS and CAS, and negative after CT sampling. CT was noninhibitory in both phosphate buffer solution and the modified disc-diffusion assay. Using scanning electron microscopy, Listeria cells were observed on stainless steel plates sampled with each sampling device except CT. The CT device, which is inexpensive and easy to use, represents a major improvement over other methods in quantifying L. monocytogenes on stainless steel surfaces and is likely applicable to enrichment of environmental samples.

Comparative evaluation of three chromogenic media combined with broth enrichment and the real-time PCR-based Xpert MRSA assay for screening of methicillin-resistant Staphylococcus aureus in nasal swabs.

PubMed

Lee, Seungok; Park, Yeon-Joon; Park, Kang-Gyun; Jekarl, Dong Wook; Chae, Hyojin; Yoo, Jin-Kyung; Seo, Sin Won; Choi, Jung Eun; Lim, Jung Hye; Heo, Seon Mi; Seo, Ju Hee

2013-07-01

We evaluated the performance of three chromogenic media (Brilliance agar I [Oxoid, UK], Brilliance agar II [Oxoid], and ChromID MRSA [Biomérieux, France]) combined with broth enrichment and the Xpert MRSA assay for screening of methicillin-resistant Staphylococcus aureus (MRSA). We obtained 401 pairs of duplicate nasal swabs from 321 patients. One swab was suspended overnight in tryptic soy broth; 50-µL aliquots of suspension were inoculated on the three chromogenic media. Brilliance agar I and II were examined after 24 hr, and ChromID MRSA, after 24 and 48 hr. The paired swab was processed directly using real-time PCR-based Xpert MRSA assay. True positives, designated as MRSA growth in any of the culture media, were detected with the prevalence of 17% in our institution. We report the sensitivity, specificity, positive predictive value, and negative predictive value of MRSA growth as follows: 92.3%, 94.0%, 75.9%, and 98.4% in Brilliance agar I (24 hr); 92.7%, 97.9%, 90.0%, and 98.5% in Brilliance agar II (24 hr); 95.6%, 95.8%, 82.3%, and 99.1% in ChromID MRSA (24 hr); 100%, 92.5%, 73.1%, and 100% in ChromID MRSA (48 hr); 92.6%, 96.7%, 85.1%, and 98.5% in Xpert MRSA assay. The agreement between the enriched culture and Xpert MRSA assay was 96.0%. Three chromogenic culture media combined with enrichment and Xpert MRSA assay demonstrated similar capabilities in MRSA detection. The Xpert MRSA assay yielded results comparable to those of culture methods, saving 48-72 hr, thus facilitating earlier detection of MRSA in healthcare settings.

Surprisal analysis of genome-wide transcript profiling identifies differentially expressed genes and pathways associated with four growth conditions in the microalga Chlamydomonas.

PubMed

Bogaert, Kenny A; Manoharan-Basil, Sheeba S; Perez, Emilie; Levine, Raphael D; Remacle, Francoise; Remacle, Claire

2018-01-01

The usual cultivation mode of the green microalga Chlamydomonas is liquid medium and light. However, the microalga can also be grown on agar plates and in darkness. Our aim is to analyze and compare gene expression of cells cultivated in these different conditions. For that purpose, RNA-seq data are obtained from Chlamydomonas samples of two different labs grown in four environmental conditions (agar@light, agar@dark, liquid@light, liquid@dark). The RNA seq data are analyzed by surprisal analysis, which allows the simultaneous meta-analysis of all the samples. First we identify a balance state, which defines a state where the expression levels are similar in all the samples irrespectively of their growth conditions, or lab origin. In addition our analysis identifies additional constraints needed to quantify the deviation with respect to the balance state. The first constraint differentiates the agar samples versus the liquid ones; the second constraint the dark samples versus the light ones. The two constraints are almost of equal importance. Pathways involved in stress responses are found in the agar phenotype while the liquid phenotype comprises ATP and NADH production pathways. Remodeling of membrane is suggested in the dark phenotype while photosynthetic pathways characterize the light phenotype. The same trends are also present when performing purely statistical analysis such as K-means clustering and differentially expressed genes.

Oxytetracycline-Resistant Coliforms in Commercial Poultry Products

PubMed Central

Corey, R. Reece; Byrnes, Joseph M.

1963-01-01

The presence of oxytetracycline-resistant bacteria was investigated with commercially frozen chicken thighs and drumsticks. Bacterial flora were surveyed by means of total and coliform counts with Tryptone Glucose Extract Agar and Desoxycholate Agar, respectively. After counting, the Desoxycholate Agar plates were replicated on the same medium containing 25, 50, 75, and 100 ppm of oxytetracycline. Resistant colonies were found on all samples that were replicated. Of 2613 colonies isolated on Desoxycholate Agar, 47.8% grew in the presence of 25 ppm of oxytetracycline. From 50 to 100 ppm, the number of resistant isolates remained essentially the same, near 34%. Of 812 colonies of antibiotic-resistant bacteria identified with dulcitol-lactose-iron-agar, 82.5% were paracolons, 13.7% were pseudomonads, and 3.8% were Escherichia or Aerobacter. Bacteria resistant to oxytetracycline were shown to be present on commercially processed chicken. The origin of the resistance to oxytetracycline was not established; however, since the antibiotic was not used during processing, it appeared that these antibiotic-resistant bacteria arose in the intestines of the chickens as a result of feed which contained antibiotic. This is supported by a comparison with the antibiotic resistance of coliforms from chickens raised on feed both with and without oxytetracycline, for the percentages of resistant colonies are similar in both commercial chicken and chicken raised on feed containing the antibiotic. PMID:14075046

Study of methods for the improvement of bacterial transport media

NASA Technical Reports Server (NTRS)

Gardner, R. L.; Beakley, J. W.

1973-01-01

A series of 500 transport media recipes was tested for ability to hold pure cultures of Streptococcus equisimilus, Corynebacterium equi, Neisseria perflava, and Haemophilus parainfluenzae for 21 days. Stuart Medium Base with 0.4% agar was used as the control medium for this and the other experiments in the investigation. At the end of the holding period inoculated transport media were quantitatively assayed, and the control media were assayed immediately after inoculation. Three vials of each medium were inoculated with an organism, and each vial's medium was diluted and spread on duplicate plates. Assay media for this experiment included Brain Heart Infusion,(BHIA) Tryptic Soy Agar, and BHIA with 1% Isovitalex enrichment.

Two-Piece Screens for Decontaminating Granular Material

NASA Technical Reports Server (NTRS)

Backes, Douglas; Poulter, Clay; Godfrey, Max; Dutton, Melinda; Tolman, Dennis

2009-01-01

Two-piece screens have been designed specifically for use in filtering a granular material to remove contaminant particles that are significantly wider or longer than are the desired granules. In the original application for which the twopiece screens were conceived, the granular material is ammonium perchlorate and the contaminant particles tend to be wires and other relatively long, rigid strands. The basic design of the twopiece screens can be adapted to other granular materials and contaminants by modifying critical dimensions to accommodate different grain and contaminant- particle sizes. A two-piece screen of this type consists mainly of (1) a top flat plate perforated with circular holes arranged in a hexagonal pattern and (2) a bottom plate that is also perforated with circular holes (but not in a pure hexagonal pattern) and is folded into an accordion structure. Fabrication of the bottom plate begins with drilling circular holes into a flat plate in a hexagonal pattern that is interrupted, at regular intervals, by parallel gaps. The plate is then folded into the accordion structure along the gaps. Because the folds are along the gaps, there are no holes at the peaks and valleys of the accordion screen. The top flat plate and the bottom accordion plate are secured within a metal frame. The resulting two-piece screen is placed at the bottom opening of a feed hopper containing the granular material to be filtered. Tests have shown that such long, rigid contaminant strands as wires readily can pass through a filter consisting of the flat screen alone and that the addition of the accordion screen below the flat screen greatly increases the effectiveness of removal of wires and other contaminant strands. Part of the reason for increased effectiveness is in the presentation of the contaminant to the filter surface. Testing has shown that wire type contamination will readily align itself parallel to the material direction flow. Since this direction of flow is nearly always perpendicular to the filter surface holes, the contamination is automatically aligned to pass through. The two-filter configuration reduces the likelihood that a given contaminant strand will be aligned with the flow of material by eliminating the perpendicular presentation angle. Thus, for wires of a certain diameter, a two-piece screen is 20 percent more effective than is the corresponding flat perforated plate alone, even if the holes in the flat plate are narrower. An accordion screen alone is similarly effective in catching contaminants, but lumps of agglomerated granules of the desired material often collect in the valleys and clog the screen. The addition of a flat screen above the accordion screen prevents clogging of the accordion screen. Flat wire screens have often been used to remove contaminants from granular materials, and are about as effective as are the corresponding perforated flat plates used alone.

Roles of curli, cellulose and BapA in Salmonella biofilm morphology studied by atomic force microscopy

PubMed Central

Jonas, Kristina; Tomenius, Henrik; Kader, Abdul; Normark, Staffan; Römling, Ute; Belova, Lyubov M; Melefors, Öjar

2007-01-01

Background Curli, cellulose and the cell surface protein BapA are matrix components in Salmonella biofilms. In this study we have investigated the roles of these components for the morphology of bacteria grown as colonies on agar plates and within a biofilm on submerged mica surfaces by applying atomic force microscopy (AFM) and light microscopy. Results AFM imaging was performed on colonies of Salmonella Typhimurium grown on agar plates for 24 h and on biofilms grown for 4, 8, 16 or 24 h on mica slides submerged in standing cultures. Our data show that in the wild type curli were visible as extracellular material on and between the cells and as fimbrial structures at the edges of biofilms grown for 16 h and 24 h. In contrast to the wild type, which formed a three-dimensional biofilm within 24 h, a curli mutant and a strain mutated in the global regulator CsgD were severely impaired in biofilm formation. A mutant in cellulose production retained some capability to form cell aggregates, but not a confluent biofilm. Extracellular matrix was observed in this mutant to almost the same extent as in the wild type. Overexpression of CsgD led to a much thicker and a more rapidly growing biofilm. Disruption of BapA altered neither colony and biofilm morphology nor the ability to form a biofilm within 24 h on the submerged surfaces. Besides curli, the expression of flagella and pili as well as changes in cell shape and cell size could be monitored in the growing biofilms. Conclusion Our work demonstrates that atomic force microscopy can efficiently be used as a tool to monitor the morphology of bacteria grown as colonies on agar plates or within biofilms formed in a liquid at high resolution. PMID:17650335

Performance of CHROMAGAR candida and BIGGY agar for identification of yeast species

PubMed Central

Yücesoy, Mine; Marol, Serhat

2003-01-01

Background The importance of identifying the pathogenic fungi rapidly has encouraged the development of differential media for the presumptive identification of yeasts. In this study two differential media, CHROMagar Candida and bismuth sulphite glucose glycine yeast agar, were evaluated for the presumptive identification of yeast species. Methods A total number of 270 yeast strains including 169 Candida albicans, 33 C. tropicalis, 24 C. glabrata, 18 C. parapsilosis, 12 C. krusei, 5 Trichosporon spp., 4 C. kefyr, 2 C. lusitaniae, 1 Saccharomyces cerevisiae and 1 Geotrichum candidum were included. The strains were first identified by germ tube test, morphological characteristics on cornmeal tween 80 agar and Vitek 32 and API 20 C AUX systems. In parallel, they were also streaked onto CHROMagar Candida and bismuth sulphite glucose glycine yeast agar plates. The results were read according to the color, morphology of the colonies and the existance of halo around them after 48 hours of incubation at 37°C. Results The sensitivity and specificity values for C. albicans strains were found to be 99.4, 100% for CHROMagar Candida and 87.0, 75.2% for BiGGY agar, respectively. The sensitivity of CHROMagar Candida to identify C. tropicalis, C. glabrata and C. krusei ranged between 90.9 and 100% while the specificity was 100%. The sensitivity rates for BiGGY agar were 66.6 and 100% while the specificity values were found to be 95.4 and 100% for C. tropicalis and C. krusei, respectively. Conclusions It can be concluded that the use of CHROMagar Candida is an easy and reliable method for the presumptive identification of most commonly isolated Candida species especially C. albicans, C. tropicalis and C. krusei. The lower sensitivity and specificity of BiGGY agar to identify commonly isolated Candida species potentially limits the clinical usefulness of this agar. PMID:14613587

Differential enumeration of subpopulations in concentrated frozen and lyophilized cultures of Lactobacillus delbrueckii ssp. bulgaricus.

PubMed

Shao, Yuyu; Wang, Zhaoxia; Bao, Qiuhua; Zhang, Heping

2017-11-01

Differential enumeration of subpopulations in concentrated frozen and lyophilized cultures of Lactobacillus delbrueckii ssp. bulgaricus ND02 derived from 2 propagation procedures was determined. The subpopulations consisted of 3 categories (physiological states): viable cells capable of forming colonies on agar plates (VC+), viable cells incapable of forming colonies on agar plates (VC-), widely referred to as viable but nonculturable (VBNC) cells, and nonviable or dead cells (NVC). Counts of VC+ were recorded using a conventional plate count procedure. A fluorescent vital staining procedure that discriminates between viable (VC+ and VC-) and NVC cells was used to determine the number of viable and nonviable cells. Both propagation procedures had 2 variables: in procedure (P)1, the propagation medium was rich in yeast extract (4.0%) and the pH was maintained at 5.7; in P2, the medium was devoid of yeast extract and the pH was maintained at 5.1. The results showed that post-propagation operations-concentration of cells by centrifugation and subsequent freezing or lyophilization of cell concentrate-induced different degrees of transience from VC+ to VC- states in cells derived from P1 and P2. Compared with cells derived from P2, cells from P1 were more labile to stress associated with centrifugation, freezing, and lyophilization, as revealed by differential counting. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

DEMONSTRATION OF INDUCED SYNERGISTIC HEMOLYSIS BY “NONHEMOLYTIC” STAPHYLOCOCCUS SPECIES1

PubMed Central

Smith, Doyle C.; Foltz, V. D.; Lord, T. H.

1964-01-01

Smith, Doyle C. (Kansas State University, Manhattan), V. D. Foltz, and T. H. Lord. Demonstration of induced synergistic hemolysis by “non-hemolytic” Staphylococcus species. J. Bacteriol. 87:188–195. 1964.—The synergistic hemolysis of “normally nonhemolytic” staphylococci on blood-agar incubated in, or adjacent to, a secondary zone of certain hemolytic staphylococci is described. The synergism apparently results from the combining of two factors, Z, produced by hemolytic staphylococci that elaborate a secondary zone, and T, produced by “nonhemolytic” staphylococci. The production of the two factors is substantiated by (i) the absence of clear hemolysis around nonhemolytic colonies and in the secondary zone of hemolytic colonies, and by (ii) evidence of clear hemolysis when the secondary zone reaches to within a few millimeters, and finally surrounds, the nonhemolytic colonies. It was possible to show on a blood-agar plate containing a mixture of both hemolytic and nonhemolytic cultures that all colonies exhibited a zone of clear hemolysis. When 400 colonies were selected from such a blood plate, 280 were nonhemolytic on subsequent studies. The remaining 120 colonies were hemolytic. Thus, there is danger of confusing non-hemolytic Staphylococcus colonies, showing induced hemolysis, with truly hemolytic colonies. If a similar situation occurred on a diagnostic blood plate, it could very likely result in failure to identify a possibly pathogenic staphylococcus. Images PMID:14102853

In Vitro Effect of Zingiber officinale Extract on Growth of Streptococcus mutans and Streptococcus sanguinis.

PubMed

Azizi, Arash; Aghayan, Shabnam; Zaker, Saeed; Shakeri, Mahdieh; Entezari, Navid; Lawaf, Shirin

2015-01-01

Background and Objectives. Tooth decay is an infectious disease of microbial origin. Considering the increasing prevalence of antibiotic resistance due to their overuse and also their side effects, medicinal plants are now considered for use against bacterial infections. This study aimed to assess the effects of different concentrations of Zingiber officinale extract on proliferation of Streptococcus mutans and Streptococcus sanguinis in vitro. Materials and Methods. In this experimental study, serial dilutions of the extract were prepared in two sets of 10 test tubes for each bacterium (total of 20). Standard amounts of bacterial suspension were added; 100ƛ of each tube was cultured on prepared solid agar plates and incubated at 37°C for 24 hours. Serial dilutions of the extract were prepared in another 20 tubes and 100ƛ of each tube was added to blood agar culture medium while being prepared. The mixture was transferred to the plates. The bacteria were inoculated on plates and incubated as described. Results. The minimum inhibitory concentration (MIC) was 0.02 mg/mL for S. mutans and 0.3 mg/mL for S. sanguinis. The minimum bactericidal concentration (MBC) was 0.04 mg for S. mutans and 0.6 mg for S. sanguinis. Conclusion. Zingiber officinale extract has significant antibacterial activity against S. mutans and S. sanguinis cariogenic microorganisms.

In Vitro Effect of Zingiber officinale Extract on Growth of Streptococcus mutans and Streptococcus sanguinis

PubMed Central

Azizi, Arash; Aghayan, Shabnam; Zaker, Saeed; Shakeri, Mahdieh; Entezari, Navid; Lawaf, Shirin

2015-01-01

Background and Objectives. Tooth decay is an infectious disease of microbial origin. Considering the increasing prevalence of antibiotic resistance due to their overuse and also their side effects, medicinal plants are now considered for use against bacterial infections. This study aimed to assess the effects of different concentrations of Zingiber officinale extract on proliferation of Streptococcus mutans and Streptococcus sanguinis in vitro. Materials and Methods. In this experimental study, serial dilutions of the extract were prepared in two sets of 10 test tubes for each bacterium (total of 20). Standard amounts of bacterial suspension were added; 100ƛ of each tube was cultured on prepared solid agar plates and incubated at 37°C for 24 hours. Serial dilutions of the extract were prepared in another 20 tubes and 100ƛ of each tube was added to blood agar culture medium while being prepared. The mixture was transferred to the plates. The bacteria were inoculated on plates and incubated as described. Results. The minimum inhibitory concentration (MIC) was 0.02 mg/mL for S. mutans and 0.3 mg/mL for S. sanguinis. The minimum bactericidal concentration (MBC) was 0.04 mg for S. mutans and 0.6 mg for S. sanguinis. Conclusion. Zingiber officinale extract has significant antibacterial activity against S. mutans and S. sanguinis cariogenic microorganisms. PMID:26347778

Investigation of microbial community isolated from indoor artworks and air environment: identification, biodegradative abilities, and DNA typing.

PubMed

Pangallo, Domenico; Chovanová, Katarina; Simonovicová, Alexandra; Ferianc, Peter

2009-03-01

This study deals with establishing the characteristics of a microbial community isolated from indoor artworks and the surrounding air environment. It is one of the few studies on microbial degradation of indoor artworks. It shows the potential biodegradative risk that can occur if artworks are not exhibited and conserved in an appropriate environment. The microbial community isolated from the indoor artworks and air environment was examined by cultural and molecular methods. Different plate assays were used to screen the biodegradative activity of the isolated microflora: Remazol Brilliant Blue R, phenol red, and Azure B for the ligninolytic properties; Ostazin brilliant red H-3B for cellulose degradation; CaCO3 glucose agar for solubilization activity; and B4 agar for biomineralization. To type the bacterial and fungal isolates, 2 PCR methods, repetitive extragenic palindromes (REP) and random amplified microsatellite polymorphisms (RAMP) were used. The art objects were principally colonized by fungi. The most commonly isolated strains were represented by hyphomycetes of the genera Penicillium, Aspergillus, Cladosporium, and Chaetomium. Members of these genera showed intensive biodegradation activity, both on wood and on stone. Bacteria were predominant in the air, exhibiting complex communities, both in the air and on the artworks. The most frequently isolated genera were Bacillus and Staphylococcus with extensive biodegradation abilities. REP-PCR revealed high variability within strains belonging to the same genus. RAMP is a new PCR-based method, used in this research for the first time to cluster the microfilamentous fungi and to characterize and select especially Penicillium and Aspergillus strains, which were isolated in a large number.

