Migration of Chemotactic Bacteria in Soft Agar: Role of Gel Concentration

PubMed Central

Croze, Ottavio A.; Ferguson, Gail P.; Cates, Michael E.; Poon, Wilson C.K.

2011-01-01

We study the migration of chemotactic wild-type Escherichia coli populations in semisolid (soft) agar in the concentration range C = 0.15–0.5% (w/v). For C≲0.35%, expanding bacterial colonies display characteristic chemotactic rings. At C = 0.35%, however, bacteria migrate as broad circular bands rather than sharp rings. These are growth/diffusion waves arising because of suppression of chemotaxis by the agar and have not been previously reported experimentally to our knowledge. For C = 0.4–0.5%, expanding colonies do not span the depth of the agar and develop pronounced front instabilities. The migration front speed is weakly dependent on agar concentration at C < 0.25%, but decreases sharply above this value. We discuss these observations in terms of an extended Keller-Segel model for which we derived novel transport parameter expressions accounting for perturbations of the chemotactic response by collisions with the agar. The model makes it possible to fit the observed front speed decay in the range C = 0.15–0.35%, and its solutions qualitatively reproduce the observed transition from chemotactic to growth/diffusion bands. We discuss the implications of our results for the study of bacteria in porous media and for the design of improved bacteriological chemotaxis assays. PMID:21806920

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.

Performance of the EUCAST Disk Diffusion Method, the CLSI Agar Screen Method, and the Vitek 2 Automated Antimicrobial Susceptibility Testing System for Detection of Clinical Isolates of Enterococci with Low- and Medium-Level VanB-Type Vancomycin Resistance: a Multicenter Study

PubMed Central

Giske, Christian G.; Haldorsen, Bjørg; Matuschek, Erika; Schønning, Kristian; Leegaard, Truls M.; Kahlmeter, Gunnar

2014-01-01

Different antimicrobial susceptibility testing methods to detect low-level vancomycin resistance in enterococci were evaluated in a Scandinavian multicenter study (n = 28). A phenotypically and genotypically well-characterized diverse collection of Enterococcus faecalis (n = 12) and Enterococcus faecium (n = 18) strains with and without nonsusceptibility to vancomycin was examined blindly in Danish (n = 5), Norwegian (n = 13), and Swedish (n = 10) laboratories using the EUCAST disk diffusion method (n = 28) and the CLSI agar screen (n = 18) or the Vitek 2 system (bioMérieux) (n = 5). The EUCAST disk diffusion method (very major error [VME] rate, 7.0%; sensitivity, 0.93; major error [ME] rate, 2.4%; specificity, 0.98) and CLSI agar screen (VME rate, 6.6%; sensitivity, 0.93; ME rate, 5.6%; specificity, 0.94) performed significantly better (P = 0.02) than the Vitek 2 system (VME rate, 13%; sensitivity, 0.87; ME rate, 0%; specificity, 1). The performance of the EUCAST disk diffusion method was challenged by differences in vancomycin inhibition zone sizes as well as the experience of the personnel in interpreting fuzzy zone edges as an indication of vancomycin resistance. Laboratories using Oxoid agar (P < 0.0001) or Merck Mueller-Hinton (MH) agar (P = 0.027) for the disk diffusion assay performed significantly better than did laboratories using BBL MH II medium. Laboratories using Difco brain heart infusion (BHI) agar for the CLSI agar screen performed significantly better (P = 0.017) than did those using Oxoid BHI agar. In conclusion, both the EUCAST disk diffusion and CLSI agar screening methods performed acceptably (sensitivity, 0.93; specificity, 0.94 to 0.98) in the detection of VanB-type vancomycin-resistant enterococci with low-level resistance. Importantly, use of the CLSI agar screen requires careful monitoring of the vancomycin concentration in the plates. Moreover, disk diffusion methodology requires that personnel be trained in interpreting zone edges

Standard Nutrient Agar 1 as a substitute for blood-supplemented Müller-Hinton agar for antibiograms in developing countries.

PubMed

Niederstebruch, N; Sixt, D

2013-02-01

In the industrial world, the agar diffusion test is a standard procedure for the susceptibility testing of bacteria isolates. Beta-hemolytic Streptococcus spp. are tested with Müller-Hinton agar supplemented with 5 % blood, a so-called blood agar. The results are interpreted using standardized tables, which only exist for this type of nutrient matrix. Because of a number difficulties, both with respect to technical issues and to manual skills, blood agar is not a feasible option in many developing countries. Beta-hemolytic Streptococcus spp. also grow on Standard Nutrient Agar 1 (StNA1). This suggests using that type of nutrient medium for running agar diffusion tests. However, there are no standardized tables that can be used for interpreting the diameters of the zones of inhibition on StNA1 1. Using the existing standardized tables for blood agar to interpret cultures on StNA1 1 would be of great benefit under such circumstances where blood agar is not available. With this in mind, we conducted comparative tests to evaluate the growth characteristics of beta-hemolytic Streptococcus spp. on StNA1 1 compared to Müller-Hinton agar supplemented with 5 % sheep blood. In this study, we were able to show that beta-hemolytic Streptococcus spp. develop similar zones of inhibition on blood agar and on StNA1 1. Therefore, it is suggested that, for the interpretation of antibiograms of beta-hemolytic Streptococcus spp. performed on StNA1 1, the standard tables for blood agar can be used.

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria.