The Mediterranean non-indigenous ascidian Polyandrocarpa zorritensis: Microbiological accumulation capability and environmental implications.

PubMed

Stabili, Loredana; Licciano, Margherita; Longo, Caterina; Lezzi, Marco; Giangrande, Adriana

2015-12-15

We investigated the bacterial accumulation and digestion capability of Polyandrocarpa zorritensis, a non-indigenous colonial ascidian originally described in Peru and later found in the Mediterranean. Microbiological analyses were carried out on homogenates from "unstarved" and "starved" ascidians and seawater from the same sampling site (Adriatic Sea, Italy). Culturable heterotrophic bacteria (22 °C), total culturable bacteria (37 °C) and vibrios abundances were determined on Marine Agar 2216, Plate Count Agar and TCBS Agar, respectively. Microbial pollution indicators were measured by the most probable number method. All the examined microbiological groups were accumulated by ascidians but differently digested. An interesting outcome is the capability of P. zorritensis to digest allochthonous microorganisms such as coliforms as well as culturable bacteria at 37 °C, counteracting the effects of microbial pollution. Thus, the potential exploitation of these filter feeders to restore polluted seawater should be taken into consideration in the management of this alien species. Copyright © 2015 Elsevier Ltd. All rights reserved.

Light chain typing of immunoglobulins in small samples of biological material

PubMed Central

Rádl, J.

1970-01-01

A method is described for the typing of the light chains of immunoglobulins in small samples of sera or external secretions and without their previous isolation. It consists of immunoelectrophoresis in agar plates which contain specific antisera against one of the light chain types. All immunoglobulins of this type are thus selected by precipitation in the central area during the electrophoretic phase. Immunoglobulins of the opposite light chain type diffuse through the agar and react with the class specific antisera from the troughs. This results in the precipitin lines as in conventional immunoelectrophoresis. This technique has proved most useful for typing heterogenous or homogeneous immunoglobulins in normal and low concentration. The antisera used for incorporation in the agar should fulfil special requirements. They should contain a high level of antibodies against common surface determinants of the immunoglobulin light chains. The further possibilities of this immunoselection technique for typing different protein mixtures is discussed. ImagesFIG. 1FIG. 2FIG. 3FIG. 4FIG. 5FIG. 6 PMID:4098592

Mutations and Misconceptions: The Isolation and Study of Mutant Bacteria.

ERIC Educational Resources Information Center

Corner, Thomas R.

1992-01-01

Describes simple, inexpensive activities for teaching students about mutants and mutations in bacteria. Explains how to isolate bacteria from soil and leaves and how to grow bacteria on agar or in broth. Describes how to construct a gradient plate for finding the minimum inhibitory concentration of a substance and how to use this set up to find…

Fun Microbiology: Using a Plant Pathogenic Fungus To Demonstrate Koch's Postulates.

ERIC Educational Resources Information Center

Mitchell, James K.; Orsted, Kathy M.; Warnes, Carl E.

1997-01-01

Describes an experiment using a plant pathogenic fungus in which students learn to follow aseptic techniques, grow and produce spores of a fungus, use a hemacytometer for enumerating spores, prepare serial dilutions, grow and inoculate plants, isolate a pure culture using agar streak plates, and demonstrate the four steps of Koch's postulates.…

Selection for Spontaneous "Escherichia coli" Streptomycin Mutants Using Basic Fuchsin.

ERIC Educational Resources Information Center

Walkosz, Ronald

1991-01-01

An exercise that uses a common bacterium, E. coli, in great numbers, to detect a demonstrable change in the ability of some cells to become resistant to the common antibiotic streptomycin is presented. The procedure for preparing and pouring the gradient antibiotic plates is provided. The advantages of using the Basic Fuchsin in the agar are…

In vitro response of Heterobasidion annosum to manganese

Treesearch

W.J. Otrosina; B.L. Illman

1994-01-01

Manganese (Mn) is postulated to play a role in wood decay caused by certain basidiomycetes. We determined the in vitro response of Heterobasidion annosum (Fr.) Bref. to Mn and compared differences between three isolates each of the S and the P intersterility groups from the Western United States.On manganese-amended malt agar plates, H. annosum produced a brownish-...

Significance of Hemolytic Colonies in Throat Cultures

PubMed Central

Quinn, Robert W.; Lowry, P. Nye

1969-01-01

These studies indicate that a single strain of hemolytic streptococci almost exclusively predominates the bacterial flora in patients with streptococcal infections and in the carrier state. One can proceed with confidence that, in isolating streptococci from throat swabs cultured on blood-agar plates, only a single hemolytic colony need be picked for serological grouping and typing. PMID:4888863

Light scattering sensor for real-time identification of Vibrio parahaemolyticus, V. vulnificus and V. cholera colonies on solid agar plates

USDA-ARS?s Scientific Manuscript database

The three most common pathogenic species of Vibrio, V. cholerae, V. parahemolyticus and V. vulnificus, are of major concern as water- and food-borne pathogens because of an increasing incidence of water and seafood related outbreaks and illnesses worldwide. Current methods are time-consuming and req...

An HTS-compatible 3D colony formation assay to identify tumor-specific chemotherapeutics.

PubMed

Horman, Shane R; To, Jeremy; Orth, Anthony P

2013-12-01

There has been increasing interest in the development of cellular behavior models that take advantage of three-dimensional (3D) cell culture. To enable assessment of differential perturbagen impacts on cell growth in 2D and 3D, we have miniaturized and adapted for high-throughput screening (HTS) the soft agar colony formation assay, employing a laser-scanning cytometer to image and quantify multiple cell types simultaneously. The assay is HTS compatible, providing high-quality, image-based, replicable data for multiple, co-cultured cell types. As proof of concept, we subjected colorectal carcinoma colonies in 3D soft agar to a mini screen of 1528 natural product compounds. Hit compounds from the primary screen were rescreened in an HTS 3D co-culture matrix containing colon stromal cells and cancer cells. By combining tumor cells and normal, nontransformed colon epithelial cells in one primary screening assay, we were able to obtain differential IC50 data, thereby distinguishing tumor-specific compounds from general cytotoxic compounds. Moreover, we were able to identify compounds that antagonized tumor colony formation in 3D only, highlighting the importance of this assay in identifying agents that interfere with 3D tumor structural growth. This screening platform provides a fast, simple, and robust method for identification of tumor-specific agents in a biologically relevant microenvironment.

Evaluation of different methods to detect methicillin resistance in Staphylococcus aureus (MRSA).

PubMed

Alipour, Farzad; Ahmadi, Malahat; Javadi, Shahram

2014-01-01

The studies suggest that dogs living with human are potential risk of becoming MRSA carrier and increased risk of infections caused by MRSA. Phenotypic methods to detect methicillin resistance in Staphylococcus aureus (MRSA) are inadequate. The objective of the present study was to determine methicillin resistance in S. aureus by phenotypic susceptibility test (oxacillin disk diffusion, cefoxitin disk diffusion, oxacillin screen agar) and molecular methods (PCR as a gold standard) and the latex agglutination test for the detection of PBP2a and to evaluate the results of these tests for its sensitivity and specificity. A total of 100 swab samples were taken from muzzle site, in more contact with human, of dogs and MRSA were isolated. Oxacillin (1 μg), cefoxitin (30 μg) disk diffusion and oxacillin screen agar method were used. The isolates were also subjected to latex agglutination test for detection of PBP2a and PCR to detect mecA gene. By PCR 37% of isolates show the presence of mecA. Latex agglutination was found to be the most sensitive (97.29%) and cefoxitin disk diffusion to be the most specific (96.82%) tests for detection of MRSA. Our finding showed that combining oxacillin screen agar or cefoxitin disk diffusion with latex agglutination improves sensitivity and specificity to detect methicillin resistance S. aureus (MRSA) isolates. Copyright © 2014 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

Development of a Novel Screening Method for the Isolation of “Cronobacter” spp. (Enterobacter sakazakii)▿

PubMed Central

Iversen, Carol; Druggan, Patrick; Schumacher, Sandra; Lehner, Angelika; Feer, Claudia; Gschwend, Karl; Joosten, Han; Stephan, Roger

2008-01-01

A differential medium, “Cronobacter” screening broth, has been designed to complement agars based on hydrolysis of chromogenic α-glucopyranoside substrates. The broth was evaluated using 329 Enterobacteriaceae strains (229 target isolates), spiked/naturally contaminated samples, and a parallel comparison with current methods for raw materials, line/end products, and factory environment samples. PMID:18310415

The bacteriological screening of donated human milk: laboratory experience of British Paediatric Association's published guidelines.

PubMed

Wright, K C; Feeney, A M

1998-01-01

This study was undertaken to assess the application of the British Paediatric Association's (BPA) published guidelines to the bacteriological screening of breast milk donated to a District General Hospital milk bank. Samples of donated milk were subjected to bacterial counts and provisional identification after both 24 and 48 h incubation on cysteine lactose electrolyte-deficient (CLED) and Columbia blood agar. 21.8% (76 out of 348) donations of milk failed to reach the BPA acceptable criteria. The organisms responsible for the rejection of these samples were all evident within 24 h incubation, and were not significantly confined to one medium. A large percentage of rejected samples originated from a small number of donor mothers; 63.2% came from one donor. In applying BPA guidelines, both CLED and Columbia blood agar were found to be equally effective in screening for unacceptable organisms in prepasteurization donated breast milk. The 24 h period allowed for bacteriological screening, prior to pasteurization of milk samples, was sufficient to allow the growth of all potentially pathogenic bacteria in this study. To prevent the donation of consistently contaminated milk, more active communication between the milk bank staff and the donor is recommended.

Performance of chromogenic media for Candida in rapid presumptive identification of Candida species from clinical materials

PubMed Central

Pravin Charles, M. V.; Kali, Arunava; Joseph, Noyal Mariya

2015-01-01

Background: In perspective of the worldwide increase in a number of immunocompromised patients, the need for identification of Candida species has become a major concern. The development of chromogenic differential media, introduced recently, facilitate rapid speciation. However, it can be employed for routine mycology workup only after an exhaustive evaluation of its benefit and cost effectiveness. This study was undertaken to evaluate the benefit and cost effectiveness of chromogenic media for speciation of Candida clinical isolates. Materials and Methods: Sputum samples of 382 patients were screened for the presence of Candida spp. by Gram stain and culture on sabouraud dextrose agar. Candida species were identified using Gram stain morphology, germ tube formation, cornmeal agar with Tween-80, sugar fermentation tests and morphology on HiCrome Candida differential agar. All the Candida isolates were inoculated on HiCrome Candida agar (HiMedia, Mumbai, India). Results: The sensitivity and specificity of HiCrome agar for identification of Candida albicans were 90% and 96.42%, respectively whereas sensitivity and specificity of carbohydrate fermentation test were 86.67% and 74.07%, respectively. Sensitivity and specificity values of HiCrome agar for detection of C. albicans, Candida parapsilosis and Candida glabrata were above 90%. Conclusions: We found HiCrome agar has high sensitivity and specificity comparable to that of the conventional method. In addition, use of this differential media could significantly cut down the turnaround time as well as cost of sample processing. PMID:26109791

Performance of chromogenic media for Candida in rapid presumptive identification of Candida species from clinical materials.

PubMed

Pravin Charles, M V; Kali, Arunava; Joseph, Noyal Mariya

2015-06-01

In perspective of the worldwide increase in a number of immunocompromised patients, the need for identification of Candida species has become a major concern. The development of chromogenic differential media, introduced recently, facilitate rapid speciation. However, it can be employed for routine mycology workup only after an exhaustive evaluation of its benefit and cost effectiveness. This study was undertaken to evaluate the benefit and cost effectiveness of chromogenic media for speciation of Candida clinical isolates. Sputum samples of 382 patients were screened for the presence of Candida spp. by Gram stain and culture on sabouraud dextrose agar. Candida species were identified using Gram stain morphology, germ tube formation, cornmeal agar with Tween-80, sugar fermentation tests and morphology on HiCrome Candida differential agar. All the Candida isolates were inoculated on HiCrome Candida agar (HiMedia, Mumbai, India). The sensitivity and specificity of HiCrome agar for identification of Candida albicans were 90% and 96.42%, respectively whereas sensitivity and specificity of carbohydrate fermentation test were 86.67% and 74.07%, respectively. Sensitivity and specificity values of HiCrome agar for detection of C. albicans, Candida parapsilosis and Candida glabrata were above 90%. We found HiCrome agar has high sensitivity and specificity comparable to that of the conventional method. In addition, use of this differential media could significantly cut down the turnaround time as well as cost of sample processing.

Strain Library Imaging Protocol for high-throughput, automated single-cell microscopy of large bacterial collections arrayed on multiwell plates.

PubMed

Shi, Handuo; Colavin, Alexandre; Lee, Timothy K; Huang, Kerwyn Casey

2017-02-01

Single-cell microscopy is a powerful tool for studying gene functions using strain libraries, but it suffers from throughput limitations. Here we describe the Strain Library Imaging Protocol (SLIP), which is a high-throughput, automated microscopy workflow for large strain collections that requires minimal user involvement. SLIP involves transferring arrayed bacterial cultures from multiwell plates onto large agar pads using inexpensive replicator pins and automatically imaging the resulting single cells. The acquired images are subsequently reviewed and analyzed by custom MATLAB scripts that segment single-cell contours and extract quantitative metrics. SLIP yields rich data sets on cell morphology and gene expression that illustrate the function of certain genes and the connections among strains in a library. For a library arrayed on 96-well plates, image acquisition can be completed within 4 min per plate.

Isolation and Purification of Antibiotic Material from Physarum gyrosum

PubMed Central

Schroeder, H. R.; Mallette, M. F.

1973-01-01

The myxomycete Physarum gyrosum was cultured in its plasmodial stage on agar plates containing 0.025 M phosphate buffer at pH 6.5, 2% bakers' yeast, and 0.2% glucose and was supplemented with live Escherichia coli. Extracts of these plasmodia contained several antibiotic substances. Antibiotic materials were partially purified by dialysis of the agar medium-mold mixture, evaporation of the dialyzate, and butanol extraction of the residue. Further purification in two paper and two thin-layer chromatographic systems gave one product which was pure in six thin-layer chromatographic systems. Antibiotic activity against some gram-positive and gram-negative bacteria and yeasts was demonstrated with partially purified extracts and a paper-chromatographically separated fraction. One pure antibiotic was effective against Bacillus cereus. PMID:4799591

Proposed quality control guidelines for antimicrobial susceptibility tests using tilmicosin.

PubMed Central

Shryock, T R; White, D W; Werner, C S; Staples, J M

1995-01-01

Quality control guidelines for tilmicosin, a novel veterinary-use-only macrolide, were developed in a multi-laboratory study according to established National Committee for Clinical Laboratory Standards (NCCLS) procedures (M23-T2). Tilmicosin was incorporated into Sensititre plates for broth microdilution endpoint testing and into two lots of 15-micrograms disks for Kirby-Bauer agar disk diffusion testing. One common lot and five unique lots of Mueller-Hinton media were used. (Broth was cation adjusted, and agar was supplemented with 5% defibrinated sheep blood.) Bacteria used for reference strains included Pasteurella haemolytica 128K, Pasteurella multocida ATCC 43137, and Staphylococcus aureus ATCC 29213 (microdilution) and ATCC 25923 (disk). Replicate tests were conducted. Disk diffusion and broth microdilution quality control ranges are proposed. PMID:7714188

Environmental Isolation of Balamuthia mandrillaris Associated with a Case of Amebic Encephalitis

PubMed Central

Schuster, Frederick L.; Dunnebacke, Thelma H.; Booton, Gregory C.; Yagi, Shigeo; Kohlmeier, Candice K.; Glaser, Carol; Vugia, Duc; Bakardjiev, Anna; Azimi, Parvin; Maddux-Gonzalez, Mary; Martinez, A. Julio; Visvesvara, Govinda S.

2003-01-01

This report describes the first isolation of the ameba Balamuthia mandrillaris from an environmental soil sample associated with a fatal case of amebic encephalitis in a northern California child. Isolation of the ameba into culture from autopsied brain tissue confirmed the presence of Balamuthia. In trying to locate a possible source of infection, soil and water samples from the child's home and play areas were examined for the presence of Balamuthia. The environmental samples (plated onto nonnutrient agar with Escherichia coli as a food source) contained, in addition to the ameba, a variety of soil organisms, including other amebas, ciliates, fungi, and nematodes, as contaminants. Presumptive Balamuthia amebas were recognized only after cultures had been kept for several weeks, after they had burrowed into the agar. These were transferred through a succession of nonnutrient agar plates to eliminate fungal and other contaminants. In subsequent transfers, axenic Naegleria amebas and, later, tissue cultures (monkey kidney cells) served as the food source. Finally, the amebas were transferred to cell-free axenic medium. In vitro, the Balamuthia isolate is a slow-growing organism with a generation time of ∼30 h and produces populations of ∼2 × 105 amebas per ml. It was confirmed as Balamuthia by indirect immunofluorescence staining with rabbit anti-Balamuthia serum and human anti-Balamuthia antibody-containing serum from the amebic encephalitis patient. The environmental isolate is similar in its antimicrobial sensitivities and identical in its 16S ribosomal DNA sequences to the Balamuthia isolate from the deceased patient. PMID:12843060

Nematophagous fungi from decomposing cattle faeces in Argentina.

PubMed

Saumell, Carlos Alfredo; Fernández, Alicia Silvina; Fusé, Luis Alberto; Rodríguez, Manuela; Sagüés, María Federica; Iglesias, Lucía Emilia

2015-01-01

Biological control of gastrointestinal nematodes of ruminants by use of nematophagous fungi would become part of any livestock parasite integral control system. Identifying autochthonous species that could then be selected for mass production is an important phase in the practical use of biological control. To search for nematophagous fungi with potential use as biological control agents against gastrointestinal nematodes in Argentina. Decomposing cattle faeces sampled in different locations were incubated in water agar 2% with Panagrellus sp. The developed nematophagous fungi were transferred to new water agar 2% plates and then to corn meal agar plates in order to carry out their identification. Fungal diversity and richness were also assessed. Seventeen species from nine genera of nematophagous fungi were found. Twelve species were nematode-trapping fungi and three species plus two fungi identified to genus level corresponded to endoparasitic fungi. Arthrobotrys conoides, Arthrobotrys oligospora, Duddingtonia flagrans, Monacrosporium doedycoides, Arthrobotrys robusta and Drechmeria coniospora were the most frequently isolated species overall in the whole study (6.6%, 5.7%, 5.7%, 5.7%, 4.7% and 4.7%, respectively) although other species were more frequently recorded at local levels such as Arthrobotrys pyriformis (18.8%). Only A. conoides has been previously isolated from ruminant faecal samples in Argentina. Five nematode-trapping fungal species are mentioned for the first time in the Americas D. flagrans and A. conoides, both identified in the present study, are among the most promising ones as biological control agents against gastrointestinal nematodes of ruminants. Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

Screening the Biosphere: The Fungicolous Fungus Trichoderma phellinicola, a Prolific Source of Hypophellins, New 17-, 18-, 19-, and 20-Residue Peptaibiotics1)

PubMed Central

Röhrich, Christian René; Iversen, Anita; Jaklitsch, Walter Michael; Voglmayr, Hermann; Vilcinskas, Andreas; Nielsen, Kristian Fog; Thrane, Ulf; von Döhren, Hans; Brückner, Hans; Degenkolb, Thomas

2013-01-01

To investigate the significance of antibiotics for the producing organism(s) in the natural habitat, we screened a specimen of the fungicolous fungus Trichoderma phellinicola (syn. Hypocrea phellinicola) growing on its natural host Phellinus ferruginosus. Results revealed that a particular group of non-ribosomal antibiotic polypeptides, peptaibiotics, which contain the non-proteinogenic marker amino acid, α-aminoisobutyric acid, was biosynthesized in the natural habitat by the fungicolous producer and, consequently, released into the host. By means of liquid chromatography coupled to electrospray high-resolution time-of-flight mass spectrometry, we detected ten 20-residue peptaibols in the specimen. Sequences of peptaibiotics found in vivo were independently confirmed by analyzing the peptaibiome of an agar plate culture of T. phellinicola CBS 119283 (ex-type) grown under laboratory conditions. Notably, this strain could be identified as a potent producer of 39 new 17-, 18-, and 19-residue peptaibiotics, which display the same building scheme as the 20-residue peptaibols found in the specimen. Two of the 19-residue peptaibols are tentatively assigned to carry tyrosinol, a novel C-terminal residue, as deduced from high-resolution tandem mass-spectrometry data. For the new peptaibiotics produced by T. phellinicola, the name ‘hypophellin(s)’, based on the teleomorph name, is introduced. PMID:23681726

Antimicrobial Activity of Bacillus Persicus 24-DSM Isolated from Dead Sea Mud.

PubMed

Al-Karablieh, Nehaya

2017-01-01

Dead Sea is a hypersaline lake with 34% salinity, gains its name due to the absence of any living macroscopic creatures. Despite the extreme hypersaline environment, it is a unique ecosystem for various halophilic microorganisms adapted to this environment. Halophilic microorganisms are known for various potential biotechnological applications, the purpose of the current research is isolation and screening of halophilic bacteria from Dead Sea mud for potential antimicrobial applications. Screening for antagonistic bacteria was conducted by bacterial isolation from Dead Sea mud samples and agar plate antagonistic assay. The potential antagonistic isolates were subjected to biochemical characterization and identification by 16S-rRNA sequencing. Among the collected isolates, four isolates showed potential antagonistic activity against Bacillus subtilis 6633 and Escherichia coli 8739. The most active isolate (24-DSM) was subjected for antagonistic activity and minimal inhibitory concentration against different gram positive and negative bacterial strains after cultivation in different salt concentration media. Results: The results of 16S-rRNA analysis revealed that 24-DSM is very closely related to Bacillus persicus strain B48, which was isolated from hypersaline lake in Iran. Therefore, the isolate 24-DSM is assigned as a new strain of B. persicusi isolated from the Dead Sea mud. B. persicusi 24-DSM showed higher antimicrobial activity, when it was cultivated with saline medium, against all tested bacterial strains, where the most sensitive bacterial strain was Corynebacterium diphtheria 51696.

Direct and transdentinal (indirect) antibacterial activity of commercially available dental gel formulations against Streptococcus mutans.

PubMed

Tüzüner, Tamer; Ulusoy, Ayça Tuba; Baygin, Ozgul; Yahyaoglu, Gorkem; Yalcin, Ilkay; Buruk, Kurtulus; Nicholson, John

2013-01-01

To evaluate the direct and transdentinal (indirect) agar diffusion antibacterial activity of different commercially available antibacterial dental gel formulations against Streptococcus mutans. The commercially available dental gel formulations were Corsodyl® (COG, 1% chlorhexidine), Cervitec® (CEG, 0.2% chlorhexidine + 0.2% sodium fluoride), Forever Bright® (FOB, aloe vera), Gengigel® (GEG, 0.2% hyaluronic acid), 35% phosphoric acid gel and distilled water (control). Direct agar diffusion was performed by isolating three wells from brain-heart infusion agar plates using sterile glass pipettes attached to a vacuum pump and adding 0.1 ml of the gels to each well. Transdentinal (indirect) agar diffusion was performed by applying gel to 0.2- and 0.5-mm-thick human dentin discs previously etched with phosphoric acid and rinsed with distilled water. Zones formed around the wells and the dentin discs were measured and analyzed using Kruskal-Wallis and Mann-Whitney U tests with Bonferroni correction (p < 0.01). Direct agar diffusion tests showed significant differences among all gel formulations (p < 0.01) except for COG and CEG (p > 0.01). COG and CEG exhibited higher antibacterial effects compared to FOB and GEG (p < 0.01) in both direct and transdentinal (indirect) testing procedures. GEG did not show any antimicrobial activity in transdentinal (indirect) testing. Commercially available dental gels inhibited S. mutans, which may indicate their potential as cavity disinfectants. Copyright © 2013 S. Karger AG, Basel.

Cellobiohydrolase (CBH) Activity Assays.

PubMed

Sharma, Hem Kanta; Qin, Wensheng; Xu, Chunbao Charles

2018-01-01

Cellulosic biomass is the most abundant biopolymer on the earth. It has great potential to quench the thirst of liquid energy by producing biofuels and thus help to mitigate human reliance on fossil fuels. Although several cellulase activity assay methods have been used to disintegrate the glycosidic bonds, the appropriate selection of substrates and synergistic involvement of multiple enzymes in hydrolytic activity is not yet fully understood. The proper quantification of hydrolytic enzymes and hydrolysates is challenging because of the heterogeneity of cellulose, changes in enzyme-substrate ratio and the presence of some inhibitory compounds like cellobiose and cellodextran. In the glycosyl hydrolase (GH) family, cellobiohydrolase (CBH) is expected to disrupt the crystalline cellulose and release the sugar molecules. Several methods have been proposed for CBH assay with slight modification in substrate and quantification of hydrolysates. However, the Avicel method is still considered as the most promising and efficient hydrolytic technique so far. The most commonly used CBH assays including Avicel and other recent methods for proper quantification are outlined in this chapter. Also a qualitative screening of CBH producing bacteria using carboxymethyl cellulose (CMC) agar plates is described.

Molecular diagnosis of strongyloidiasis in a population of an endemic area through nested-PCR.

PubMed

Sharifdini, Meysam; Keyhani, Amir; Eshraghian, Mohammad Reza; Beigom Kia, Eshrat

2018-01-01

This study is aimed to diagnose and analyze strongyloidiasis in a population of an endemic area of Iran using nested-PCR, coupled with parasitological methods. Screening of strongyloidiasis infected people using reliable diagnostic techniques are essential to decrease the mortality and morbidity associated with this infection. Molecular methods have been proved to be highly sensitive and specific for detection of Strongyloides stercoralis in stool samples. A total of 155 fresh single stool samples were randomly collected from residents of north and northwest of Khouzestan Province, Iran. All samples were examined by parasitological methods including formalin-ether concentration and nutrient agar plate culture, and molecular method of nested-PCR. Infections with S. stercoralis were analyzed according to demographic criteria. Based on the results of nested-PCR method 15 cases (9.7%) were strongyloidiasis positive. Nested-PCR was more sensitive than parasitological techniques on single stool sampling. Elderly was the most important population index for higher infectivity with S. stercoralis . In endemic areas of S. stercoralis , old age should be considered as one of the most important risk factors of infection, especially among the immunosuppressed individuals.

Cloning, Sequencing, and Role in Virulence of Two Phospholipases (A1 and C) from Mesophilic Aeromonas sp. Serogroup O:34

PubMed Central

Merino, Susana; Aguilar, Alicia; Nogueras, Maria Mercedes; Regue, Miguel; Swift, Simon; Tomás, Juan M.

1999-01-01

Two different representative recombinant clones encoding Aeromonas hydrophila lipases were found upon screening on tributyrin (phospholipase A1) and egg yolk agar (lecithinase-phospholipase C) plates of a cosmid-based genomic library of Aeromonas hydrophila AH-3 (serogroup O34) introduced into Escherichia coli DH5α. Subcloning, nucleotide sequencing, and in vitro-coupled transcription-translation experiments showed that the phospholipase A1 (pla) and C (plc) genes code for an 83-kDa putative lipoprotein and a 65-kDa protein, respectively. Defined insertion mutants of A. hydrophila AH-3 defective in either pla or plc genes were defective in phospholipase A1 and C activities, respectively. Lecithinase (phospholipase C) was shown to be cytotoxic but nonhemolytic or poorly hemolytic. A. hydrophila AH-3 plc mutants showed a more than 10-fold increase in their 50% lethal dose on fish and mice, and complementation of the plc single gene on these mutants abolished this effect, suggesting that Plc protein is a virulence factor in the mesophilic Aeromonas sp. serogroup O:34 infection process. PMID:10417167

The isolation of salmonellas from British pork sausages and sausage meat.

PubMed Central

Roberts, D.; Boag, K.; Hall, M. L.; Shipp, C. R.

1975-01-01

Between 1969 and 1974, 1467 packets (3309 samples) of pork sausages and sausage meat produced by two large and two medium sized manufacturers and several local butchers were examined for the presence of salmonellas. Of these, 435 packets (786 samples) were found to contain salmonellas, but there was a wide variation in the isolation rates according to the producer. The salmonella incidence in samples from several small and two medium sized producers was low (0-11%) while the results from the two large producers investigated showed a striking difference, the rate of salmonella contamination in the product of one was low (about 2%) and in that of the other consistently high (40-60%). A comparison of liquid enrichment media, incubation temperatures and selective agar media was also carried out to determine the most efficient combination for the isolation of salmonellas from minced meat products. The results showed that (a) incubation of enrichment cultures at 43 degrees C. yielded a consistently greater number of salmonella isolations that at 37 degrees C., regardless of plating medium, (b) tetrathionate broth A (Rolfe) was superior to selenite broth as en enrichment medium at both 37 and 43 degrees C. and (c) brilliant green agar gave better results than deoxycholate citrate sucrose agar and bismuth sulphite agar as a selective medium. PMID:1100710

The isolation of salmonellas from British pork sausages and sausage meat.

PubMed

Roberts, D; Boag, K; Hall, M L; Shipp, C R

1975-10-01

Between 1969 and 1974, 1467 packets (3309 samples) of pork sausages and sausage meat produced by two large and two medium sized manufacturers and several local butchers were examined for the presence of salmonellas. Of these, 435 packets (786 samples) were found to contain salmonellas, but there was a wide variation in the isolation rates according to the producer. The salmonella incidence in samples from several small and two medium sized producers was low (0-11%) while the results from the two large producers investigated showed a striking difference, the rate of salmonella contamination in the product of one was low (about 2%) and in that of the other consistently high (40-60%). A comparison of liquid enrichment media, incubation temperatures and selective agar media was also carried out to determine the most efficient combination for the isolation of salmonellas from minced meat products. The results showed that (a) incubation of enrichment cultures at 43 degrees C. yielded a consistently greater number of salmonella isolations that at 37 degrees C., regardless of plating medium, (b) tetrathionate broth A (Rolfe) was superior to selenite broth as en enrichment medium at both 37 and 43 degrees C. and (c) brilliant green agar gave better results than deoxycholate citrate sucrose agar and bismuth sulphite agar as a selective medium.

Rapid isolation of gluten-digesting bacteria from human stool and saliva by using gliadin-containing plates

PubMed Central

Sarantopoulos, Christos; Ongchangco, Deryn; Sry, Jeremy; Cesario, Thomas

2014-01-01

The number of individuals with gluten intolerance has increased dramatically over the last years. To date, the only therapy for gluten intolerance is the complete avoidance of dietary gluten. To sustain a strictly gluten-free diet, however, is very challenging. Therefore, there is need for a non-dietary therapy. Any such treatment must appreciate that the immunogenic part of gluten are gliadin peptides which are poorly degraded by the enzymes of the gastrointestinal tract. Probiotic therapy and oral enzyme therapy containing gluten-degrading bacteria (GDB) and their gliadin-digesting enzymes are possible new approaches for the treatment of gluten intolerance, however effectively isolating GDB for these treatments is problematic. The goal of this study was to develop an easy technique to isolate GDB rapidly and efficiently with the hope it might lead to newer ways of developing either probiotics or traditional medicines to treat gluten intolerance. Several researchers have already isolated successfully GDB by using gluten minimal or limited agar plates. Although these plates can be used to isolate bacteria which can tolerate gluten, further assays are needed to investigate if the same bacteria can also digest gluten. The agar plates we developed can detect bacteria which cannot only tolerate gluten but are able to digest it as well. Therefore, we were able to combine two steps into one step. Using such technologies, we were able to isolate five GDB from saliva and stool, and identified three bacterial reference strains with gluten-degrading activity. The technique we developed to isolate bacteria with gluten-degrading activity is fast, effective, and easy to use. The GDB isolated by our technology could have potential as part of a probiotic or enzymatic therapy for people with gluten intolerance. PMID:25519429

Terrestrial microbes in martian and chondritic meteorites

NASA Astrophysics Data System (ADS)

Airieau, S.; Picenco, Y.; Andersen, G.

2007-08-01

Introduction: The best extraterrestrial analogs for microbiology are meteorites. The chemistry and mineralogy of Asteroid Belt and martian (SNC) meteorites are used as tracers of processes that took place in the early solar system. Meteoritic falls, in particular those of carbonaceous chondrites, are regarded as pristine samples of planetesimal evolution as these rocks are primitive and mostly unprocessed since the formation of the solar system 4.56 billion years ago. Yet, questions about terrestrial contamination and its effects on the meteoritic isotopic, chemical and mineral characteristics often arise. Meteorites are hosts to biological activity as soon as they are in contact with the terrestrial biosphere, like all rocks. A wide biodiversity was found in 21 chondrites and 8 martian stones, and was investigated with cell culture, microscopy techniques, PCR, and LAL photoluminetry. Some preliminary results are presented here. The sample suite included carbonaceous chondrites of types CR, CV, CK, CO, CI, and CM, from ANSMET and Falls. Past studies documented the alteration of meteorites by weathering and biological activity [1]-[4]. Unpublished observations during aqueous extraction for oxygen isotopic analysis [5], noted the formation of biofilms in water in a matter of days. In order to address the potential modification of meteoritic isotopic and chemical signatures, the culture of microbial contaminating species was initiated in 2005, and after a prolonged incubation, some of the species obtained from cell culture were analyzed in 2006. The results are preliminary, and a systematic catalog of microbial contaminants is developing very slowly due to lack of funding. Methods: The primary method was cell culture and PCR. Chondrites. Chondritic meteorite fragments were obtained by breaking stones of approximately one gram in sterile mortars. The core of the rocks, presumably less contaminated than the surface, was used for the present microbial study, and the remaining fragments of the samples were used for amino acid and isotopic analyses [6]. Some samples were fragments of dried and wet meteorites isolated in centrifuge tubes after a 10-day water extraction. Sabouraud Dextrose (dilutions 1:10 and 1:1000), Bacto Agar, LB Broth Miller (dilutions 1:10 and 1:1000), and R2A agar (1:1 and 1:1000), were autoclaved and cooled in culture plates inside a clean hood for cell culture. Some controls retained sterile moist agar still adhering to the perimeter of the plates for up to 18 months, and validated the sterile technique. Cell culture, PCR and microscopy documented a diversity of archea, prokaryotes and eukaryotes in these samples [7]. The plates displaying microbial growth at room temperature after 6 weeks or less were used to produce streak plates and isolate colonies of individual species for long term freezing in Eppendorf tubes. Any plate with biological growth along the perimeter of the plate was discarded. The plates without microbial activity after 6 weeks were stored in a fridge for 18 months. Control plates, exposed to the clean hood, laboratory room, used gloves, and weighing paper used in the analyses, sustained the prolonged storage with no sign of microbial activity that could be related to the analysis method. Dust grains and water extracts from the meteorites were spread on agar surfaces in cell culture Petri dishes in a clean hood. SNC samples.In early 2005, the surface of SNC stones in the USNM curation facility were brushed with sterile swabs. Fallen dust grains were collected on weighing paper and isolated in sterile tubes. The sample suite included Zagami USNM 6545, Lafayette USNM 1505, Los Angeles USNM 7052, Shergotty USNM 321, Nakhla USNM 5892, Nakhla USNM 426 (117.4 g) and Nakhla USNM 426 (18.2 g), and Chassigny USNMMNHN 2524. The controls, worker's gloves, blank swabs, and weighing paper exhibited no microbial activity in subsequent months. The cell culture was conducted with Sabouraud Dextrose and R2A only, by deposition of dry grains onto the surface of agar in culture plates that were incubated at 20 Celsius 2 weeks, then at 35 Celsius 3 days, and finally at 4 Celsius for 18 months. Limulus Amoebocyte Lysate (LAL Assay) measurements of the swabs [7] revealed low biological activity in all SNCs, except in a small piece of Nakhla USNM 426 (18.2 g) (activity below detection limit) and Chassigny (data unavailable). The LAL technique assesses gram negative bacterial equivalents. Culture sample material was recovered from the agar plates using sterile pipette tips. The material was added to 100 uL sterile water. This material was boiled for 15 min. in a water bath to lyse the cells and inactivate enzymes. 10 uL of the boiled lysate was used as template in PCR. Universal bacterial primers (27F and 1492R) were used to amplify a major portion of the 16S rRNA gene. PCR product was purified using a Qiagen MinElute PCR Purification kit and then sent for sequencing. Results and Comments: GRA 95229. Biological growth out of dust grains deposited on the agar surface was visible with the naked eye, and occurred after incubation in a fridge for 10 months. Leoville. Biological growth occurred at the contact of a dust grain and the agar surface after 1 month. It continued to expand outside a dust grain deposited on Sabouraud Dextrose 1:1000, while stored at 20 Celsius for 12 months. The fan shaped outgrowth reached about 1 cm on either side of the meteorite grain and dried. DNA was recovered, and PCR products yielded sequences of a Bacillus spp. Bacillus is a common soil bacterial genus, and the close sequence relatives of this isolate were no exception. They were rock or soil Bacillus, and have been found in Allende [4]. EET 87770. Rock fragments were wet for 8 months (Millipore water) in a sterile centrifuge tube, and were used to make a spread plate that dried over a period of 10 months. The yellow dried colonies yielded good PCR product and the sequences were compared to other GenBank sequences using the BLAST program. The closest matches were in the genus Microbacterium. Soil and plant isolates were close relatives by sequence comparison. Los Angeles. After 11 months of incubation in a fridge, a yellow colony grew at the center of a culture plate of Los Angeles dust grains (1:1000 R2A). There was no cell activity in the other agars. A DNA extraction yielded no usable results [7]. Sequencing was not performed because the culture plate became contaminated with outside organisms that overtook the colony of interest. Conclusions: The sequences for EET 87770 and Leoville were of a good quality and the sequence reads were long, so the data are clear that these are typical soil and/or plant-related bacteria commonly found in Earth habitats. Microbial species present in a dozen chondritic samples from isolates are not yet identified, and the contaminant in Los Angeles needs to be recovered. In addition, isotopic analyses of samples with various amounts of microbial contamination could help quantified isotopic impact of microbes on protoplanetary chemistry in these rocks. References : [1] Gounelle, M. and Zolensky M. LPS, (2001) LPS XXXII, Abstract #999. [2] Fries, M. et al. (2005) Meteoritical Society Meeting 68, Abstract # 5201. [3] Burckle, L. H. and Delaney, J. S (1999) Meteoritics & Planet. Sci., 32, 475-478. [4] Whitby, C. et al. (2000) ) LPS XXXI, Abstract #1732. [5] Airieau, S. A. et al (2005) Geochim. Cosmochim. Acta, 69, 4166-4171. [6] Unpublished data, with H. J. Cleaves, A. Aubrey, J. Bada (Scripps Institution of Oceanography), M. Thiemens (UC San Diego) and M. Fogel (Carnegie Institution of Washington). [7] Unpublished data, with A. Steele (CIW), and N. Wainwright (Marine Biological Laboratory). Acknowledgements: Lisa Welleberger for access to SNC samples at USNM; Ralph Harvey for organizing ANSMET; Denise C. Thiry and Andrew Steele for long term storage of samples, NormWainwright for LAL measurements. A small portion of this work was funded with a NASA Cosmochemistry grant, ( P. I. Thiemens).