PubMed

Gold, Ben; Roberts, Julia; Ling, Yan; Lopez Quezada, Landys; Glasheen, Jou; Ballinger, Elaine; Somersan-Karakaya, Selin; Warrier, Thulasi; Nathan, Carl

2016-12-14

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 °C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to ≥99% bacterial kill (MBC≥99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

Studies on antimicrobial activity, in vitro, of Physalis angulata L. (Solanaceae) fraction and physalin B bringing out the importance of assay determination.

PubMed

Silva, Melissa T G; Simas, Sonia M; Batista, Terezinha G F M; Cardarelli, Paola; Tomassini, Therezinha C B

2005-11-01

Complex physalin metabolites present in the capsules of the fruit of Physalis angulata L. have been isolated and submitted to a series of assays of antimicrobial activity against Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, S. aureus ATCC 25923, S. aureus ATCC 6538P, Neisseria gonorrhoeae ATCC 49226, Escherichia coli ATCC 8739; E. coli ATCC 25922, Candida albicans ATCC 10231 applying different methodologies such as: bioautography, dilution broth, dilution agar, and agar diffusion techniques. A mixture of physalins (pool) containing physalins B, D, F, G inhibit S. aureus ATCC 29213, S. aureus ATCC 25923, S. aureus ATCC 6538P, and N. gonorrhoeae ATCC 49226 at a concentration of 200 mg/microl, using agar dilution assays. The mixture was inactive against P. aeruginosa ATCC27853, E. coli ATCC 8739; E. coli ATCC 25922, C. albicans ATCC 10231 when applying bioautography assays. Physalin B (200 microg/ml) by the agar diffusion assay inhibited S. aureus ATCC 6538P by +/- 85%; and may be considered responsible for the antimicrobial activity.

Immobilization of pectin degrading enzyme from Bacillus licheniformis KIBGE IB-21 using agar-agar as a support.

PubMed

Rehman, Haneef Ur; Aman, Afsheen; Zohra, Raheela Rahmat; Qader, Shah Ali Ul

2014-02-15

Pectinase from Bacillus licheniformis KIBGE IB-21 was immobilized in agar-agar matrix using entrapment technique. Effect of different concentrations of agar-agar on pectinase immobilization was investigated and it was found that maximum immobilization was achieved at 3.0% agar-agar with 80% enzyme activity. After immobilization, the optimum temperature of enzyme increased from 45 to 50 °C and reaction time from 5 to 10 minutes as compared to free enzyme. Due to the limited diffusion of high molecular weight substrate, K(m) of immobilized enzyme slightly increased from 1.017 to 1.055 mg ml(-1), while Vmax decreased from 23,800 to 19,392 μM min(-1) as compared to free enzyme. After 120 h entrapped pectinase retained their activity up to 82% and 71% at 30 °C and 40 °C, respectively. The entrapped pectinase showed activity until 10th cycle and maintain 69.21% activity even after third cycle. Copyright © 2013 Elsevier Ltd. All rights reserved.

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)…

Serotyping reanalysis of unserotypable Actinobacillus pleuropneumoniae isolates by agar gel diffusion test.

PubMed

Morioka, Ayako; Shimazaki, Yoko; Uchiyama, Mariko; Suzuki, Shoko

2016-05-03

We observed increasing unserotypable (UT) Actinobacillus pleuropneumoniae isolates using agar gel diffusion (AGD) test. To reanalyze their serovar, we performed rapid slide agglutination (RSA) test and multiplex PCR for 47 UT isolates. Of these, 25 were serovar 1 (UT-serovar 1), 20 were serovar 2 (UT-serovar 2) and 2 were serovar 15 (UT-serovar 15). We examined serotyping antigen extraction temperature to determine heat influence. UT-serovar 1 and 15 were influenced by heat, because their precipitation lines were observed in the case of low antigen extraction temperature. To investigate the relationship between antigenicity and genotype, we performed pulsed-field gel electrophoresis (PFGE) analysis using UT-serovar 2 and 15. The predominant PFGE pattern of UT-serovar 2 was identical to that of serovar 2.

Serotyping reanalysis of unserotypable Actinobacillus pleuropneumoniae isolates by agar gel diffusion test

PubMed Central

MORIOKA, Ayako; SHIMAZAKI, Yoko; UCHIYAMA, Mariko; SUZUKI, Shoko

2016-01-01

We observed increasing unserotypable (UT) Actinobacillus pleuropneumoniae isolates using agar gel diffusion (AGD) test. To reanalyze their serovar, we performed rapid slide agglutination (RSA) test and multiplex PCR for 47 UT isolates. Of these, 25 were serovar 1 (UT-serovar 1), 20 were serovar 2 (UT-serovar 2) and 2 were serovar 15 (UT-serovar 15). We examined serotyping antigen extraction temperature to determine heat influence. UT-serovar 1 and 15 were influenced by heat, because their precipitation lines were observed in the case of low antigen extraction temperature. To investigate the relationship between antigenicity and genotype, we performed pulsed-field gel electrophoresis (PFGE) analysis using UT-serovar 2 and 15. The predominant PFGE pattern of UT-serovar 2 was identical to that of serovar 2. PMID:26726101

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

Comparison of disk diffusion and agar dilution methods for gentamicin susceptibility testing of Neisseria gonorrhoeae.