Clinical test to detect mecA and antibiotic resistance in Staphylococcus aureus, based on novel biotechnological methods.

PubMed

Shahmohammadi, Mohammad Reza; Nahaei, Mohammad Reza; Akbarzadeh, Abolfazl; Milani, Morteza

2016-09-01

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common organisms isolated from clinical samples, and has been associated with morbidity and mortality among hospitalized patients. The aim of this study was to evaluate the prevalence and antibiotic susceptibility patterns among MRSA and methicillin-sensitive S. aureus (MSSA) isolates collected from four hospitals in Iran. A total of 183 isolates of S. aureus were collected from various clinical specimens of four hospitals in Iran. The isolates were identified by using the conventional biochemical tests. Three methods-oxacillin agar disk diffusion, oxacillin agar screening, and PCR- were applied to determine susceptibility to oxacillin. The conventional disk agar diffusion test was used to evaluate the antibiotic sensitivity of our isolates against 15 antibiotics, according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Of 183 isolates, 77 isolates (42.1%) were found to be MRSA, by the PCR method. The highest antibiotic resistance was found to be against penicillin, co-trimoxazole, erythromycin, and tetracycline respectively. All isolates were susceptible to vancomycin, according to the results of disk agar diffusion. Among other antibiotics, teicoplanin (84%) and fusidic acid (80.5%) were more active against MRSA isolates. For the different methods evaluated, the sensitivities and specificities were as follows: for disk agar diffusion (84.9% and 95.9%) and for agar screening test with oxacillin concentrations of 0.6 μg/ml (70.8% and 97.4%), 4 μg/ml (96.1%and 97.2%) and 6 μg/ml (96% and 96.3%), respectively. The results of our study showed that 47% of S. aureus isolates were MRSA. Overall, in this research study, resistance to all test antimicrobial agents in MRSA isolates were higher than that of MSSA isolates. Our results also revealed that 85% of mecA-positive isolates and 15% of mecA-negative isolates were resistant to methicillin; while 96% of mecA-negative isolates were sensitive to methicillin. Meanwhile 4% of mecA-positive isolates were also sensitive to methicillin.

Development of zinc-plated regenerator material

NASA Astrophysics Data System (ADS)

Y Xu, M.; Morie, T.; Tsuchiya, A.

2017-12-01

An effective way to improve the efficiency of a cryocooler is to improve the efficiency of the regenerator. In general, the heat capacity of materials decreases as temperature decreases. Thus, when temperature is below 40 K, lead or bismuth spheres are often used as regenerator materials. However, the pressure drop in a sphere regenerator is much larger than that in a screen regenerator. To overcome this dilemma, Xu et al. reported that cooling performance at the temperature of less than 40 K was improved when using tin-plated screens at the cold end of the regenerator. However, the reliability of tin at low temperatures is still not verified fully because of its phase transition from a normal β phase to an abnormal α phase, which may result in a significant reduction of the mechanical strength. In this paper, a zinc-plated screen is proposed as another potential alternative. A comparison test was performed with a two-stage GM cryocooler by replacing part of the first stage regenerator material, phosphorus bronze screens, with zinc-plated screens. Compared to a regenerator filled with bronze screens, the cooling capacity of the first stage increased by about 11% at 40 K and 60% at 30 K with these zinc-plated screens. The detailed experimental results are reported in this paper.

Enumeration of the contaminating bacterial microbiota in unfermented pasteurized milks enriched with probiotic bacteria.

PubMed

Champagne, C P; Raymond, Y; Gonthier, J; Audet, P

2009-04-01

Pasteurized and unfermented milks supplemented with probiotic bacteria are appearing on the market. It then becomes a challenge to ascertain the undesirable contamination microbiota in the presence of a largely superior population of probiotic bacteria. A method to enumerate the contaminating microbial microbiota in such probiotic-enriched milks was developed. The probiotic cultures, Lactobacillus rhamnosus Lb-Immuni-T and Bifidobacterium animalis subsp. lactis BB-12(R), were added to a pasteurized unfermented milk to reach a minimum of 1 billion CFU per 250 mL portion, as ascertained by plating on de Man - Rogosa - Sharpe (MRS) agar in anaerobic conditions. No growth of B. animalis subsp. lactis BB-12 was noted on plate count agar (PCA) or Petrifilm plates, and the presence of this culture did not affect standard plate counts (SPC) of contaminating bacteria. However, L. rhamnosus formed colonies on PCA and Petrifilm plates. Attempts were thus made to inhibit the growth of the probiotic lactobacilli in PCA. The addition of 2% sodium phosphate (SP) or 5% glycerophosphate (GP) inhibited the growth of the lactobacilli in broths, but pin-point colonies of L. rhamnosus Lb-Immuni-T nevertheless appeared on PCA supplemented with phosphates. SPC could be obtained on PCA + 2% SP by only counting the large colonies, but this resulted in a significant (4.4 fold) underestimation of SPC values. On Petrifilm AC, at dilutions 0 to 2, all colonies were considered as being contaminants, while at dilutions 3 and 4, only large colonies were counted for SPC determinations. There was a direct correlation (R2 = 0.99) between SPC values with Petrifilm in uninoculated milks and those obtained on probiotic-enriched milks. The high correlation obtained over the 102 to 106 CFU/mL range of SPC values show that this Petrifilm method is appropriate to evaluate the microbiological quality of pasteurized milks enriched with L. rhamnosus Lb-Immuni-T and B. animalis subsp. lactis BB-12.

Microbial Populations in Two Swamp Soils of South Carolina

Treesearch

David S. Priester; William R. Harms

1971-01-01

Microbial populations were counted in agar-plated samples of two swamp soils collected in summer and winter. Number of aerobic and anaerobic microorganisms differed significantly among the soils and between seasons. Alluvial soil from the river swamp was high in organic matter, N, K, Ca, and pH and averaged 88 million microorganisms per gram over the growing season....

Microbiology of Fresh Comminuted Turkey Meat

DTIC Science & Technology

1976-04-12

823 Reprinted from J. Milk Food Technol. Vol. 39. No. 12. Pages 823-829 (December. 1976) Cooyright 1976, International Association of Milk , Food, and...most probable number (MPN) method (13). niacin, and riboflavin (29). Canadian officials have proposed microbial standards The literature contains...onto Mueller-Hinton agar plates containing 5% various retail markets in the San Francisco Bay Area. They were defibrinated sheep blood. Representative

Phytophthora species associated with stem cankers on tanoak in southwestern Oregon

Treesearch

Paul Reeser; Wendy Sutton; Everett Hansen

2008-01-01

In effort to eradicate Phytophthora ramorum from Oregon forests, tanoak over its entire range in southwestern Oregon is surveyed intensively for stem disease. Pieces of bark from the leading edge of tanoak stem cankers were plated on cornmeal agar amended with 10 ppm natamycin, 200 ppm a-ampicillin, and 10 ppm rifamycin SV (CARP) to favor the...

Activity of two strobilurin fungicides against three species of decay fungi in agar plate tests

Treesearch

Juliet D. Tang; Tina Ciaramitaro; Maria Tomaso-Peterson; Susan V. Diehl

2017-01-01

The objective of this study was to examine the toxicity of strobilurin fungicides against wood decay fungi in order to assess their potential to act as a co-biocide for copper-based wood protection. Two strobilurin fungicides, Heritage (50% azoxystrobin active ingredient) and Insignia (20% pyraclostrobin active ingredients), and copper sulfate pentahydrate were tested...

Behavioral differences of Heterodera glycines and Meloidogyne incognita infective juveniles exposed to root extracts in vitro

USDA-ARS?s Scientific Manuscript database

The in vitro behaviors of infective juveniles (J2) of Heterodera glycines and Meloidogyne incognita were compared in the presence and absence of plant root extracts. In an agar plate attraction-retention assay, H. glycines was 15-fold more responsive to a chemical attractant (CaCl2; P < 0.05) than w...

Rapid staining and enumeration of small numbers of total bacteria in water by solid-phase laser cytometry

NASA Technical Reports Server (NTRS)

Broadaway, Susan C.; Barton, Stephanie A.; Pyle, Barry H.

2003-01-01

The nucleic acid stain SYBR Green I was evaluated for use with solid-phase laser cytometry to obtain total bacterial cell counts from several water sources with small bacterial numbers. Results were obtained within 30 min and exceeded or equaled counts on R2A agar plates incubated for 14 days at room temperature.

Interactions between stream fungi and bacteria associated with decomposing leaf litter at different levels of nutrient availability

Treesearch

Vladislav Gulis; Keller Suberkropp

2003-01-01

We examined the potential for interactions between aquatic hyphomycetes and bacteria isolated from leaves decaying in a headwater stream. In agar plate assays, culture filtrates of each of 28 aquatic hyphomycete isolates tested (5 species) inhibited bacterial growth (16 Gram-negative bacterial isolates belonging to 6 colony morphotypes were tested). Inhibition of...

Identification of Viable Helicobacter pylori in Drinking Water Supplies by Cultural and Molecular Techniques.

PubMed

Santiago, Paula; Moreno, Yolanda; Ferrús, M Antonía

2015-08-01

Helicobacter pylori is one of the most common causes of chronic bacterial infection in humans, directly related to peptic ulcer and gastric cancer. It has been suggested that H. pylori can be acquired through different transmission routes, including water. In this study, culture and qPCR were used to detect and identify the presence of H. pylori in drinking water. Furthermore, the combined techniques PMA-qPCR and DVC-FISH were applied for detection of viable cells of H. pylori. Among 24 drinking water samples, 16 samples were positive for the presence of H. pylori, but viable cells were only detected in six samples. Characteristic colonies, covered by a mass of bacterial unspecific growth, were observed on selective agar plates from an only sample, after enrichment. The mixed culture was submitted to DVC-FISH and qPCR analysis, followed by sequencing of the amplicons. Molecular techniques confirmed the growth of H. pylori on the agar plate. Our results demonstrate for the first time that H. pylori can survive and be potentially infective in drinking water, showing that water distribution systems could be a potential route for H. pylori transmission. © 2015 John Wiley & Sons Ltd.

Isolation and characterization of pathogenic Vibrio alginolyticus from diseased cobia Rachycentron canadum.

PubMed

Liu, Ping-Chung; Lin, Ji-Yang; Hsiao, Pei-Tze; Lee, Kuo-Kau

2004-01-01

Outbreaks of serious mortality among cultured juvenile cobia Rachycentron canadum L. (weighing 8-10 g) characterized by lethargy, dark skin and ascites in the peritoneal cavity while some fish possessing damaged eyes occurred in July and August of 2001 in Taiwan. Fifteen motile bacterial strains were isolated from head kidney and/or the ascites on tryptic soy agar supplemented with 1% NaCl (TSA1) and/or thiosulphate citrate bile salt (TCBS) sucrose agar plates during the two outbreaks. All the isolates were characterized and identified as Vibrio alginolyticus on the basis of biochemical characteristics, and comparisons with those of the reference strain V. alginolyticus ATCC 17749. The strain C3c01 (a representative of the 15 similar field isolates), was virulent to the cobia with an LD50 value of 3.28 x 10(4) colony forming units/g fish body weight. All the moribund/dead fish exhibited lethargy, dark skin and ascites in the peritoneal cavity as that observed in natural outbreaks. The same bacteria could be reisolated from kidney and the ascites of fish after bacterial challenge using TSA1 and TCBS plates. The results reveal that V. alginolyticus is an infectious agent of vibriosis in the cobia.

Agrobacterium tumefasciens-mediated transformation of the aquatic fungus Blastocladiella emersonii.

PubMed

Vieira, André L G; Camilo, César M

2011-08-01

Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class. Copyright © 2011 Elsevier Inc. All rights reserved.

Clinical and economic evaluation of BBL CHROMagar Salmonella (CHROMSal) versus subculture after selenite broth enrichment to CHROMSal and Hektoen enteric agars to detect enteric Salmonella in a large regional microbiology laboratory.

PubMed

Church, Deirdre L; Emshey, Diana; Lloyd, Tracie; Pitout, Johann

2010-09-01

Stool culture for enteric pathogens is one of the most labor-intensive clinical microbiology procedures. Direct plating of stool to BBL CHROMagar Salmonella (CHROMSal) (BD Diagnostics, Sparks, MD) versus subculture after selenite broth enrichment (Sel) to CHROMSal (Sel-CHROMSal) and Hektoen enteric agar (Sel-Hek) (PML Microbiologicals, Eugene, OR) to detect Salmonella were compared. The number of colony picks and biochemical/serotyping tests per plate was recorded. A cost comparison was done. Fifty-one of 2999 (1.7%) stools yielded Salmonella sp., and 80% of isolates grew on CHROMSal by 24 h. CHROMSal demonstrated much less false-positive growth compared to Sel-Hek (P < 0.0001), which reduced biochemical and serotyping tests by 85% and 20%, respectively. Sel-CHROMSal and CHROMSal versus Sel-Hek improved enteric Salmonella detection when compared to a true positive "gold standard" (i.e., recovery by any culture method) with a sensitivity, specificity, positive predictive value, and negative predictive value of 100% and 94.12%, 100% and 99.97%, 100% and 97.96%, and 100% and 99.90%, respectively. CHROMSal use would result in substantial cost and labor savings.

Gene disruption in Salmonella typhimurim by modified λ Red disruption system.

PubMed

Ahani Azari, A; Zahraei Salehi, T; Nayeri Fasaei, B; Alebouyeh, M

2015-01-01

There are many techniques to knock out directed genes in bacteria, some of which have been described in Salmonella species. In this study, a combination of SOEing PCR method and the λ Red disruption system were used to disrupt phoP gene in wild type and standard strains of Salmonella typhimurium. Three standards PCR and one fusion PCR reactions were performed to construct a linear DNA including upstream and downstream of phoP gene and Kanamycin cassette. As a template plasmid, we used pKD4 which carries kanamycin gene flanked by FRT (FLP recognition target) sites. The resulting construct was electroporated into prepared competent cells of S. typhimurium. The transformants colonies related to the standard strain appeared on the LB-Km-agar plates after incubation, but there was no colony on LB-Km-agar plates corresponding to the wild type strain. The failure in transformation of the wild type strain may be because of inflexibility of the λ Red disruption system in this strain or its unique restriction-modification system. However, by this construct we are able to generate phoP mutant in many of the Salmonella species due to high homology of the phoP gene which exists in different species.

Waving and skewing: how gravity and the surface of growth media affect root development in Arabidopsis.

PubMed

Oliva, Michele; Dunand, Christophe

2007-01-01

Arabidopsis seedlings growing on inclined agar surfaces exhibit characteristic root behaviours called 'waving' and 'skewing': the former consists of a series of undulations, whereas the latter is a deviation from the direction of gravity. Even though the precise basis of these growth patterns is not well understood, both gravity and the contact between the medium and the root are considered to be the major players that result in these processes. The influence of these forces on root surface-dependent behaviours can be verified by growing seedlings at different gel pitches: plants growing on vertical plates present roots with slight waving and skewing when compared with seedlings grown on plates held at minor angles of < 90 degrees . However, other factors are thought to modulate root growth on agar; for instance, it has been demonstrated that the presence and concentration of certain compounds in the medium (such as sucrose) and of drugs able to modify the plant cell cytoskeleton also affect skewing and waving. The recent discovery of an active role of ethylene on surface-dependent root behaviour, and the finding of new mutants showing anomalous growth, pave the way for a more detailed description of these phenomena.

Hair sheep blood, citrated or defibrinated, fulfills all requirements of blood agar for diagnostic microbiology laboratory tests.

PubMed

Yeh, Ellen; Pinsky, Benjamin A; Banaei, Niaz; Baron, Ellen Jo

2009-07-03

Blood agar is used for the identification and antibiotic susceptibility testing of many bacterial pathogens. In the developing world, microbiologists use human blood agar because of the high cost and inhospitable conditions for raising wool sheep or horses to supply blood. Many pathogens either fail to grow entirely or exhibit morphologies and hemolytic patterns on human blood agar that confound colony recognition. Furthermore, human blood can be hazardous to handle due to HIV and hepatitis. This study investigated whether blood from hair sheep, a hardy, low-maintenance variety of sheep adapted for hot climates, was suitable for routine clinical microbiology studies. Hair sheep blood obtained by jugular venipuncture was anticoagulated by either manual defibrination or collection in human blood bank bags containing citrate-phosphate-dextrose. Trypticase soy 5% blood agar was made from both forms of hair sheep blood and commercial defibrinated wool sheep blood. Growth characteristics, colony morphologies, and hemolytic patterns of selected human pathogens, including several streptococcal species, were evaluated. Specialized identification tests, including CAMP test, reverse CAMP test, and satellite colony formation with Haemophilus influenzae and Abiotrophia defectiva were also performed. Mueller-Hinton blood agar plates prepared from the three blood types were compared in antibiotic susceptibility tests by disk diffusion and E-test. The results of all studies showed that blood agar prepared from citrated hair sheep blood is suitable for microbiological tests used in routine identification and susceptibility profiling of human pathogens. The validation of citrated hair sheep blood eliminates the labor-intensive and equipment-requiring process of manual defibrination. Use of hair sheep blood, in lieu of human blood currently used by many developing world laboratories and as an alternative to cost-prohibitive commercial sheep blood, offers the opportunity to dramatically improve the safety and accuracy of laboratory diagnosis of pathogenic bacteria in resource-poor countries.

Anticlostridial agent 8-hydroxyquinoline improves the isolation of faecal bifidobacteria on modified Wilkins-Chalgren agar with mupirocin.

PubMed

Novakova, J; Vlkova, E; Salmonova, H; Pechar, R; Rada, V; Kokoska, L

2016-04-01

The need for suitable selective cultivation media for the isolation of Bifidobacterium spp. continues to be a real concern in the field of intestinal microbiology. Isolation of bifidobacteria from human and animal faecal samples using selective agar plating may be problematic especially in samples with increased clostridial counts than bifidobacterial counts. Due to the absence of anticlostridial agents in existing selective media, clostridia can displace bifidobacteria resulting in incorrect estimation of their counts. Therefore, we supplemented the existing selective medium 'modified Wilkins Chalgren agar with mupirocin' (MWM) with 90 mg l(-1) of 8-hydroxyquinoline (8HQ), which was recently proved to act selectively against clostridia. The newly composed 'modified Wilkins-Chalgren agar with 8HQ' (MWMQ) was tested on pure bifidobacterial and clostridial strains, their mixtures, and using faecal samples of mammalian origin; its selectivity was evaluated by genus-specific identification of isolates. The results demonstrated that the presence of 8HQ in this agar eliminated the growth of nonbifidobacterial strains on MWMQ compared to that on MWM, whereas the recovery of bifidobacterial counts was at satisfactory levels. In conclusion, MWMQ could be recommended for bifidobacterial isolation from human and animal faeces especially when bifidobacteria are not numerically dominant and there are chances of clostridial contamination. Routine isolation of bifidobacteria from mammalian faeces does not use a reliable selective agar with an anticlostridial agent. Overgrowth of clostridia may result in incorrect estimation of bifidobacterial counts. Thus, in order to improve the selectivity of existing media for bifidobacterial isolation, we chose the modified Wilkins-Chalgren agar with mupirocin and supplemented it with 8-hydroxyquinoline (8HQ), a molecule that shows anticlostridial activity without affecting the growth of bifidobacteria. This newly composed medium showed enhanced selectivity and specificity compared to the original medium and therefore, can be recommended for the isolation of bifidobacteria from mammal faeces. © 2016 The Society for Applied Microbiology.

Microbial contamination in intraoral phosphor storage plates: the dilemma.

PubMed

de Souza, Tricia Murielly Pereira Andrade; de Castro, Ricardo Dias; de Vasconcelos, Laís César; Pontual, Andréa Dos Anjos; de Moraes Ramos Perez, Flávia Maria; Pontual, Maria Luiza Dos Anjos

2017-01-01

The aims of this study were to evaluate microbial contamination in phosphor storage plates in dental radiology services and discuss the possible origin of this contamination. The sample comprised 50 phosphor plates: 14 plates from service A, 30 from service B, and 6 in the control group, consisting of plates never used. Damp sterile swabs were rubbed on the phosphor plates, and then transferred to tests tubes containing sterile saline solution. Serial dilutions were made, and then inoculated in triplicate on Mueller Hinton agar plates and incubated at 37 °C/48 h, before counting the colony-forming units (CFU). The samples were also seeded in brain-heart infusion medium to confirm contamination by turbidity of the culture medium. All solutions, turbid and clean, were seeded in selective and non-selective media. At service A and B, 50 and 73.3 % of the phosphor plates were contaminated, respectively. This contamination was mainly due to bacteria of the genus Staphylococcus. CFU counts ranged from 26.4 to 80.0 CFU/plate. Most of the phosphor plates evaluated shown to be contaminated, mainly by Staphylococcus ssp. Quantitatively, this contamination occurred at low levels, possibly arising from handling of the plates. The use of a second plastic barrier may have diminished contamination by microorganisms from the oral cavity. There is a risk of cross-contamination by phosphor storage plates used in dental radiology services.

Selective enumeration of propionibacteria in Emmental-type cheese using Petrifilm™ aerobic count plates added to lithium glycerol broth.

PubMed

de Freitas, Rosângela; Luiz, Lívia M Pinheiro; Alves, Maura Pinheiro; Valence-Bertel, Florence; Nero, Luís Augusto; de Carvalho, Antônio Fernandes

2013-08-01

Propionibacteria derived from dairy products are relevant starter cultures for the production of Swiss and Emmental-type cheeses, and the monitoring of which is mandatory for proper quality control. This study aimed to evaluate an alternative procedure to enumerate propionibacteria, in order to develop a reliable and practical methodology to be employed by dairy industries. 2,3,5-triphenyltetrazolium chloride (TTC) inhibitory activity was tested against five reference strains (CIRM 09, 38, 39, 40 and 116); TTC at 0·0025% (w/v) was not inhibitory, with the exception of one strain (CIRM 116). Subsequently, the four TTC-resistant strains, three commercial starter cultures (PS-1, PB-I, and CHOO) and twelve Emmental-type cheese samples were subjected to propionibacteria enumeration using Lithium Glycerol (LG) agar, and Petrifilm™ Aerobic Count (AC) plates added to LG broth (anaerobic incubation at 30 °C for 7 d). Petrifilm™ AC added to LG broth presented high counts than LG agar (P<0·05) for only two reference strains (CIRM 39, and 40) and for all commercial starter cultures. Cheese sample counts obtained by both procedures did not show significant differences (P<0·05). Significant correlation indexes were observed between the counts recorded by both methods (P<0·05). These results demonstrate the reliability of Petrifilm™ AC plates added to LG broth in enumerating select Propionibacterium spp., despite some limitations observed for specific commercial starter cultures.

Evaluation of a pulsed xenon ultraviolet light device for isolation room disinfection in a United Kingdom hospital.

PubMed

Hosein, Ian; Madeloso, Rosie; Nagaratnam, Wijayaratnam; Villamaria, Frank; Stock, Eileen; Jinadatha, Chetan

2016-09-01

Pathogen transmission from contaminated surfaces can cause hospital-associated infections. Although pulsed xenon ultraviolet (PX-UV) light devices have been shown to decrease hospital room bioburden in the United States, their effectiveness in United Kingdom (UK) hospitals is less understood. Forty isolation rooms at the Queens Hospital (700 beds) in North London, UK, were sampled for aerobic bacteria after patient discharge, after manual cleaning with a hypochlorous acid-troclosene sodium solution, and after PX-UV disinfection. PX-UV device efficacy on known organisms was tested by exposing inoculated agar plates in a nonpatient care area. Turnaround times for device usage were recorded, and a survey of hospital staff for perceptions of the device was undertaken. After PX-UV disinfection, the bacterial contamination measured in colony forming units (CFU) decreased by 78.4%, a 91% reduction from initial bioburden levels prior to terminal cleaning. PX-UV exposure resulted in a 5-log CFU reduction for multidrug-resistant organisms (MDROs) on spiked plates. The average device turnaround time was 1 hour, with minimal impact on patient throughput. Ward staff were enthusiastic about device deployment, and device operators reported physical comfort in usage. PX-UV use decreased bioburden in patient discharge rooms and on agar plates spiked with MDROs. The implementation of the PX-UV device was well received by hospital cleaning and ward staff, with minimal disruption to patient flow. Copyright © 2016 Association for Professionals in Infection Control and Epidemiology, Inc. All rights reserved.

A method for detecting fungal contaminants in wall cavities.

PubMed

Spurgeon, Joe C

2003-01-01

This article describes a practical method for detecting the presence of both fungal spores and culturable fungi in wall cavities. Culturable fungi were collected in 25 mm cassettes containing 0.8 microm mixed cellulose ester filters using aggressive sampling conditions. Both culturable fungi and fungal spores were collected in modified slotted-disk cassettes. The sample volume was 4 L. The filters were examined microscopically and dilution plated onto multiple culture media. Collecting airborne samples in filter cassettes was an effective method for assessing wall cavities for fungal contaminants, especially because this method allowed the sample to be analyzed by both microscopy and culture media. Assessment criteria were developed that allowed the sample results to be used to classify wall cavities as either uncontaminated or contaminated. As a criterion, wall cavities with concentrations of culturable fungi below the limit of detection (LOD) were classified as uncontaminated, whereas those cavities with detectable concentrations of culturable fungi were classified as contaminated. A total of 150 wall cavities was sampled as part of a field project. The concentrations of culturable fungi were below the LOD in 34% of the samples, whereas Aspergillus and/or Penicillium were the only fungal genera detected in 69% of the samples in which culturable fungi were detected. Spore counting resulted in the detection of Stachybotrys-like spores in 25% of the samples that were analyzed, whereas Stachybotrys chartarum colonies were only detected on 2% of malt extract agar plates and on 6% of corn meal agar plates.

Effects of H3O+, OH-, \\text{O}_{2}^{-} , \\text{NO}_{\\text{x}}^{-} and NO x for Escherichia coli inactivation in atmospheric pressure DC corona discharges

NASA Astrophysics Data System (ADS)

Sekimoto, Kanako; Gonda, Rena; Takayama, Mitsuo

2015-08-01

The effects of ionic and neutral species such as H3O+, OH-, \\text{O}2- , \\text{NO}x- (x = 2, 3), and NO x on Escherichia coli (E. coli) inactivation in gas and liquid phases was investigated using atmospheric pressure DC corona discharges with point-to-plane electrodes. The above chemical species as well as OH and O3 were selectively irradiated onto E. coli suspensions on agar plates using a needle angle of 45° with respect to the plates, airflow, and a grid plate. Irradiation with the positive ion H3O+ did not inactivate E. coli, while the negative ions OH-/\\text{O}2- resulted in bactericidal inactivation, in both gas and liquid phases. In contrast, the negative ions \\text{NO}x- and neutral species NO x in the gas phase had quite strong bactericidal effects on E. coli compared to those species in the liquid phase. These results suggest that liquid-phase HNO3, formed primarily via the reaction of gas-phase \\text{NO}x- and NO x with H2O in agar, has only a weak inactivation effect on E. coli. Furthermore, using naphthylethylenediamine spectrophotometry, the threshold amount of gas-phase \\text{NO}x- and NO x for E. coli inactivation was determined to be  ≈1.3   ×   10-9 mol mm-1.

Prevalence of Candida albicans and carriage of Candida non-albicans in the saliva of preschool children, according to their caries status.

PubMed

Lozano Moraga, Carla Paola; Rodríguez Martínez, Gonzalo Andrés; Lefimil Puente, Claudia Andrea; Morales Bozo, Irene Cecilia; Urzúa Orellana, Blanca Regina

2017-01-01

This study was conducted to establish associations among the Candida carriage rate, the diversity of Candida species carried and the different caries status of preschool children. Sixty-one children between 2 and 5 years of age were examined by a single expert examiner and were divided into three groups, the caries-free, moderate caries and severe caries groups, according to the criteria of the International Caries Detection and Assessment System II (ICDAS). Saliva samples were obtained from the members of each group and were plated on Sabouraud agar plates to assess the Candida carriage rates. CHROMagar Candida medium was used for the preliminary screening. Biochemical testing or PCR/sequencing was conducted to identify the different Candida species in the samples. The differences observed were considered significant if the p value was <0.05. The Candida carriage rate and the number of species of this fungus carried were higher in the group with the highest level of caries severity (p < 0.05). Whereas Candida albicans was the most predominant Candida species in the saliva of all of the children, C. dubliniensis was identified only in the most caries-affected group in addition to other rare species of Candida non-albicans. A high salivary Candida carriage rate and the presence of specific species of this fungus (such as C. albicans and C. dubliniensis) appear to be related to the severity of caries experienced by preschool children.

Application of bacterial cytological profiling to crude natural product extracts reveals the antibacterial arsenal of Bacillus subtilis.

PubMed

Nonejuie, Poochit; Trial, Rachelle M; Newton, Gerald L; Lamsa, Anne; Ranmali Perera, Varahenage; Aguilar, Julieta; Liu, Wei-Ting; Dorrestein, Pieter C; Pogliano, Joe; Pogliano, Kit

2016-05-01

Although most clinically used antibiotics are derived from natural products, identifying new antibacterial molecules from natural product extracts is difficult due to the complexity of these extracts and the limited tools to correlate biological activity with specific molecules. Here, we show that bacterial cytological profiling (BCP) provides a rapid method for mechanism of action determination on plates and in complex natural product extracts and for activity-guided purification. We prepared an extract from Bacillus subtilis 3610 that killed the Escherichia coli lptD mutant and used BCP to observe two types of bioactivities in the unfractionated extract: inhibition of translation and permeablization of the cytoplasmic membrane. We used BCP to guide purification of the molecules responsible for each activity, identifying the translation inhibitors bacillaene and bacillaene B (glycosylated bacillaene) and demonstrating that two molecules contribute to cell permeabilitization, the bacteriocin subtilosin and the cyclic peptide sporulation killing factor. Our results suggest that bacillaene mediates translational arrest, and show that bacillaene B has a minimum inhibitory concentration 10 × higher than unmodified bacillaene. Finally, we show that BCP can be used to screen strains on an agar plate without the need for extract preparation, greatly saving time and improving throughput. Thus, BCP simplifies the isolation of novel natural products, by identifying strains, crude extracts and fractions with interesting bioactivities even when multiple activities are present, allowing investigators to focus labor-intensive steps on those with desired activities.

Application of bacterial cytological profiling to crude natural product extracts reveals the antibacterial arsenal of Bacillus subtilis

PubMed Central

Nonejuie, Poochit; Trial, Rachelle M.; Newton, Gerald L.; Lamsa, Anne; Perera, Varahenage Ranmali; Aguilar, Julieta; Liu, Wei-Ting; Dorrestein, Pieter C.; Pogliano, Joe; Pogliano, Kit

2016-01-01

Although most clinically used antibiotics are derived from natural products, identifying new antibacterial molecules from natural product extracts is difficult due to the complexity of these extracts and the limited tools to correlate biological activity with specific molecules. Here, we show that bacterial cytological profiling (BCP) provides a rapid method for mechanism of action determination on plates and in complex natural product extracts and for activity-guided purification. We prepared an extract from Bacillus subtilis 3610 that killed the Escherichia coli lptD mutant and used BCP to observe two types of bioactivities in the unfractionated extract: inhibition of translation and permeablization of the cytoplasmic membrane. We used BCP to guide purification of the molecules responsible for each activity, identifying the translation inhibitors bacillaene and bacillaene B (glycosylated bacillaene) and demonstrating that two molecules contribute to cell permeabilitization, the bacteriocin subtilosin and the cyclic peptide sporulation killing factor. Our results suggest that bacillaene mediates translational arrest, and show that bacillaene B has a minimum inhibitory concentration 10 × higher than unmodified bacillaene. Finally, we show that BCP can be used to screen strains on an agar plate without the need for extract preparation, greatly saving time and improving throughput. Thus, BCP simplifies the isolation of novel natural products, by identifying strains, crude extracts and fractions with interesting bioactivities even when multiple activities are present, allowing investigators to focus labor-intensive steps on those with desired activities. PMID:26648120

Characterization of Metarhizium species and varieties based on molecular analysis, heat tolerance and cold activity

USGS Publications Warehouse

Fernandes, E.K.K.; Keyser, C.A.; Chong, J.P.; Rangel, D.E.N.; Miller, M.P.; Roberts, D.W.

2010-01-01

Aims: The genetic relationships and conidial tolerances to high and low temperatures were determined for isolates of several Metarhizium species and varieties. Methods and Results: Molecular-based techniques [AFLP and rDNA (ITS1, ITS2 and 5??8S) gene sequencing] were used to characterize morphologically identified Metarhizium spp. isolates from a wide range of sources. Conidial suspensions of isolates were exposed to wet heat (45 ?? 0??2??C) and plated on potato dextrose agar plus yeast extract (PDAY) medium. After 8-h exposure, the isolates divided clearly into two groups: (i) all isolates of Metarhizium anisopliae var. anisopliae (Ma-an) and Metarhizium from the flavoviride complex (Mf) had virtually zero conidial relative germination (RG), (ii) Metarhizium anisopliae var. acridum (Ma-ac) isolates demonstrated high heat tolerance (c. 70-100% RG). Conidial suspensions also were plated on PDAY and incubated at 5??C for 15 days, during which time RGs for Ma-an and Ma-ac isolates were virtually zero, whereas the two Mf were highly cold active (100% RG). Conclusions: Heat and cold exposures can be used as rapid tools to tentatively identify some important Metarhizium species and varieties. Significance and Impact of the Study: Identification of Metarhizium spp. currently relies primarily on DNA-based methods; we suggest a simple temperature-based screen to quickly obtain tentative identification of isolates as to species or species complexes. ?? 2009 The Society for Applied Microbiology.

New methods for isolation of keratolytic bacteria inducing intractable hoof wall cavity (Gidoh) in a horse; double screening procedures of the horn powder agar-translucency test and horn zymography

PubMed Central

KUWANO, Atsutoshi; NIWA, Hidekazu; ARAI, Katsuhiko

2017-01-01

ABSTRACT To establish a new system to isolate keratolytic bacteria from the hoof wall cavity (Gidoh) of a racehorse, we invented the horn powder agar-translucency (HoPAT) test and horn zymography (HZ). Using routine bacteriological techniques and these methods, we isolated five strains of keratolytic soil bacteria, which were then identified by means of 16S ribosomal RNA (rRNA) gene sequencing analysis. The findings from the study on the horse suggested that Brevibacterium luteolum played the main role in the local fragility of the hoof, eventually forming a Gidoh in coordination with four other strains of keratolytic bacteria. The double screening procedures of the HoPAT test and HZ were useful and easy techniques for isolating the keratolytic bacteria from the horn lesions. PMID:28400703

Phytate degrading activities of lactic acid bacteria isolated from traditional fermented food

NASA Astrophysics Data System (ADS)

Damayanti, Ema; Ratisiwi, Febiyani Ndaru; Istiqomah, Lusty; Sembiring, Langkah; Febrisiantosa, Andi

2017-03-01

The objective of this study was to determine the potential of LAB with phytate degrading activity from fermented traditional food grain-based and legume-based. Lactic acid bacteria were isolated from different sources of traditional fermented food from Gunungkidul Yogyakarta Indonesia such as gembus tempeh (tofu waste), soybean tempeh, lamtoro tempeh (Leucaena bean) and kara tempeh. Isolation of LAB was performed using Total Plate Count (TPC) on de Man Rogosa Sharpe Agar (MRSA) medium supplemented with CaCO3. They were screened for their ability to degrade myo-inositol hexaphosphate or IP6 by using qualitative streak platemethod with modified de Man Rogosa-MorpholinoPropanesulfonic Acid Sharpe (MRS-MOPS) medium contained sodium salt of phytic acid as substrate and cobalt chloride staining (plate assay) method. The selected isolates were further assayed for phytase activities using quantitative method with spectrophotometer and the two selected isolates growth were optimized. Furthermore, thhe isolates that shown the highest phytase activity was characterized and identified using API 50 CH kitand 16S rRNA gene sequencing. The results showed that there were 18 LAB isolates obtained from samplesand 13 isolates were able to degrade sodium phytate based on qualitative screening. According to quantitative assay, the highest phytate degrading activities were found in TG-2(23.562 U/mL) and TG-1 (19.641 U/mL) isolated from gembus tempeh. The phytate activity of TG-2 was optimum at 37 °C with agitation, while the phytate activity of TG-1 was optimum at 45 °C without agitation. Characterization and identification of TG-2 isolate with the highest phytate degrading activity using API 50 CH and 16S rRNA showed that TG-2had homology with Lactobacillus fermentum. It could be concluded that LAB from from fermented traditional food grain-based and legume-based produced the extracellular phytase. Keywords: lactic acid bacteria, tempeh, phytatedegrading activity

Improving culture media for the isolation of Clostridium difficile from compost.