PubMed

Gianecini, Ricardo; Oviedo, Claudia; Irazu, Lucia; Rodríguez, Marcelo; Galarza, Patricia

2018-03-29

Gentamicin is a promising antibiotic for the treatment of multidrug-resistant gonorrhea. The aim of this study was to analyze the suitability and reliably of disk diffusion to monitor the susceptibility to gentamicin. We studied 237 Neisseria gonorrhoeae isolates obtained in 2013 and 2015. Reference MICs were correlated with inhibition zone diameters (in millimeters) of gentamicin 10 µg disks manufactured by BBL and Oxoid. The Pearson correlation between disk diffusion and agar dilution was r = -.68 (P < 0.001) for BBL disk and r = -.71 (P < 0.001) for Oxoid disk. No very major or major discrepancies were detected. However, a high percentage of minor discrepancies was observed (44.7%, BBL disk) and (21.9%, Oxoid disk). By adjusting the susceptible breakpoint to S ≥ 17 mm, the minor discrepancies rate was reduced to 19.4% (BBL disk) and 10.1% (Oxoid disk). The disk diffusion may be a screening method in clinical laboratories to detect the gentamicin susceptibility of N. gonorrhoeae. Copyright © 2018 Elsevier Inc. All rights reserved.

21 CFR 582.7115 - Agar-agar.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Agar-agar. 582.7115 Section 582.7115 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7115 Agar-agar. (a...

21 CFR 582.7115 - Agar-agar.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Agar-agar. 582.7115 Section 582.7115 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7115 Agar-agar. (a...

Comparison of Agar Dilution, Disk Diffusion, MicroScan, and Vitek Antimicrobial Susceptibility Testing Methods to Broth Microdilution for Detection of Fluoroquinolone-Resistant Isolates of the Family Enterobacteriaceae

PubMed Central

Steward, Christine D.; Stocker, Sheila A.; Swenson, Jana M.; O’Hara, Caroline M.; Edwards, Jonathan R.; Gaynes, Robert P.; McGowan, John E.; Tenover, Fred C.

1999-01-01

Fluoroquinolone resistance appears to be increasing in many species of bacteria, particularly in those causing nosocomial infections. However, the accuracy of some antimicrobial susceptibility testing methods for detecting fluoroquinolone resistance remains uncertain. Therefore, we compared the accuracy of the results of agar dilution, disk diffusion, MicroScan Walk Away Neg Combo 15 conventional panels, and Vitek GNS-F7 cards to the accuracy of the results of the broth microdilution reference method for detection of ciprofloxacin and ofloxacin resistance in 195 clinical isolates of the family Enterobacteriaceae collected from six U.S. hospitals for a national surveillance project (Project ICARE [Intensive Care Antimicrobial Resistance Epidemiology]). For ciprofloxacin, very major error rates were 0% (disk diffusion and MicroScan), 0.9% (agar dilution), and 2.7% (Vitek), while major error rates ranged from 0% (agar dilution) to 3.7% (MicroScan and Vitek). Minor error rates ranged from 12.3% (agar dilution) to 20.5% (MicroScan). For ofloxacin, no very major errors were observed, and major errors were noted only with MicroScan (3.7% major error rate). Minor error rates ranged from 8.2% (agar dilution) to 18.5% (Vitek). Minor errors for all methods were substantially reduced when results with MICs within ±1 dilution of the broth microdilution reference MIC were excluded from analysis. However, the high number of minor errors by all test systems remains a concern. PMID:9986809

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

Abilities of the mCP Agar Method and CRENAME Alpha Toxin-Specific Real-Time PCR Assay To Detect Clostridium perfringens Spores in Drinking Water

PubMed Central

Maheux, Andrée F.; Bérubé, Ève; Boudreau, Dominique K.; Villéger, Romain; Cantin, Philippe; Boissinot, Maurice; Bissonnette, Luc

2013-01-01

We first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of a Clostridium perfringens-specific real-time PCR (rtPCR) assay based on the cpa gene (cpa rtPCR) by using a bacterial strain panel composed of C. perfringens and non-C. perfringens Clostridium strains. All non-C. perfringens Clostridium strains tested negative, whereas all C. perfringens strains tested positive with the cpa rtPCR, for an analytical specificity and ubiquity of 100%. The cpa rtPCR assay was then used to confirm the identity of 116 putative C. perfringens isolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar and cpa rtPCR were identified by sequencing the 16S rRNA and cpa genes. Four mCP−/rtPCR+ colonies were identified as C. perfringens, whereas 3 mCP+/rtPCR− colonies were identified as non-C. perfringens. The cpa rtPCR was negative with all 51 non-C. perfringens strains and positive with 64 of 65 C. perfringens strains. Finally, we compared mCP agar and a CRENAME (concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment) procedure plus cpa rtPCR (CRENAME + cpa rtPCR) for their abilities to detect C. perfringens spores in drinking water. CRENAME + cpa rtPCR detected as few as one C. perfringens CFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME + cpa rtPCR also allows the simultaneous and sensitive detection of Escherichia coli and C. perfringens from the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection. PMID:24077714

Thermal characterization of magnetically aligned carbonyl iron/agar composites.