PubMed

Dharmasena, Muthu; Jiang, Xiuping

2018-06-01

This study was to optimize the detection methods for Clostridium difficile from the animal manure-based composts. Both autoclaved and unautoclaved dairy composts were inoculated with a 12-h old suspension of a non-toxigenic C. difficile strain (ATCC 43593) and then plated on selected agar for vegetative cells and endospores. Six types of enrichment broths supplemented with taurocholate and l-cysteine were assessed for detecting a low level of artificially inoculated C. difficile (ca. 5 spores/g) from dairy composts. The efficacy of selected enrichment broths was further evaluated by isolating C. difficile from 29 commercial compost samples. Our results revealed that using heat-shock was more effective than using ethanol-shock for inducing endospore germination, and the highest endospore count (p < 0.05) was yielded at 60 °C for 25 min. C. difficile agar base, supplemented with 0.1% l-cysteine, 7% defibrinated horse blood, and cycloserine-cefoxitin (CDA-CYS-H-CC agar) was the best medium (p < 0.05) for recovering vegetative cells from compost. C. difficile endospore populations from both types of composts enumerated on both CDA-CYS-H-CC agar supplemented with 0.1% sodium taurocholate (CDA-CYS-H-CC-T agar) and brain heart infusion agar supplemented with 0.5% yeast extract, 0.1% l-cysteine, cycloserine-cefoxitin, and 0.1% sodium taurocholate (BHIA-YE-CYS-CC-T agar) media were not significantly different from each other (p > 0.05). Overall, enrichment of inoculated compost samples in broths containing moxalactam-norfloxacin (MN) produced significantly higher (p < 0.05) spore counts than in non-selective broths or broths supplemented with CC. Enrichment in BHIB-YE-CYS-MN-T broth followed by culturing on an agar containing 7% horse blood and 0.1% taurocholate provided a more sensitive and selective combination of media for detecting a low population of C. difficile from environmental samples with high background microflora. Copyright © 2018 Elsevier Ltd. All rights reserved.

Enterococcus in surface waters from the Des Moines River (Iowa) watershed: location, persistence and vancomycin resistance.

PubMed

Larsen, Bryan; Essmann, Michael K; Geletta, Simon; Duff, Barbara

2012-01-01

The object of this study was to quantify vancomycin-resistant enterococci in surface water from Central Iowa obtained from April 2007 to August 2007. Water from established sampling sites in four watersheds was plated on bile-esculin agar. Presumptively identified enterococci were categorized as "above the level of concern" if the sample contained ≥ 107 CFU per 100 ml. Confirmation of isolates as enterococci was based on growth at elevated temperature in high salt and on Enterococcus agar. Isolates that grew on 6 μg/ml vancomycin agar were deemed resistant. PCR analysis of resistant strains characterized vancomycin resistance genes. 77.2% of surface water samples from Central Iowa contained enterococci. Among enterococcal isolates, 10.4% grew on media containing 6 μg/ml vancomycin. PCR analysis of resistance genes showed a preponderance of VanC2/C3 in the area studied and VanB was not detected. Vancomycin-resistant Enterococcus is present in Central Iowa surface waters but resistance rarely involved VanA genotypes. Nevertheless, the potential for community-acquired infections remains a risk.

Comparative study on the microbiological features of angular cheilitis in HIV seropositive and HIV seronegative patients from South India

PubMed Central

Krishnan, P Anitha; Kannan, Ranganathan

2013-01-01

Objective: This study was designed to compare the microbiological features of angular cheilitis (AC) in human immunodeficiency virus (HIV) seropositive and HIV seronegative individuals, in a group of south Indians. Materials and Methods: Swabs from oral commissures of 46 patients were obtained and inoculated on to Sabouraud's dextrose agar (SDA) supplemented with chloramphenicol, blood agar (BA) and MacConkey's agar (MCA) plates and cultured. α-hemolytic Streptococci, Staphylococcus albus, Staphylococcus aureus, Candida species, Klebsiella species and Pseudomonas species were cultured. Candidal colonies were further speciated by the conventional biotyping technique. Results: In AC of HIV seropositive patients Candida albicans and Staphylococcus aureus were more prevalent than that in HIV seronegative patients. Incidentally in patients with CD4 cell count less than 200 there was an increase in the incidence of Candidal and Staphylococcus aureus colonization when compared to patients with CD4 cell count higher than 200. Conclusion: The present study suggests a definite difference in the microbial flora of AC in HIV seropositive patients than that of HIV seronegative population. PMID:24574650

New culture medium for the presumptive identificaion of Candida albicans and Cryptococcus neoformans.

PubMed Central

Fleming, W H; Hopkins, J M; Land, G A

1977-01-01

A new medium composed of Tween 80, oxgall, caffeic acid, and Davis agar (TOC) that provides for the rapid presumptive identification of Candida albicans and Cryptococcus neoformans is described herein. C. albicans is differentiated from other yeasts by the sequential production of germ tubes and chlamydospores. In a comparison with cormeal agar control plates, there was an increase of chlamydospore-forming strains of C. albicans (97.1% versus 87.2%) and a decrease in the time required for chlamydospore formation (24 h versus 48 h). C. neoformans produced a brown pigment of TOC, which is specific for its identification, thus differentiating it from the other yeasts. A comparison of 24-h pigment production by C. neoformans on TOC with that of birdseed agar showed a dark, coffee brown color in the former cultures and a light brown color in the latter. The change in pigmentation of C. neoformans, as well as morphological changes in C. albicans, can be induced within 3 to 12 h and in not more than 24 h on the TOC medium. Images PMID:321472

Comparative study of 6-APA production by free and agar immobilized bacteria in nutrient broth culture.

PubMed

Dolui, A K; Das, S

2011-04-01

In the present study different bacterial samples were isolated from soil of different places of Dibrugarh and screened for biotransformation ability to produce 6-Aminopenicillanic acid. Among ten isolated bacterial samples, three gram positive bacterial samples designated as AKDD-2, AKDD-4 and AKDD-6 showed the production of 6-APA from penicillin G. Assessment of production of 6-APA after incubation in penicillin G (2 mg/ml) by three different samples separately in free and agar immobilization state was done by HPLC analysis. Reusability of immobilized cells was found successful up to 14 days.

Prevalence of Candida dubliniensis among the stored vaginal Candida isolates in a Turkish hospital.

PubMed

Acikgoz, Z C; Sancak, B; Gamberzade, S; Misirlioglu, M

2004-10-01

In this study, 600 stored Candida species, isolated from vaginal samples of immunocompetent women, and phenotypically identified as C. albicans on the basis of a positive germ tube test, were screened for the presence of C. dubliniensis by three phenotypical methods. Only one strain (0.17%) failed to grow at 45 degrees C, and produced abundant chlamydospores on both the cornmeal-Tween 80 agar and the Staib agar. This strain was identified as C. dubliniensis by using the ID-32C kit (bioMerieux Vitek) and confirmed by DNA sequencing of internal transcript spacer (ITS) region.

Assay for adhesion and agar invasion in S. cerevisiae.

PubMed

Guldal, Cemile G; Broach, James

2006-11-08

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8 The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar. Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

Assay for Adhesion and Agar Invasion in S. cerevisiae

PubMed Central

Guldal, Cemile G; Broach, James

2006-01-01

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8 The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar. Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation. PMID:18704175

Occurrence of yeasts, enterococci and other enteric bacteria in subgingival biofilm of HIV-positive patients with chronic gingivitis and necrotizing periodontitis

PubMed Central

Gaetti-Jardim Júnior, Elerson; Nakano, Viviane; Wahasugui, Thais C.; Cabral, Fátima C.; Gamba, Rosa; Avila-Campos, Mario Julio

2008-01-01

The purpose of this study was to determine the prevalence of enteric bacteria and yeasts in biofilm of 80 HIV-positive patients with plaque-associated gingivitis or necrotizing periodontitis. Patients were subjected to extra, intra oral and radiographic examinations. The oral hygiene, bleeding on probing, gingival conditions, and attachment loss were evaluated. Clinical specimens were collected from gingival crevices or periodontal pockets, transferred to VMGA III, diluted and transferred to Sabouraud Dextrose agar with 100 μg/ml of chloramphenicol, peptone water, EVA broth, EMB agar, SS agar, Bile esculin agar and Brilliant green agar. Isolation of yeasts was carried out at room temperature, for 3-7 days; and for the isolation of enteric microorganisms plates were incubated at 37°C, for 24-48 h. The yeasts identification was performed according to the carbon and nitrogen assimilation, fermentation of carbohydrates and germ tube formation. Bacteria were identified according to their colonial and cellular morphologies and biochemical tests. Yeasts were identified as Candida albicans and its occurrence was more common in patients with CD4+ below 200/mm3 and was affected by the extension of periodontal involvement (P = 0.0345). Enteric bacteria recovered from clinical specimens were identified as Enterobacter sakazakii, Enterobacter cloacae, Serratia liquefaciens, Klebsiella oxytoca and Enterococcus sp. Enterobacteriaceae and enterococci were detected in 32.5% of clinical samples from patients with necrotizing periodontitis. In conclusion, non-oral pathogenic bacteria and C. albicans were more prevalent in periodontal sites of HIV-positive patients with necrotizing periodontitis and chronic gingivitis. PMID:24031212

Preliminary Evaluation of the Control of Microbial Fouling by Laboratory and Pilot-Scale Air-Stripping Columns

DTIC Science & Technology

1985-03-01

used to remove trichloroethylene (TCE) from contaminated well water. 7 MATERIALS Chemicals: Trichloroethylene (Aldrich chemical, Milwaukee, WI), sodium ...Cleveland, OH), sodium hydrcxide (J.T. Baker, Phillipsburg, NJ), potassium dichloroisocyanurate (Dorex Inc., Frankfort, IL), potassium iodide starch...NJ). Media and Reagents: Plate count agar (Difco Laboratories, Detroit, MI), lauryl tryptose broth (Difco Laboratories, Detroit, MI), motility medium

A Direct Quantitative Agar-Plate Based Assay for Analysis of Pseudomonas protegens PF-5 Degradation of Polyurethane Films (Postprint)

DTIC Science & Technology

2014-10-02

Journal article published in International Biodeterioration & Biodegradation 95 (2014) 311-319. The U.S. Government is joint author of the work and...SUBJECT TERMS pseudomonas biofilms, polyurethane, biodegradation , FTIR spectroscopy, citrate, impranil 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF...International Biodeterioration & Biodegradation 95 (2014) 311e319Contents lists avaiInternational Biodeterioration & Biodegradation journal homepage

Bacterial Chemotaxis to Naphthalene and Nitroarene Compounds

DTIC Science & Technology

2008-07-31

Qualitative capillary assays showing chemotaxis of succinate-grown 17 (uninduced) and induced (succinate plus salicylate -grown) Acidovorax sp. JS42...succinate plus 2NT- or succinate plus salicylate -grown) wild-type Acidovorax sp. JS42 cells List of Tables Table 1. Summary of chemotaxis...mM salicylate , or naphthalene crystals. Noble agar (1.8%; Difco) was used to solidify MSB medium for plates. For plasmid selection and maintenance

Survival of Phytophthora ramorum Chlamydospores at high and low temperatures

Treesearch

Paul W. Tooley; Marsha Browning

2008-01-01

Chlamydospores were produced as described by Colburn and Shishkoff (Phytopathology 96:S25). Samples (5cc) of chlamydospores in sand inoculum were placed in 15 ml conical plastic test tubes and incubated at selected temperatures for 1, 2, 3, 4, and 7 days. Following incubation, tube contents were resuspended in 0.2 percent water agar and 1 ml was plated onto PARPH...

Comparison of Antibacterial Efficacy of Turmeric Extract, Morinda Citrifolia and 3% Sodium Hypochlorite on Enterococcus faecalis: An In-vitro Study.

PubMed

Chaitanya, Bathula Vimala; Somisetty, Kusum Valli; Diwan, Abhinav; Pasha, Shiraz; Shetty, Nandaprasad; Reddy, Yashwanth; Nadigar, Shankar

2016-10-01

Sodium hypochlorite (NaOCl), the most commonly used irrigant, has many potential properties like its unique ability to dissolve pulp tissue, excellent antimicrobial activity, but has a cytotoxic effect when injected into periapical tissues. It is also known to produce allergic reactions, foul smell and taste, and potential for corrosion. Facultative organisms such as Enterococcus faecalis and aerobes like Staphylococcus aureus are considered to be the most resistant species and one of the possible causes of root canal treatment failure. So there is a need to find an alternative to sodium hypochlorite to act against these resistant microorganisms. To evaluate and compare the antibacterial efficacy of morinda citrifolia and turmeric extract with 3% NaOCl as a root canal irrigant, against E. faecalis and S.aureus . The antimicrobial efficacy was assessed in vitro using agar well diffusion method. Agar plates were prepared using Brain-Heart Infusion (BHI) agar. Cultures of E.faecalis and S.aureus were grown in nutrient broth at 37°C. Plates were incubated for 24 hours at 37°C and microbial zones of inhibition were recorded. Statistical analysis was performed using ANOVA. NaOCl (3%) showed larger zones of inhibition than herbal irrigants against both the microorganisms. Among the herbal irrigants, morinda citrifolia showed larger zones of inhibition than turmeric hydro-alcoholic extract and turmeric water extract which was statistically significant (p<0.05). NaOCl (3%) showed maximum antibacterial activity against E. faecalis , followed by morinda citrifolia and turmeric extracts. Considering the potential for undesirable properties of NaOCl, use of herbal alternatives in endodontics might prove to be advantageous.

Comparison of Antibacterial Efficacy of Turmeric Extract, Morinda Citrifolia and 3% Sodium Hypochlorite on Enterococcus faecalis: An In-vitro Study

PubMed Central

Somisetty, Kusum Valli; Diwan, Abhinav; Pasha, Shiraz; Shetty, Nandaprasad; Reddy, Yashwanth; Nadigar, Shankar

2016-01-01

Introduction Sodium hypochlorite (NaOCl), the most commonly used irrigant, has many potential properties like its unique ability to dissolve pulp tissue, excellent antimicrobial activity, but has a cytotoxic effect when injected into periapical tissues. It is also known to produce allergic reactions, foul smell and taste, and potential for corrosion. Facultative organisms such as Enterococcus faecalis and aerobes like Staphylococcus aureus are considered to be the most resistant species and one of the possible causes of root canal treatment failure. So there is a need to find an alternative to sodium hypochlorite to act against these resistant microorganisms. Aim To evaluate and compare the antibacterial efficacy of morinda citrifolia and turmeric extract with 3% NaOCl as a root canal irrigant, against E. faecalis and S.aureus. Materials and Methods The antimicrobial efficacy was assessed in vitro using agar well diffusion method. Agar plates were prepared using Brain-Heart Infusion (BHI) agar. Cultures of E.faecalis and S.aureus were grown in nutrient broth at 37°C. Plates were incubated for 24 hours at 37°C and microbial zones of inhibition were recorded. Statistical analysis was performed using ANOVA. Results NaOCl (3%) showed larger zones of inhibition than herbal irrigants against both the microorganisms. Among the herbal irrigants, morinda citrifolia showed larger zones of inhibition than turmeric hydro-alcoholic extract and turmeric water extract which was statistically significant (p<0.05). Conclusion NaOCl (3%) showed maximum antibacterial activity against E. faecalis, followed by morinda citrifolia and turmeric extracts. Considering the potential for undesirable properties of NaOCl, use of herbal alternatives in endodontics might prove to be advantageous. PMID:27891459

Antibiotic resistance and biofilm formation among coagulase-negative staphylococci isolated from clinical samples at a tertiary care hospital of eastern Nepal.

PubMed

Shrestha, Lok Bahadur; Bhattarai, Narayan Raj; Khanal, Basudha

2017-01-01

Coagulase negative staphylococci were long regarded non-pathogenic as they are the commensals of human skin and mucosa but the recent changes in the medical practice and changes in underlying host populations, they are being considered significant pathogens associated with number of nosocomial infections. The objective of the study was to determine the species, antimicrobial susceptibility pattern, biofilm forming ability of the clinically significant CoNS isolates and to compare the different methods for the detection of biofilm formation. A total of 52 clinically significant CoNS isolates obtained from different units during a year period were studied. Characterization was done using standard microbiological guidelines and antimicrobial susceptibility was done following CLSI guidelines. Biofilm formation was detected by using three methods i.e. tissue culture plate method, congo red agar method and tube adherence method. Among 52 isolates , S. epidermidis (52%) was the most common species which was followed by S. saprophyticus (18%) and S. haemolyticus (14%). Antimicrobial susceptibility pattern of CoNS documented resistance of 80% to ampicillin. Resistance to cefoxitin and ceftriaxone was observed in 58% of the isolates. Biofilm formation was observed in 65.38% of the isolates. The accuracy of Congo red agar and tube adherence method for the detection of biofilm formation was 82% and 76% respectively. CoNS isolates obtained from clinical samples should be processed routinely and antimicrobial susceptibility testing should be performed. Multidrug-resistant CoNS are prevalent. All the three methods i.e. tissue culture plate, Congo red agar and tube adherence method can be used in detecting biofilm formation.

AUTORADIOGRAPHIC ANALYSIS ON AGAR PLATES OF ANTIGENS FROM SUB CELLULAR FRACTIONS OF RAT LIVER SLICES

PubMed Central

Morgan, W. S.; Perlmann, P.; Hultin, T.

1961-01-01

Slices of rat livers were incubated with 14C amino acids, homogenized, and subjected to differential centrifugation. The microsomes were further extracted with the non-ionic detergent Lubrol W and with EDTA. These extracts and the microsome free "cell sap," freed from the pH 5 precipitable fraction, were subsequently reacted with antisera using agar diffusion techniques. The antisera employed were obtained from rabbits injected with different subcellular fractions of rat liver or with rat serum proteins. When the agar diffusion plates were autoradiographed it was found that some of the precipitates were radioactive while others were not. Control experiments indicated that this labeling was due to the specific incorporation of 14C amino acids into various rat liver antigens during incubation of the slices rather than to a non-specific adsorption of radioactive material to the immunological precipitates. When the slices were incubated with the isotope for up to 30 minutes, the serum proteins which could be extracted from the microsomes with the detergent were strongly labeled, as were a number of additional microsomal antigens of unknown significance. In contrast, the serum proteins present in the cell sap were only weakly labeled. Most of the typical cell sap proteins, both those precipitable and those soluble at pH 5, seemed to remain unlabeled. No consistently reproducible results were obtained with the EDTA extracts of the ribosomal residues remaining after extraction of the microsomes with the detergent. Incubation of the liver slices for longer periods (up to 120 minutes) led to a strong labeling of the serum proteins in the cell sap as well as to the appearance of labeling in additional cell sap proteins. The results are discussed with regard to the subcellular site of synthesis and the metabolism of the different antigens. PMID:13772607

Estimation method for serial dilution experiments.

PubMed

Ben-David, Avishai; Davidson, Charles E

2014-12-01

Titration of microorganisms in infectious or environmental samples is a corner stone of quantitative microbiology. A simple method is presented to estimate the microbial counts obtained with the serial dilution technique for microorganisms that can grow on bacteriological media and develop into a colony. The number (concentration) of viable microbial organisms is estimated from a single dilution plate (assay) without a need for replicate plates. Our method selects the best agar plate with which to estimate the microbial counts, and takes into account the colony size and plate area that both contribute to the likelihood of miscounting the number of colonies on a plate. The estimate of the optimal count given by our method can be used to narrow the search for the best (optimal) dilution plate and saves time. The required inputs are the plate size, the microbial colony size, and the serial dilution factors. The proposed approach shows relative accuracy well within ±0.1log10 from data produced by computer simulations. The method maintains this accuracy even in the presence of dilution errors of up to 10% (for both the aliquot and diluent volumes), microbial counts between 10(4) and 10(12) colony-forming units, dilution ratios from 2 to 100, and plate size to colony size ratios between 6.25 to 200. Published by Elsevier B.V.

Comparison of methods for isolation and enumeration of thermophilic actinomycetes from dust.