PubMed

Diaz-Bleis, D; Vales-Pinzón, C; Freile-Pelegrín, Y; Alvarado-Gil, J J

2014-01-01

Composites of magnetic particles into polymeric matrices have received increasing research interest due to their capacity to respond to external magnetic or electromagnetic fields. In this study, agar from Gelidium robustum has been chosen as natural biocompatible polymer to build the matrix of the magnetic carbonyl iron particles (CIP) for their uses in biomedical fields. Heat transfer behavior of the CIP-agar composites containing different concentrations (5, 10, 15, 20, 25 and 30% w/w) of magnetically aligned and non-aligned CIP in the agar matrix was studied using photothermal radiometry (PTR) in the back-propagation emission configuration. The morphology of the CIP-agar composites with aligned and non-aligned CIP under magnetic field was also evaluated by scanning electron microscopy (SEM). The results revealed a dominant effect of CIP concentration over the alignment patterns induced by the magnetic field, which agrees with the behavior of the thermal diffusivity and thermal conductivity. Agar served as a perfect matrix to be used with CIP, and CIP-agar composites magnetically aligned at 20% CIP concentration can be considered as promising 'smart' material for hyperthermia treatments in the biomedical field. Copyright © 2013 Elsevier Ltd. All rights reserved.

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.

Antibiotic susceptibility of methicillin-resistant staphylococci (MRS) of food origin: A comparison of agar disc diffusion method and a commercially available miniaturized test.

PubMed

Buzón-Durán, Laura; Capita, Rosa; Alonso-Calleja, Carlos

2018-06-01

Methicillin-resistant staphylococci (MRS) are a major concern to public and animal health. Thirty MRS (Staphylococcus aureus, S. cohnii, S. epidermidis, S. haemolyticus, S. hominis, S. lentus, S. lugdunensis, S. sciuri, and S. xylosus) isolates from meat and poultry preparations were tested for antimicrobial susceptibility to 11 antimicrobials (belonging to seven different categories) of clinical significance using both the standard agar disc diffusion method and a commercially available miniaturized system (Sensi Test Gram-positive). It is worth stressing that 16 isolates (53.33%) exhibited an extensively drug-resistant phenotype (XDR). The average number of resistances per strain was 4.67. These results suggest that retail meat and poultry preparations are a likely vehicle for the transmission of multi-drug resistant MRS. Resistance to erythromycin was the commonest finding (76.67% of strains), followed by tobramycin, ceftazidime (66.67%), ciprofloxacin (56.67%) and fosfomycin (53.33%). An agreement (kappa coefficient) of 0.64 was found between the two testing methods. Using the agar disc diffusion as the reference method, the sensitivity, specificity and accuracy of the miniaturized test were 98.44%, 69.44% and 83.33%, respectively. Most discrepancies between the two methods were due to isolates that were susceptible according to the disc diffusion method but resistant according to the miniaturized test (false positives). Copyright © 2017. Published by Elsevier Ltd.

21 CFR 184.1115 - Agar-agar.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Agar-agar. 184.1115 Section 184.1115 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) DIRECT FOOD SUBSTANCES AFFIRMED AS GENERALLY RECOGNIZED AS SAFE Listing of Specific...

21 CFR 184.1115 - Agar-agar.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Agar-agar. 184.1115 Section 184.1115 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) DIRECT FOOD SUBSTANCES AFFIRMED AS GENERALLY RECOGNIZED AS SAFE Listing of Specific...

Morphological identification of Candida species on glucose agar, rice extract agar and corn meal agar with and without Tween-80.

PubMed

Joshi, K R; Solanki, A; Prakash, P

1993-01-01

A comparative study for the identification of 32 known strains of Candida species on the basis of morphology on glucose agar, rice extract agar and corn meal agar with and without Tween 80 revealed that when Tween 80 is incorporated in the media identification is possible for 96.8% of the species within 48 hours on rice extract agar and for 96.8% of the species within 48 hours on rice extract agar and for 90.6% of the species on glucose agar. The germ tubes and chlamydospores were also produced more on rice extract agar than on 0.1% glucose agar. Rice extract agar with Tween 80 can be used as single medium for morphologic identification of Candida species. The inoculated medium is first incubated at 37 degrees C for 3 hours and examined for germ tube formation and then incubated at 25 degrees C for 24 to 72 hours and examined for appearance of chlamydospores and mycelial morphology.

Modeling of the Bacillus subtilis Bacterial Biofilm Growing on an Agar Substrate

PubMed Central

Wang, Xiaoling; Wang, Guoqing; Hao, Mudong

2015-01-01

Bacterial biofilms are organized communities composed of millions of microorganisms that accumulate on almost any kinds of surfaces. In this paper, a biofilm growth model on an agar substrate is developed based on mass conservation principles, Fick's first law, and Monod's kinetic reaction, by considering nutrient diffusion between biofilm and agar substrate. Our results show biofilm growth evolution characteristics such as biofilm thickness, active biomass, and nutrient concentration in the agar substrate. We quantitatively obtain biofilm growth dependence on different parameters. We provide an alternative mathematical method to describe other kinds of biofilm growth such as multiple bacterial species biofilm and also biofilm growth on various complex substrates. PMID:26355542

Modeling of the Bacillus subtilis Bacterial Biofilm Growing on an Agar Substrate.

PubMed

Wang, Xiaoling; Wang, Guoqing; Hao, Mudong

2015-01-01

Bacterial biofilms are organized communities composed of millions of microorganisms that accumulate on almost any kinds of surfaces. In this paper, a biofilm growth model on an agar substrate is developed based on mass conservation principles, Fick's first law, and Monod's kinetic reaction, by considering nutrient diffusion between biofilm and agar substrate. Our results show biofilm growth evolution characteristics such as biofilm thickness, active biomass, and nutrient concentration in the agar substrate. We quantitatively obtain biofilm growth dependence on different parameters. We provide an alternative mathematical method to describe other kinds of biofilm growth such as multiple bacterial species biofilm and also biofilm growth on various complex substrates.

Comparison of commercial enzyme-linked immunosorbent assay kits with agar gel precipitation and hemagglutination-inhibition tests for detecting antibodies to avian influenza viruses.

PubMed

Shiraishi, Rikiya; Nishiguchi, Akiko; Tsukamoto, Kenji; Muramatsu, Masatake

2012-09-01

We evaluated the utility of 5 commercial enzyme-linked immunosorbent assay (ELISA) kits for detecting antibodies to avian influenza viruses. The sensitivities and specificities of the ELISA kits were compared with those of the agar gel precipitation (AGP) and hemagglutination-inhibition (HI) tests. The results suggest that some ELISA kits might not be suitable for monitoring during the early stages of avian influenza virus infections. Therefore, ELISA kits should only be used in conjunction with a profound knowledge about monitoring of avian influenza.

A new effective assay to detect antimicrobial activity of filamentous fungi.

PubMed

Pereira, Eric; Santos, Ana; Reis, Francisca; Tavares, Rui M; Baptista, Paula; Lino-Neto, Teresa; Almeida-Aguiar, Cristina

2013-01-15

The search for new antimicrobial compounds and the optimization of production methods turn the use of antimicrobial susceptibility tests a routine. The most frequently used methods are based on agar diffusion assays or on dilution in agar or broth. For filamentous fungi, the most common antimicrobial activity detection methods comprise the co-culture of two filamentous fungal strains or the use of fungal extracts to test against single-cell microorganisms. Here we report a rapid, effective and reproducible assay to detect fungal antimicrobial activity against single-cell microorganisms. This method allows an easy way of performing a fast antimicrobial screening of actively growing fungi directly against yeast. Because it makes use of an actively growing mycelium, this bioassay also provides a way for studying the production dynamics of antimicrobial compounds by filamentous fungi. The proposed assay is less time consuming and introduces the innovation of allowing the direct detection of fungal antimicrobial properties against single cell microorganisms without the prior isolation of the active substance(s). This is particularly useful when performing large screenings for fungal antimicrobial activity. With this bioassay, antimicrobial activity of Hypholoma fasciculare against yeast species was observed for the first time. Copyright © 2012 Elsevier GmbH. All rights reserved.

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…

A study of electrochemical devices based on Agar-Agar-NH4I biopolymer electrolytes

NASA Astrophysics Data System (ADS)

Selvalakshmi, S.; Mathavan, T.; Selvasekarapandian, S.; Premalatha, M.

2018-04-01

A polymer electrolyte system has been developed using a biopolymer namely, Agar-Agar in combination with ammonium iodide in different weight percentages by solution casting technique. The films were characterized electrically by AC Impedance Spectroscopy for its conductivity. The highest conductivity achieved at room temperature was for 50 wt. % agar-agar: 50 wt. % NH4I with a conductivity value of 1.20 × 10-4 Scm-1. An electrochemical cell was fabricated in the configuration of: Zn + ZnSO4.7H2O + graphite (anode) | 50 wt. % (Agar-agar): 50 wt. % NH4I (electrolyte) | PbO2 + V2O5 + graphite (cathode) and it produced a maximum open circuit voltage of 1.73 V. A single PEM fuel cell was constructed with the highest conducting sample (50 wt. % (Agar-agar): 50 wt. % NH4I) and it exhibited an output voltage of 408mV.

Visualizing repetitive diffusion activity of double-strand RNA binding proteins by single molecule fluorescence assays.

PubMed

Koh, Hye Ran; Wang, Xinlei; Myong, Sua

2016-08-01

TRBP, one of double strand RNA binding proteins (dsRBPs), is an essential cofactor of Dicer in the RNA interference pathway. Previously we reported that TRBP exhibits repetitive diffusion activity on double strand (ds)RNA in an ATP independent manner. In the TRBP-Dicer complex, the diffusion mobility of TRBP facilitates Dicer-mediated RNA cleavage. Such repetitive diffusion of dsRBPs on a nucleic acid at the nanometer scale can be appropriately captured by several single molecule detection techniques. Here, we provide a step-by-step guide to four different single molecule fluorescence assays by which the diffusion activity of dsRBPs on dsRNA can be detected. One color assay, termed protein induced fluorescence enhancement enables detection of unlabeled protein binding and diffusion on a singly labeled RNA. Two-color Fluorescence Resonance Energy Transfer (FRET) in which labeled dsRBPs is applied to labeled RNA, allows for probing the motion of protein along the RNA axis. Three color FRET reports on the diffusion movement of dsRBPs from one to the other end of RNA. The single molecule pull down assay provides an opportunity to collect dsRBPs from mammalian cells and examine the protein-RNA interaction at single molecule platform. Copyright © 2016 Elsevier Inc. All rights reserved.

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...

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.

Structural, morphological, optical and biological properties of pure ZnO and agar/zinc oxide nanocomposites.