PubMed Central

Treuhaft, M W; Arden Jones, M P

1982-01-01

Thermophilic actinomycetes are the primary sensitizing agents in farmer's lung disease. We compared dilution pour-plate and spread-plate methods for their usefulness in enumerating thermophilic actinomycetes in moldy silage dust and evaluated the ability of a nonquantitative gravity settling technique to recover thermophilic actinomycetes from moldy silage. Spread plates and pour plates yielded similar estimates of total thermophiles. Higher counts were observed on spread plates (P less than 0.05) for Thermoactinomyces candidus, Micropolyspora faeni, and Saccharomonospora viridis. M. faeni and S. viridis were less efficient than T. candidus in breaking through the agar of pour plates to form colonies which could be identified. Coefficients of variability were less than 10% for the two methods. The relative proportion of organisms recovered by the settling method correlated well with that recovered on spread plates for M. faeni (r = 0.79), S. viridis (r = 0.88), and Thermomonospora spp. (r = 0.79), but not well for T. candidus (r = 0.28). When sophisticated air-sampling equipment is not available, dilution spread plates of dust washings provide a reproducible method for enumerating a broad range of thermophilic actinomycetes of interest. The gravity settling method is a simple, rapid alternative when isolation is all that is required. PMID:6761363

The planning and establishment of a sample preparation laboratory for drug discovery

PubMed Central

Dufresne, Claude

2000-01-01

Nature has always been a productive source of new drugs. With the advent of high-throughput screening, it has now become possible to rapidly screen large sample collections. In addition to seeking greater diversity from natural product sources (micro-organisms, plants, etc.), fractionation of the crude extracts prior to screening is becoming a more important part of our efforts. As sample preparation protocols become more involved, automation can help to achieve and maintain a desired sample throughput. To address the needs of our screening program, two robotic systems were designed. The first system processes crude extracts all the way to 96-well plates, containing solutions suitable for screening in biological and biochemical assays. The system can dissolve crude extracts, fractionate them on solid-phase extraction cartridges, dry and weigh each fraction, re-dissolve them to a known concentration, and prepare mother plates. The second system replicates mother plates into a number of daughter plates. PMID:18924691

Auxin, ethylene and the regulation of root growth under mechanical impedance

NASA Astrophysics Data System (ADS)

Sharma, Rameshwar; Santisree, Parankusam; Nongmaithem, Sapana; Sreelakshmi, Yellamaraju

2012-07-01

Among the multitude functions performed by plant roots, little information is available about the mechanisms that allow roots to overcome the soil resistance, in order to grow in the soil to obtain water and nutrient. Tomato (Solanum lycopersicum) seedlings grown on horizontally placed agar plates showed a progressive decline in the root length with the increasing impedance of agar media. The incubation with 1-methylcyclopropane (1-MCP), an inhibitor of ethylene perception, led to aerial growth of roots. In contrast, in absence of 1-MCP control roots grew horizontally anchored to the agar surface. Though 1-MCP-treated and control seedlings showed differential ability to penetrate in the agar, the inhibition of root elongation was nearly similar for both treatments. While increased mechanical impedance also progressively impaired hypocotyl elongation in 1-MCP treated seedlings, it did not affect the hypocotyl length of control seedlings. The decline in root elongation was also associated with increased expression of DR5::GUS activity in the root tip signifying accumulation of auxin at the root tip. The increased expression of DR5::GUS activity in the root tip was also observed in 1-MCP treated seedlings, indicating independence of this response from ethylene signaling. Our results indicate operation of a sensing mechanism in root that likely operates independently of ethylene but involves auxin to determine the degree of impedance of the substratum.

Nitrate analogs as attractants for soybean cyst nematode.

PubMed

Hosoi, Akito; Katsuyama, Tsutomu; Sasaki, Yasuyuki; Kondo, Tatsuhiko; Yajima, Shunsuke; Ito, Shinsaku

2017-08-01

Soybean cyst nematode (SCN) Heterodera glycines Ichinohe, a plant parasite, is one of the most serious pests of soybean. In this paper, we report that SCN is attracted to nitrate and its analogs. We performed attraction assays to screen for novel attractants for SCN and found that nitrates were attractants for SCN and SCN recognized nitrate gradients. However, attraction of SCN to nitrates was not observed on agar containing nitrate. To further elucidate the attraction mechanism in SCN, we performed attraction assays using nitrate analogs ([Formula: see text], [Formula: see text], [Formula: see text]). SCN was attracted to all nitrate analogs; however, attraction of SCN to nitrate analogs was not observed on agar containing nitrate. In contrast, SCN was attracted to azuki root, irrespective of presence or absence of nitrate in agar media. Our results suggest that the attraction mechanisms differ between plant-derived attractant and nitrate.

Induction of a global stress response during the first step of Escherichia coli plate growth.

PubMed

Cuny, Caroline; Lesbats, Maïalène; Dukan, Sam

2007-02-01

We have investigated the first events that occur when exponentially grown cells are transferred from a liquid medium (Luria-Bertani [LB]) to a solid medium (LB agar [LBA]). We observed an initial lag phase of 180 min for the wild type MG1655 without any apparent growth. This lack of growth was independent of the bacterial physiological state (either the stationary or the exponential phase), the solid medium composition, or the number of cells on the plate, but it was dependent on the bacterial genotype. Using lacZ-reporter fusions and two-dimensional electrophoresis analysis, we observed that when cells from exponential-phase cultures were plated on LBA, several global regulons, like heat shock regulons (RpoH, RpoE, CpxAR) and oxidative-stress regulons (SoxRS, OxyR, Fur), were immediately induced. Our results indicate that in order to grow on plates, bacteria must not only adapt to new conditions but also perceive a real stress.

Comparison of Dry Medium Culture Plates for Mesophilic Aerobic Bacteria in Milk, Ice Cream, Ham, and Codfish Fillet Products

PubMed Central

Park, Junghyun; Kim, Myunghee

2013-01-01

This study was performed to compare the performance of Sanita-Kun dry medium culture plate with those of traditional culture medium and Petrifilm dry medium culture plate for the enumeration of the mesophilic aerobic bacteria in milk, ice cream, ham, and codfish fillet. Mesophilic aerobic bacteria were comparatively evaluated in milk, ice cream, ham, and codfish fillet using Sanita-Kun aerobic count (SAC), Petrifilm aerobic count (PAC), and traditional plate count agar (PCA) media. According to the results, all methods showed high correlations of 0.989~1.000 and no significant differences were observed for enumerating the mesophilic aerobic bacteria in the tested food products. SAC method was easier to perform and count colonies efficiently as compared to the PCA and PAC methods. Therefore, we concluded that the SAC method offers an acceptable alternative to the PCA and PAC methods for counting the mesophilic aerobic bacteria in milk, ice cream, ham, and codfish fillet products. PMID:24551829

bdhA-patD operon as a virulence determinant, revealed by a novel large-scale approach for identification of Legionella pneumophila mutants defective for amoeba infection.

PubMed

Aurass, P; Pless, B; Rydzewski, K; Holland, G; Bannert, N; Flieger, A

2009-07-01

Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular parasite of eukaryotic cells. In the environment, it colonizes amoebae. After being inhaled into the human lung, the bacteria infect and damage alveolar cells in a way that is mechanistically similar to the amoeba infection. Several L. pneumophila traits, among those the Dot/Icm type IVB protein secretion machinery, are essential for exploiting host cells. In our search for novel Legionella virulence factors, we developed an agar plate assay, designated the scatter screen, which allowed screening for mutants deficient in infecting Acanthamoeba castellanii amoebae. Likewise, an L. pneumophila clone bank consisting of 23,000 transposon mutants was investigated here, and 19 different established Legionella virulence genes, for example, dot/icm genes, were identified. Importantly, 70 novel virulence-associated genes were found. One of those is L. pneumophila bdhA, coding for a protein with homology to established 3-hydroxybutyrate dehydrogenases involved in poly-3-hydroxybutyrate metabolism. Our study revealed that bdhA is cotranscribed with patD, encoding a patatin-like protein of L. pneumophila showing phospholipase A and lysophospholipase A activities. In addition to strongly reduced lipolytic activities and increased poly-3-hydroxybutyrate levels, the L. pneumophila bdhA-patD mutant showed a severe replication defect in amoebae and U937 macrophages. Our data suggest that the operon is involved in poly-3-hydroxybutyrate utilization and phospholipolysis and show that the bdhA-patD operon is a virulence determinant of L. pneumophila. In summary, the screen for amoeba-sensitive Legionella clones efficiently isolated mutants that do not grow in amoebae and, in the case of the bdhA-patD mutant, also human cells.

Screening and Crystallization Plates for Manual and High-throughput Protein Crystal Growth

NASA Technical Reports Server (NTRS)

Thorne, Robert E. (Inventor); Berejnov, Viatcheslav (Inventor); Kalinin, Yevgeniy (Inventor)

2010-01-01

In one embodiment, a crystallization and screening plate comprises a plurality of cells open at a top and a bottom, a frame that defines the cells in the plate, and at least two films. The first film seals a top of the plate and the second film seals a bottom of the plate. At least one of the films is patterned to strongly pin the contact lines of drops dispensed onto it, fixing their position and shape. The present invention also includes methods and other devices for manual and high-throughput protein crystal growth.

Simplifying Collection of Corneal Specimens in Cases of Suspected Bacterial Keratitis

PubMed Central

Kaye, Stephen B.; Rao, Prasad G.; Smith, Godfrey; Scott, John A.; Hoyles, Sharon; Morton, Clare E.; Willoughby, Colin; Batterbury, Mark; Harvey, Graham

2003-01-01

Identification of the causative organisms in suspected bacterial keratitis traditionally involves collecting multiple corneal scrapes, which are plated directly onto different solid agar culture media. Difficulties have been reported with this practice, so the development of a simpler diagnostic method in suspected bacterial keratitis would be useful. It is unclear whether a single corneal scrape sent to the microbiology laboratory in a liquid transport culture medium (indirect method) is as reliable for the diagnosis of bacterial keratitis as inoculation of multiple scrapes directly onto agar plates (direct method). To investigate this, bacterial recovery was assessed following transfer and transport of different concentrations and types of bacteria from an artificially contaminated surgical blade into brain heart infusion (BHI). Bacterial recovery rates between the proposed (indirect) and standard (direct) method were then compared after the in vitro inoculation of pig corneas and following specimen collection in patients with presumed bacterial ulcerative keratitis. Recovery of bacteria from contaminated surgical blades was found to be the same from both solid and liquid culture media. There was no significant difference in the numbers of positive cultures from solid (direct) and liquid (indirect) culture media, both in the experimental pig cornea inoculation study (P = 0.34) and in experiments with patients with clinical infections (P = 0.4), with an 85.2% agreement between methods (kappa = 0.61, P < 0.0001). In conclusion, therefore, the collection of two corneal scrapes, one used for Gram staining and the other transported in BHI followed by plating and subculturing in an enrichment medium, provides a simple method for the investigation of presumed bacterial keratitis. PMID:12843063

The prevalence and epidemiology of plasmid-mediated penicillin and tetracycline resistance among Neisseria gonorrhoeae isolates in Guangzhou, China, 2002-2012.

PubMed

Zheng, Heping; Wu, Xingzhong; Huang, Jinmei; Qin, Xiaolin; Xue, Yaohua; Zeng, Weiying; Lan, Yinyuan; Ou, Jiangli; Tang, Sanmei; Fang, Mingheng

2015-10-09

Gonococcal antimicrobial resistance is a global problem. Different resistance plasmids have emerged and spread among the isolates of Neisseria gonorrhoeae worldwide and in China. We conducted this study to monitor the plasmid-mediated penicillin and tetracycline resistance among N. gonorrhoeae isolates in Guangzhou from 2002 to 2012. Consecutive isolates of N. gonorrhoeae were collected from outpatients with gonorrhea attending the STD clinic in Guangdong Provincial Centre for Skin Diseases and STIs Control and Prevention. Penicillinase-producing N. gonorrhoeae (PPNG) isolates were analyzed by the paper acidometric method. Plasmid-mediated resistance to tetracycline in N. gonorrhoeae (TRNG) isolates was screened by the agar plate dilution method. Plasmid types were determined for TRNG and PPNG isolates using polymerase chain reaction (PCR). Minimum inhibitory concentrations (MICs) to penicillin and tetracycline were detected by the agar plate dilution. Of 1378 consecutive N. gonorrhoeae isolates, 429 PPNG and 639 TRNG isolates were identified. The prevalence of PPNG, TRNG, and PPNG/TRNG increased from 18.3 to 47.1 % (χ (2) = 31.57, p < 0.001), from 29.4 to 52.1 % (χ (2) = 16.28, p < 0.001) and from 10.0 to 26.2 % (χ (2) = 10.46, p < 0.001) between 2002 and 2012, respectively. Genotyping of plasmids among PPNGs showed that the majority (93.7 %) of the isolates were the Asian type plasmids, while the African type plasmid emerged in 2008 and rapidly increased to 14.0 % in 2012 (χ (2) = 25.03, p < 0.001). For TRNGs, all 639 isolates carried the Dutch type plasmid. MICs of penicillin G and tetracycline persisted at high levels and the MIC90s were 32-fold higher than the resistant cutoff point over 11 years. The prevalence rates of penicillin- and tetracycline-resistant N. gonorrhoeae varied from 90.9 to 91.1 % and from 88.3 to 89.3 % during 2002 to 2012, respectively. Resistance to penicillin and tetracycline among N. gonorrhoeae isolates remained at high levels in Guangzhou. The Asian type PPNG continued to spread and Dutch type TRNG was still the dominant strain. The African type PPNG has emerged and is spreading rapidly.

Investigation of biofilm formation on contact eye lenses caused by methicillin resistant Staphylococcus aureus.

PubMed

Khalil, M A; Sonbol, F I

2014-01-01

The objective was to investigate the biofilm-forming capacity of methicillin resistant Staphylococcus aureus (MRSA) isolated from eye lenses of infected patients. A total of 32 MRSA isolated from contact lenses of patients with ocular infections were screened for their biofilm-forming capacity using tube method (TM), Congo red agar (CRA), and microtiter plate (MtP) methods. The effect of some stress factor on the biofilm formation was studied. The biofilm-forming related genes, icaA, icaD and 10 microbial surface components that recognize adhesive matrix molecule (MSCRAMM), of the selected MRSA were also detected using polymerase chain reaction. Of 32 MRSA isolates, 34.37%, 59.37%, and 81.25% showed positive results using CRA, TM or MtP, respectively. Biofilm production was found to be reduced in the presence of ethanol or ethylenediaminetetraacetic acid and at extreme pH values. On the other hand, glucose or heparin leads to a concentration dependent increase of biofilm production by the isolates. The selected biofilm producing MRSA isolate was found to harbor the icaA, icaD and up to nine of 10 tested MSCRAMM genes, whereas the selected non biofilm producing MRSA isolate did not carry any of the tested genes. The MtP method was found to be the most effective phenotypic screening method for detection of biofilm formation by MRSA. Furthermore, the molecular approach should be taken into consideration for the rapid and correct diagnosis of virulent bacteria associated with contact eye lenses.

Eradication of Helicobacter pylori infection by the probiotic strains Lactobacillus johnsonii MH-68 and L. salivarius ssp. salicinius AP-32.

PubMed

Hsieh, Pei-Shan; Tsai, Yi-Chun; Chen, Yi-Chun; Teh, Su-Fen; Ou, Chung-Mou; King, V An-Erl

2012-12-01

The current therapy for Helicobacter pylori infection includes antimicrobial agents and proton pump inhibitors. We have examined the ability of Lactobacillus spp. to inhibit H. pylori infection. Probiotic strains isolated from samples of adult feces, infant feces, breast milk, and vaginal swab collected from healthy volunteers in Taiwan and commercially available strains were screened for antagonism toward H. pylori. Inhibition liquid culture assay was used to screen potential anti-H. pylori activity. Then, we performed agar plate inhibition assay, and assays to determine the capacity of probiotics for adhesion, and inhibition and killing of H. pylori, and measured the levels of IL-8 and IL-10. Using animal models, we studied regulation of gastric acid and histopathological changes accompanying anti-H. pylori activity. We found that six of the tested strains suppressed urease activity of H. pylori: Lactobacillus acidophilus TYCA08, L. acidophilus TYCA15, L. johnsonii MH-68, and L. salivarius subsp. salicinius AP-32 were more effective than the others. In vivo, L. johnsonii MH-68 and L. salivarius subsp. salicinius AP-32 alone or in combination, reduced the H. pylori load in the gastric mucosa, and also reduced inflammatory chemokine expression and lymphocyte infiltration. Lactobacillus johnsonii MH-68 and L. salivarius subsp. salicinius AP-32 effectively suppress H. pylori viability, and when used as probiotics, they may help decrease the occurrence of gastritis, and even reduce the risk of H. pylori infection. © 2012 Blackwell Publishing Ltd.

Volatiles from the xylarialean fungus Hypoxylon invadens.

PubMed

Dickschat, Jeroen S; Wang, Tao; Stadler, Marc

2018-01-01

The volatiles emitted by agar plate cultures of the xylarialean fungus Hypoxylon invadens were investigated by use of a closed loop stripping apparatus in combination with GC-MS. Several aromatic compounds were found that could only be identified by comparison to all possible constitutional isomers with different ring substitution patterns. For the set of identified compounds a plausible biosynthetic scheme was suggested that gives further support for the assigned structures.

Draft Genome Sequences of Two Janthinobacterium lividum Strains, Isolated from Pristine Groundwater Collected from the Oak Ridge Field Research Center

PubMed Central

Wu, Xiaoqin; Deutschbauer, Adam M.; Kazakov, Alexey E.; Wetmore, Kelly M.; Cwick, Bryson A.; Walker, Robert M.; Novichkov, Pavel S.; Arkin, Adam P.

2017-01-01

ABSTRACT We present here the draft genome sequences of two Janthinobacterium lividum strains, GW456P and GW458P, isolated from groundwater samples collected from a background site at the Oak Ridge Field Research Center. Production of a purple pigment by these two strains was observed when grown on diluted (1/10) LB agar plates. PMID:28663297

MALDI-TOF mass spectrometry for early identification of bacteria grown in blood culture bottles.

PubMed

Zabbe, Jean-Benoît; Zanardo, Laura; Mégraud, Francis; Bessède, Emilie

2015-08-01

This note reports an interesting way to rapidly identify bacteria grown from blood culture bottles. Chocolate agar plates were inoculated with 1 drop of the positive blood bottle medium. After a 3-hour incubation, the growth veil was submitted to MALDI-TOF mass spectrometry: 77% of the bacteria present have been correctly identified. Copyright © 2015 Elsevier B.V. All rights reserved.

Chromogenic agar medium for detection and isolation of Escherichia coli serogroups O26,O45,O103,O111,O121, and O145 from fresh beef and cattle feces

USDA-ARS?s Scientific Manuscript database

Non-O157 Shiga toxin–producing Escherichia coli (STEC) strains are clinically important foodborne pathogens. Unlike E. coli O157:H7, these foodborne pathogens have no unique biochemical characteristics to readily distinguish them from other E. coli strains growing on plating media. In this study, a ...