PubMed

Magesh, G; Bhoopathi, G; Nithya, N; Arun, A P; Ranjith Kumar, E

2018-05-26

In this work, ZnO nanoparticles were prepared by in situ chemical precipitation method in the presence of Agar biopolymer. The influence of Agar concentrations on the structural, morphological and optical properties of ZnO have been investigated. The XRD pattern of Pure ZnO and Agar/ZnO nanocomposites indicates the hexagonal wurtzite phase of ZnO. The crystallite size of pure ZnO and Agar/ZnO nanocomposites was found to be in the range of 35.5 to 19.73 nm. Pure ZnO and Agar/ZnO nanocomposites showed nanospheroid and nanopaddy shaped morphology from FESEM studies. The interplanar distance observed from the HRTEM image confirms the plane of the prepared material. The elemental composition of the samples were characterized by EDX. The optical properties of Pure ZnO and Agar/ZnO nanocomposites were characterized by UV, FTIR and PL. The band gap of Agar/ZnO nanocomposites were varied with the Agar concentration. Oxygen vacancy induced photoluminescence of ZnO are observed and its intensity is found to be increased linearly with the Agar concentration. The antibacterial activity of ZnO and Agar/ZnO nanocomposites was evaluated by disc diffusion method against Gram-positive (B.subtilis) and Gram-negative (P. aeruginosa) bacteria. The cytotoxicity of Agar/ZnO nanocomposites was studied against Normal (L929) and Breast cancer cell line (MB231). The result of this investigation reveals that the Agar/ZnO nanocomposites deliver a dose dependent toxicity in normal and cancer cell line. Copyright © 2018. Published by Elsevier B.V.

Assessment of Metronidazole Susceptibility in Helicobacter pylori: Statistical Validation and Error Rate Analysis of Breakpoints Determined by the Disk Diffusion Test

PubMed Central

Chaves, Sandra; Gadanho, Mário; Tenreiro, Rogério; Cabrita, José

1999-01-01

Metronidazole susceptibility of 100 Helicobacter pylori strains was assessed by determining the inhibition zone diameters by disk diffusion test and the MICs by agar dilution and PDM Epsilometer test (E test). Linear regression analysis was performed, allowing the definition of significant linear relations, and revealed correlations of disk diffusion results with both E-test and agar dilution results (r2 = 0.88 and 0.81, respectively). No significant differences (P = 0.84) were found between MICs defined by E test and those defined by agar dilution, taken as a standard. Reproducibility comparison between E-test and disk diffusion tests showed that they are equivalent and with good precision. Two interpretative susceptibility schemes (with or without an intermediate class) were compared by an interpretative error rate analysis method. The susceptibility classification scheme that included the intermediate category was retained, and breakpoints were assessed for diffusion assay with 5-μg metronidazole disks. Strains with inhibition zone diameters less than 16 mm were defined as resistant (MIC > 8 μg/ml), those with zone diameters equal to or greater than 16 mm but less than 21 mm were considered intermediate (4 μg/ml < MIC ≤ 8 μg/ml), and those with zone diameters of 21 mm or greater were regarded as susceptible (MIC ≤ 4 μg/ml). Error rate analysis applied to this classification scheme showed occurrence frequencies of 1% for major errors and 7% for minor errors, when the results were compared to those obtained by agar dilution. No very major errors were detected, suggesting that disk diffusion might be a good alternative for determining the metronidazole sensitivity of H. pylori strains. PMID:10203543

Agar Disk Diffusion and Automated Microbroth Dilution Produce Similar Antimicrobial Susceptibility Testing Results for Salmonella Serotypes Newport, Typhimurium, and 4,5,12:i-, But Differ in Economic Cost

PubMed Central

Cummings, Kevin J.; Warnick, Lorin D.; Schukken, Ynte H.; Siler, Julie D.; Gröhn, Yrjo T.; Davis, Margaret A.; Besser, Tom E.; Wiedmann, Martin

2011-01-01

Abstract Data generated using different antimicrobial testing methods often have to be combined, but the equivalence of such results is difficult to assess. Here we compared two commonly used antimicrobial susceptibility testing methods, automated microbroth dilution and agar disk diffusion, for 8 common drugs, using 222 Salmonella isolates of serotypes Newport, Typhimurium, and 4,5,12:i-, which had been isolated from clinical salmonellosis cases among cattle and humans. Isolate classification corresponded well between tests, with 95% overall category agreement. Test results were significantly negatively correlated, and Spearman's correlation coefficients ranged from −0.98 to −0.38. Using Cox's proportional hazards model we determined that for most drugs, a 1 mm increase in zone diameter resulted in an estimated 20%–40% increase in the hazard of growth inhibition. However, additional parameters such as isolation year or serotype often impacted the hazard of growth inhibition as well. Comparison of economical feasibility showed that agar disk diffusion is clearly more cost-effective if the average sample throughput is small but that both methods are comparable at high sample throughput. In conclusion, for the Salmonella serotypes and antimicrobial drugs analyzed here, antimicrobial susceptibility data generated based on either test are qualitatively very comparable, and the current published break points for both methods are in excellent agreement. Economic feasibility clearly depends on the specific laboratory settings, and disk diffusion might be an attractive alternative for certain applications such as surveillance studies. PMID:21877930

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

Use of agar agar stabilized milled zero-valent iron particles for in situ groundwater remediation

NASA Astrophysics Data System (ADS)

Schmid, Doris; Velimirović, Milica; Wagner, Stephan; Micić Batka, Vesna; von der Kammer, Frank; Hofmann, Thilo

2015-04-01

A major obstacle for use of nanoscale zero-valent iron (nZVI) particles as a nontoxic material for effective in situ degradation of chlorinated aliphatic hydrocarbons (CAHs) is the high production cost. For that reason, submicro-scale milled zero-valent iron particles were recently developed (milled ZVI, UVR-FIA, Germany) by grinding macroscopic raw materials of elementary iron as a cheaper alternative to products produced by solid-state reduction. However, milled ZVI particles tend to aggregate and due to the rather large particle size (d50= 11.9 µm) also rapidly sediment. To prevent aggregation and consequently sedimentation of milled ZVI particles and therefore improve the mobility after in situ application, the use of a stabilizer is considered in literature as a most promising option. In this study, milled ZVI particles (1 g L-1 of particle concentration) were stabilized by environmentally friendly polymer agar agar (>0.5 g L-1), which had a positive impact on the milled ZVI stability. Sedimentation rate was significantly decreased by increasing the suspension viscosity. Column transport experiments were performed for bare and agar agar stabilized milled ZVI particles in commercially available fine grained quartz sand (DORSILIT® Nr.8, Gebrüder Dorfner GmbH Co, Germany) and different porous media collected from brownfields. The experiments were carried out under field relevant injection conditions of 100 m d-1. The maximal travel distance (LT) of less than 10 cm was determined for non-stabilized suspension in fine grained quartz sand, while agar agar (1 g L-1) stabilized milled ZVI suspension revealed LT of 12 m. Similar results were observed for porous media from brownfields showing that mobility of agar agar stabilized particle suspensions was significantly improved compared to bare particles. Based on the mobility data, agar agar stabilized milled zero-valent iron particles could be used for in situ application. Finally, lab-scale batch degradation

The radiosensitivity of a murine fibrosarcoma as measured by three cell survival assays.

PubMed Central

Rice, L.; Urano, M.; Suit, H. D.

1980-01-01

The radiation sensitivity of a weakly immunogenic spontaneous fibrosarcoma of the C3Hf/Sed mouse (designated FSa-II) was assessed by three in vivo cell survival methods: end-point dilution (TD50) assay, lung colony (LC) assay, and agar diffusion chamber (ADC) assay. The hypoxic fraction of this tumour was also determined by the ADC method. Although there was a good agreement of the cell survival data between the ADC and LC methods, the TD50 method yielded a considerably less steep cell survival curve. Beneficial aspects and limitations of each assay are discussed. In addition, the use of the ADC method for the growth of xenogeneic cell lines and a preliminary experiment with human tumour cells in non-immunosuppressed hosts suggest that this method may be a valuable adjunct for studying the growth and therapeutic responses of human tumour cells. PMID:6932931

Preparation of an agar-silver nanoparticles (A-AgNp) film for increasing the shelf-life of fruits.

PubMed

Gudadhe, Janhavi A; Yadav, Alka; Gade, Aniket; Marcato, Priscyla D; Durán, Nelson; Rai, Mahendra

2014-12-01

Preparation of protective coating possessing antimicrobial properties is present day need as they increase the shelf life of fruits and vegetables. In the present study, preparation of agar-silver nanoparticle film for increasing the shelf life of fruits is reported. Silver nanoparticles (Ag-NPs) biosynthesised using an extract of Ocimum sanctum leaves, were mixed with agar-agar to prepare an agar-silver nanoparticles (A-AgNp) film. This film was surface-coated over the fruits, Citrus aurantifolium (Thornless lime) and Pyrus malus (Apple), and evaluated for the determination of antimicrobial activity of A-AgNp films using disc diffusion method, weight loss and shelf life of fruits. This study demonstrates that these A-AgNp films possess antimicrobial activity and also increase the shelf life of fruits.

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.

Comparison of Brilliant Green Agar and Hektoen Enteric Agar Media in the Isolation of Salmonellae from Food Products

PubMed Central

Goo, Velma Y. L.; Ching, George Q. L.; Gooch, John M.

1973-01-01

Brilliant Green (BG) agar and Hektoen enteric (HE) agar media were compared for their efficiency in isolating salmonellae from various food products. Of the 11,226 food specimens examined, 1,662 (or 14.9%) yielded salmonellae. Of this number, 1,475 (88.7%) were recovered from BG agar and 1,315 (79.1%) were recovered from HE agar media. The results indicate that BG agar is more effective in isolating salmonellae from food products. A smaller subsidiary study showed HE agar to be more selective than BG agar. Four hundred ten specimens yielded 92 nonlactose-fermenting isolants other than salmonellae on BG agar and only 11 such isolants on HE agar. PMID:4584576

Back to the kitchen: food-grade agar is a low-cost alternative to bacteriological agar.

PubMed

Petrovski, Steve; Tillett, Daniel

2012-10-15

Food-grade agar can be used as a low-cost substitute for bacteriological agar in the preparation of solid microbial media. No difference was observed in the colony morphology, growth rate, or viability of bacteria grown on solid media prepared using food-grade agar as compared with using bacteriological-grade agar. This simple tip can reduce the cost of the most common solid media by 80% or more. Copyright © 2012 Elsevier Inc. All rights reserved.

Extraction of agar from Gelidium sesquipedale (Rhodopyta) and surface characterization of agar based films.

PubMed

Guerrero, P; Etxabide, A; Leceta, I; Peñalba, M; de la Caba, K

2014-01-01

The chemical structure of the agar obtained from Gelidium sesquipedale (Rhodophyta) has been determined by (13)C nuclear magnetic resonance ((13)C NMR) and Fourier transform infrared spectroscopy (FTIR). Agar (AG) films with different amounts of soy protein isolate (SPI) were prepared using a thermo-moulding method, and transparent and hydrophobic films were obtained and characterized. FTIR analysis provided a detailed description of the binding groups present in the films, such as carboxylic, hydroxyl and sulfonate groups, while the surface composition was examined using X-ray photoelectron spectroscopy (XPS). The changes observed by FTIR and XPS spectra suggested interactions between functional groups of agar and SPI. This is a novel approach to the characterization of agar-based films and provides knowledge about the compatibility of agar and soy protein for further investigation of the functional properties of biodegradable films based on these biopolymers. Copyright © 2013 Elsevier Ltd. All rights reserved.