Effectiveness of Vaporous Hydrogen Peroxide for the Decontamination of Representative Military Materials

DTIC Science & Technology

2004-11-16

Agent Resistant Coating (CARC) Geobacillus stearothermophilus ATCC 7953 Flott Glass Galvanized aluminum Polyimid (Kapton) Nylon Webbing Runway...spores were harvested from 7-10 day –old cultures plated upon Lemko Agar. The spores were washed thrice in sterile distilled water (dH2O), and...NEGATIVE SURROGATE Inocula of log-phase Y. ruckeri were prepared immediately before application to sterile coupons. Cells were grown in nutrient

Rapid, Multiplexed Microfluidic Phage Display

DTIC Science & Technology

2012-01-01

affinity phage- displayed peptides for multiple targets in just a single round and without the need for bacterial infection. The chip is shown to be able...by bacterial titer and amplification, and at least two additional rounds of selection. After the final round of biopan- ning, eluted phage are grown on...agar plates, and individual plaques are selected for DNA characterization to determine the amino acid sequence of the phage-displayed peptides. While

Detection of fungal hyphae using smartphone and pocket magnifier: going cellular.

PubMed

Agarwal, Tushar; Bandivadekar, Pooja; Satpathy, Gita; Sharma, Namrata; Titiyal, Jeewan S

2015-03-01

The aim of this study was to detect fungal hyphae in a corneal scraping sample using a cost-effective assembly of smartphone and pocket magnifier. In this case report, a tissue sample was obtained by conventional corneal scraping from a clinically suspicious case of mycotic keratitis. The smear was stained with Gram stain, and a 10% potassium hydroxide mount was prepared. It was imaged using a smartphone coupled with a compact pocket magnifier and integrated light-emitting diode assembly at point-of-care. Photographs of multiple sections of slides were viewed using smartphone screen and pinch-to-zoom function. The same slides were subsequently screened under a light microscope by an experienced microbiologist. The scraping from the ulcer was also inoculated on blood agar and Sabouraud dextrose agar. Smartphone-based digital imaging revealed the presence of gram-positive organism with hyphae. Examination under a light microscope also yielded similar findings. Fusarium was cultured from the corneal scraping, confirming the diagnosis of mycotic keratitis. The patient responded to topical 5% natamycin therapy, with resolution of the ulcer after 4 weeks. Smartphones can be successfully used as novel point-of-care, cost-effective, reliable microscopic screening tools.

Screening of bacterial antagonists for biological control of Phytophthora blight of pepper.

PubMed

Rajkumar, M; Lee, Wang Hyu; Lee, Kui Jae

2005-01-01

The aim of this study was to assess the potential of bacterial antagonists to control Phytophthora blight of pepper caused by P. capsici using different screening methods. Among a collection of fluorescent pseudomonas isolated from the rhizosphere of pepper, twelve isolates were initially selected based on dual culture assay on potato dextrose agar and corn meal agar. Further, these twelve isolates were screened for the reduction of disease severity caused by P. capsici using detached leaves and seedling assay. Most of the antagonists showed varying levels of antagonism against P. capsici in both detached leaves and seedlings assay. In addition, few isolates increased shoot and root length of pepper in seedling assays. Among them, isolate PS119 showing highest ability to reduce the disease severity in the in vitro seedling assay was found to be the most efficient antagonists against P. capsici in the in vivo biological control tests. These results indicate that the in vitro seedling assay can be used as a rapid and more accurate technique for the selection of promising biocontrol agents against P. capsici. ((c) 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

Sterilization of Escherichia coli O157:H7 using micro corona ionizer.

PubMed

Chua, Beelee; Son, Ahjeong

2014-06-01

We demonstrated in vitro sterilization of Escherichia coli O157:H7 bacteria on agar by a pin-between-planes micro corona ionizer. The gap between the pin and the grid was ~1.1 mm, the length of the grid was ~2.1 mm and the height was ~1.0 mm. The effective pin radius and discharge length were both approximated to be 200 μm. Ozone generation rates of ~2.3 × 10(-3) mg/s, ~2.7 × 10(-3) mg/s and ~3.5 × 10(-3) mg/s at 1,500 V were calculated for relative humidity (RH) of 35 %, 25 % and 10 % respectively. Analytical ozone generation rate increases as RH decreases and it is consistent with experimental observations. Using target and control petri dishes with E. coli plated agar, the sterilization capability of the micro corona ionizer at 37 °C for 24 h was evaluated. A ~60 % reduction in bacterial colony was shown with plate counting and its kill radius could be tuned from ~ 20 mm to ~5 mm by reducing the duty cycle from 100 % to 50 % with 30 min pulse width. The results suggested that the micro corona ionizer might be suitable as a tunable ozone source in wound dressing for chronic wound management.

Characterization of the dominant bacterial communities during storage of Norway lobster and Norway lobster tails (Nephrops norvegicus) based on 16S rDNA analysis by PCR-DGGE.

PubMed

Bekaert, Karen; Devriese, Lisa; Maes, Sara; Robbens, Johan

2015-04-01

The aim of this study was to investigate the microbial quality of whole Norway lobster (Nephrops norvegicus) and Norway lobster tails to optimize handling conditions. This was done by assessing the total viable count (TVC) and characterizing the dominant microbiota. The cultivable microorganisms were quantified via classical microbiological plating methods. To characterize as many bacterial species present as possible, we performed advanced molecular identification techniques (PCR-DGGE). The initial TVC of fresh Norway lobster meat was high (3.0 log cfu/g) as compared to fish. No significant difference between whole Norway lobster and Norway lobster tails could be found during the storage period. From day 6 of storage, a significant difference between Plate Count Agar (PCA) and Marine Agar (MA) was observed. The microbiota of Norway lobster was dominated by members of the Gram-negative genera such as Psychrobacter spp., Pseudoalteromonas spp., Pseudomonas spp., Luteimonas spp., and Aliivibrio spp. From these bacteria, mainly Psychrobacter spp. and Pseudomonas spp. remained present until the end of the storage period. These are known spoilage organisms in fishery products. Other known spoilage organisms of crustaceans such as Photobacterium spp. could not be identified. Copyright © 2014 Elsevier Ltd. All rights reserved.

Prevalence of Staphylococcus, including Methicillin-Resistant Staphylococcus aureus, in a Physical Therapy Education Facility.

PubMed

Dhagat, Priya V; Gibbs, Karen A; Rohde, Rodney E

2015-01-01

The purpose of this study was to assess the prevalence of Staphylococcus species, including methicillin-resistant Staphylococcus aureus (MRSA), in a physical therapy (PT) education facility. The PT laboratory classrooms were routinely used by graduate PT students and faculty, undergraduate anatomy students, and licensed practitioners for continuing education purposes. A total of 88 swab samples were collected from plinths and other equipment and plated onto mannitol salt agar (MSA). Suspected S. aureus colonies were confirmed by Staphyloslide latex testing. S. aureus isolates were plated to HardyCHROM agar to identify MRSA. VITEK antibiotic susceptibility testing confirmed MRSA isolates. Forty-seven samples showed growth (47/88, 53%), and 7 tested positive for S. aureus (7/47, 15%). Of those 7, one demonstrated oxacillin resistance and was confirmed as MRSA (1/7, 2%). Remaining samples grew other species of Staphylococcus and gram-negative bacilli. Given high classroom utilization, staphylococci environmental prevalence would be expected. However, the presence of MRSA was unexpected. Results demonstrate the potential for easily transmissible and potentially harmful organisms to be present in multi-use classrooms utilized by health professions students where frequent skin-to-skin contact occurs. Strict, routine cleaning of plinths and other equipment is imperative in reducing exposure risk.

[Sporothrix globosa isolation related to a case of lymphocutaneous sporotrichosis].

PubMed

Cruz, Rodrigo; Vieille, Peggy; Oschilewski, David

2012-08-01

Sporothrix schenckii complex comprises a group of environmental dimorphic fungi that cause sporotrichosis. In Chile, isolated cases have been reported in humans, though no environmental isolates have been described. To achieve isolation of Sporothrix complex from the soil where a 75 year old patient with lymphocutaneous sporotrichosis performs horticulture work. In March and July 2011 soil and plant debris from five sectors where the patient does his work in horticulture was extracted. The soil samples were diluted and inoculated in Sabouraud agar with cycloheximide and chloramphenicol at 26 °C. The plant debris was directly inoculated in the same medium. Colonies suggestive of Sporothrix complex were reseeded in PDA agar at 26 ° C and identified as recommended by Marimon et al. Of the 10 plates from the first sampling, one colony was identified as Sporothrix globosa. In the second sampling, Sporothrix globosa grew in two plates seeded with soil, with a total of 6 colonies. There was no growth of Sporothrix complex in plant debris. The isolate from the patient was also identified as Sporothrix globosa. For the first time in Chile a species of Sporothrix complex was isolated from the environment. Sporothrix globosa was the species identified both in the ground and from the patient with sporotrichosis.

The effectiveness of processed grapefruit-seed extract as an antibacterial agent: I. An in vitro agar assay.

PubMed

Reagor, Lee; Gusman, Jean; McCoy, Lana; Carino, Edith; Heggers, John P

2002-06-01

Grapefruit-seed extract (GSE) Citricidal has, in recent reports, been reported to be successful in combating a variety of common infectious agents. In our study, drops of concentrated grapefruit-seed extract were tested for antibacterial properties against a number of gram-positive and gram-negative organisms. Sixty-seven (67) distinct biotypes were tested for their susceptibilities to the GSE as well as to 5 other topical antibacterials (Silvadene, Sulfamylon, Bactroban, Nitrofurazone, and Silvadene, Nystatin). Wells were punched into Mueller-Hinton agar plates, which were then inoculated with the organism to be tested; each well was then inoculated with one of the antibacterial agents. After an overnight incubation period, the plates were checked for zones of bacterial susceptibility around the individual wells, with a measured susceptibility zone diameter of 10 mm or more considered a positive result. The GSE was consistently antibacterial against all of the biotypes tested, with susceptibility zone diameters equal to or greater than 15 mm in each case. Our preliminary data thus suggest an antibacterial characteristic to GSE that is comparable to that of proven topical antibacterials. Although the GSE appeared to have a somewhat greater inhibitory effect on gram-positive organisms than on gram-negative organisms, its comparative effectiveness against a wide range of bacterial biotypes is significant.

A comparison of the Sensititre® MYCOTB panel and the agar proportion method for the susceptibility testing of Mycobacterium tuberculosis.

PubMed

Abuali, M M; Katariwala, R; LaBombardi, V J

2012-05-01

The agar proportion method (APM) for determining Mycobacterium tuberculosis susceptibilities is a qualitative method that requires 21 days in order to produce the results. The Sensititre method allows for a quantitative assessment. Our objective was to compare the accuracy, time to results, and ease of use of the Sensititre method to the APM. 7H10 plates in the APM and 96-well microtiter dry MYCOTB panels containing 12 antibiotics at full dilution ranges in the Sensititre method were inoculated with M. tuberculosis and read for colony growth. Thirty-seven clinical isolates were tested using both methods and 26 challenge strains of blinded susceptibilities were tested using the Sensititre method only. The Sensititre method displayed 99.3% concordance with the APM. The APM provided reliable results on day 21, whereas the Sensititre method displayed consistent results by day 10. The Sensititre method provides a more rapid, quantitative, and efficient method of testing both first- and second-line drugs when compared to the gold standard. It will give clinicians a sense of the degree of susceptibility, thus, guiding the therapeutic decision-making process. Furthermore, the microwell plate format without the need for instrumentation will allow its use in resource-poor settings.

Keratinophilic fungi and other moulds associated with air-dust particles from Egypt.

PubMed

Abdel-Hafez, S I; Moubasher, A H; Barakat, A

1990-01-01

One-hundred and eleven species and three species varieties belonging to 39 genera were collected from 50 dust samples on the five media used at 28 degrees C. Using the hair-baiting technique with horse hair, 10 species of Chrysosporium were isolated: C. asperatum, C. state of Arthroderma tuberculatum, C. indicum, C. inops, C. keratinophilum, C. merdarium, C. pannorum, C. queenslandicum, C. tropicum and C. xerophilum. True dermatophytes were isolated: Trichophyton verrucosum and Trichophyton sp. Also, numerous fungi tolerating high levels of cycloheximide were encountered, such as members of Acremonium, Aspergillus and Penicillium. On plates of glucose or cellulose Czapek-Dox agar (free from sucrose) the most frequent fungi were: Alternaria alternata, Aspergillus flavus, A. flavus var. columnaris, A. fumigatus, A. niger, A. ochraceus, A. sydowii, A. terreus, Chaetomium globosum, Cladosporium herbarum, Emericella nidulans, Fusarium oxysporum, Mucor hiemalis, Penicillium chrysogenum, P. oxalicum, Scopulariopsis brevicaulis and Ulocladium atrum. On plates of 50% sucrose or 10 and 20% NaCl-Czapek's agar, some interesting species were frequently encountered: Eurotium amstelodami, E. chevalieri, E. halophilicum, E. montevidensis, E. repens, E. rubrum and Scopulariopsis halophilica. The isolated fungi have been tested for osmophilicity and halophilicity, they showed different rates of growth on sucrose and sodium chloride-Czapek's medium of various osmotic potential.

Screening test recommendations for methicillin-resistant Staphylococcus aureus surveillance practices: A cost-minimization analysis.

PubMed

Whittington, Melanie D; Curtis, Donna J; Atherly, Adam J; Bradley, Cathy J; Lindrooth, Richard C; Campbell, Jonathan D

2017-07-01

To mitigate methicillin-resistant Staphylococcus aureus (MRSA) infections, intensive care units (ICUs) conduct surveillance through screening patients upon admission followed by adhering to isolation precautions. Two surveillance approaches commonly implemented are universal preemptive isolation and targeted isolation of only MRSA-positive patients. Decision analysis was used to calculate the total cost of universal preemptive isolation and targeted isolation. The screening test used as part of the surveillance practice was varied to identify which screening test minimized inappropriate and total costs. A probabilistic sensitivity analysis was conducted to evaluate the range of total costs resulting from variation in inputs. The total cost of the universal preemptive isolation surveillance practice was minimized when a polymerase chain reaction screening test was used ($82.51 per patient). Costs were $207.60 more per patient when a conventional culture was used due to the longer turnaround time and thus higher isolation costs. The total cost of the targeted isolation surveillance practice was minimized when chromogenic agar 24-hour testing was used ($8.54 per patient). Costs were $22.41 more per patient when polymerase chain reaction was used. For ICUs that preemptively isolate all patients, the use of a polymerase chain reaction screening test is recommended because it can minimize total costs by reducing inappropriate isolation costs. For ICUs that only isolate MRSA-positive patients, the use of chromogenic agar 24-hour testing is recommended to minimize total costs. Copyright © 2017 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Microbial susceptibility to calcium hydroxide pastes and their vehicles.

PubMed

Gomes, Brenda Paula Figueiredo de Almeida; Ferraz, Caio Cezar Randi; Garrido, Fabio Devora; Rosalen, Pedro Luiz; Zaia, Alexandre Augusto; Teixeira, Fabricio Batista; de Souza-Filho, Francisco José

2002-11-01

The aim of this study was to investigate the susceptibility of some microorganisms commonly isolated from root canals to calcium hydroxide in combination with several vehicles by the agar diffusion method. Stainless-steel cylinders were placed on each inoculated agar medium. The test medications and their controls were placed inside the cylinders. The zones of growth inhibition were measured and recorded after the incubation period for each plate, and the results were analyzed statistically. Enterococcus faecalis was most resistant, whereas the anaerobic Porphyromonas endodontalis was more susceptible to all medications, followed by P. gingivalis and Prevotella intermedial intermedia. Ca(OH)2 + CMCP + glycerin showed significantly larger mean zones of inhibition when compared with the other medications. We conclude that anaerobic Gram-negative bacteria are more susceptible to calcium hydroxide pastes than facultative Gram-positive microorganisms.

Evaluation of CP Chromo Select Agar for the enumeration of Clostridium perfringens from water.

PubMed

Manafi, Mammad; Waldherr, Kerstin; Kundi, Michael

2013-10-01

The European Directive on drinking water quality has included mCP agar as the reference method for recovering Clostridium perfringens from drinking waters. In the present study, three media (mCP, TSCF and CP Chromo Select Agar) were evaluated for recovery of C. perfringens in different surface water samples. Out of 139 water samples, using a membrane filtration technique, 131 samples (94.2%) were found to be presumptively positive for C. perfringens in at least one of the culture media. Green colored colonies on CP Chromo Select Agar (CCP agar) were counted as presumptive C. perfringens isolates. Out of 483 green colonies on CCP agar, 96.3% (465 strains, indole negative) were identified as C. perfringens, and 15 strains (3.1%) were indole positive and were identified as Clostridium sordellii, Clostridium bifermentans or Clostridium tetani. Only 3 strains (0.6%) gave false positive results and were identified as Clostridium fallax, Clostridium botulinum, and Clostridium tertium. Variance analysis of the data obtained shows statistically no significant differences in the counts obtained between media employed in this work. The mCP method is very onerous for routine screening and bacterial colonies could not be used for further biochemical testing. The colonies on CCP and TSCF were easy to count and subculture for confirmation tests. TSCF detects sulfite-reducing clostridia, including species other than C. perfringens, and in some cases excessive blackening of the agar frustrated counting of the colonies. If the contamination was too high, TSCF did not consistently produce black colonies and as a consequence, the colonies were white and gave false negative results. On the other hand, the identification of typical and atypical colonies isolated from all media demonstrated that CCP agar was the most useful medium for C. perfringens recovery in water samples. Copyright © 2013 Elsevier B.V. All rights reserved.

Methicillin-Resistant Staphylococcus aureus (MRSA) Detection: Comparison of Two Molecular Methods (IDI-MRSA PCR Assay and GenoType MRSA Direct PCR Assay) with Three Selective MRSA Agars (MRSA ID, MRSASelect, and CHROMagar MRSA) for Use with Infection-Control Swabs▿

PubMed Central

van Hal, S. J.; Stark, D.; Lockwood, B.; Marriott, D.; Harkness, J.

2007-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) is an increasing problem. Rapid detection of MRSA-colonized patients has the potential to limit spread of the organism. We evaluated the sensitivities and specificities of MRSA detection by two molecular methods (IDI-MRSA PCR assay and GenoType MRSA Direct PCR assay) and three selective MRSA agars (MRSA ID, MRSASelect, and CHROMagar MRSA), using 205 (101 nasal, 52 groin, and 52 axillary samples) samples from consecutive known MRSA-infected and/or -colonized patients. All detection methods had higher MRSA detection rates for nasal swabs than for axillary and groin swabs. Detection of MRSA by IDI-MRSA was the most sensitive method, independent of the site (94% for nasal samples, 80% for nonnasal samples, and 90% overall). The sensitivities of the GenoType MRSA Direct assay and the MRSA ID, MRSASelect, and CHROMagar MRSA agars with nasal swabs were 70%, 72%, 68%, and 75%, respectively. All detection methods had high specificities (95 to 99%), independent of the swab site. Extended incubation for a further 24 h with selective MRSA agars increased the detection of MRSA, with a corresponding decline in specificity secondary to a significant increase in false-positive results. There was a noticeable difference in test performance of the GenoType MRSA Direct assay in detection of MRSA (28/38 samples [74%]) compared with detection of nonmultiresistant MRSA (17/31 samples [55%]) (susceptible to two or more non-β-lactam antibiotics). This was not observed with selective MRSA agar plates or IDI-MRSA. Although it is more expensive, in addition to rapid turnaround times of 2 to 4 h, IDI-MRSA offers greater detection of MRSA colonization, independent of the swab site, than do conventional selective agars and GenoType MRSA Direct. PMID:17537949