In vitro antifungal susceptibility testing of Scopulariopsis brevicaulis strains using agar diffusion method.

PubMed

Skóra, Magdalena; Macura, Anna B

2011-01-01

The genus Scopulariopsis is a common soil saprotroph and has been isolated from air, organic waste and also from plant, animal and human tissues. Scopulariopsis has mainly been associated in humans with superficial mycoses, but it has also been described as the cause of subcutaneous and invasive infections. The most common aetiological agent of infections in humans is Scopulariopsis brevicaulis. This species has been reported to be resistant in vitro to broad-spectrum antifungal agents available today. The aim of the study was to establish in vitro antifungal susceptibility of 35 S. brevicaulis strains against amphotericin B (AMB), flucytosine (FC), caspofungin (CAS), terbinafine (TER), ciclopirox (CIC), voriconazole (VOR), clotrimazole (CTR), miconazole (MCZ), econazole (ECO), ketoconazole (KET), itraconazole (ITR), and fluconazole (FLU). Antifungal susceptibility tests were evaluated by an agar diffusion method (Neo-Sensitabs, Rosco, Denmark). AMB, FC, CAS, ITR and FLU showed no antifungal activity against S. brevicaulis. TER, CIC, CTR, KET, VOR, ECO, and MCZ revealed inhibitory activity for S. brevicaulis, but it varied for each of the drugs. The best antifungal effect was observed for TER and CIC. All isolates had large inhibition zones for TER and CIC. CTR was also inhibitory for all tested S. brevicaulis isolates, but the diameters of inhibition zones were smaller than for TER and CIC. Nearly 89% isolates showed inhibition zones for KET and the mean diameter of the inhibition zone was comparable to CTR. The least antifungal activity exhibited VQR, ECO and MCZ. Because of the multiresistance of S. brevicaulis, infections due to this species may not respond to particular antifungal treatment and other therapeutic approaches should be considered, e.g., combined therapy and/or surgery.

Microscopic-observation drug susceptibility and thin layer agar assays for the detection of drug resistant tuberculosis: a systematic review and meta-analysis.

PubMed

Minion, Jessica; Leung, Erika; Menzies, Dick; Pai, Madhukar

2010-10-01

Simple, rapid, and affordable tests are needed to detect drug resistance in Mycobacterium tuberculosis. We did a systematic review and meta-analysis to investigate the accuracy of microscopic-observation drug susceptibility (MODS) and thin layer agar (TLA) assays for rapid screening of patients at risk of drug-resistant tuberculosis. In accordance with protocols and methods recommended by the Cochrane Diagnostic Test Accuracy Working Group, we systematically searched PubMed, Embase, and Biosis for reports published between January, 1990, and February, 2009. We included studies investigating detection of drug resistance in M tuberculosis with the MODS or TLA assay, and in which an accepted reference standard was used. Data extracted from the studies were combined by use of bivariate random-effects regression models and hierarchical summary receiver operating characteristic curves to estimate sensitivity and specificity for detection of resistance to specific drugs. We identified 12 studies, of which nine investigated the MODS assay and three investigated the TLA assay. For the MODS assay of rifampicin resistance, pooled estimates were 98·0% (95% CI 94·5-99·3) for sensitivity and 99·4% (95·7-99·9) for specificity. For the MODS assay of isoniazid resistance with a 0·1 μg/mL cutoff, pooled sensitivity was 97·7% (94·4-99·1) and pooled specificity was 95·8% (88·1-98·6), but with a 0·4 μg/mL cutoff, sensitivity decreased to 90·0% (84·5-93·7) and specificity increased to 98·6% (96·9-99·4). All assessments of rifampicin and isoniazid resistance with the TLA assay yielded 100% accuracy. Mean turnaround time was 9·9 days (95% CI 4·1-15·8) for the MODS assay and 11·1 days (10·1-12·0) for the TLA assay. MODS and TLA assays are inexpensive, rapid alternatives to conventional methods for drug susceptibility testing of M tuberculosis. Our data and expert opinion informed WHO's recommendation for use of selected non-commercial drug susceptibility tests

A spectrophotometer-based diffusivity assay reveals that diffusion hindrance of small molecules in extracellular matrix gels used in 3D cultures is dominated by viscous effects.

PubMed

Galgoczy, Roland; Pastor, Isabel; Colom, Adai; Giménez, Alicia; Mas, Francesc; Alcaraz, Jordi

2014-08-01

The design of 3D culture studies remains challenging due to the limited understanding of extracellular matrix (ECM)-dependent hindered diffusion and the lack of simple diffusivity assays. To address these limitations, we set up a cost-effective diffusivity assay based on a Transwell plate and the spectrophotometer of a Microplate Reader, which are readily accessible to cell biology groups. The spectrophotometer-based assay was used to assess the apparent diffusivity D of FITC-dextrans with molecular weight (4-70kDa) spanning the physiological range of signaling factors in a panel of acellular ECM gels including Matrigel, fibrin and type I collagen. Despite their technical differences, D data exhibited ∼15% relative difference with respect to FRAP measurements. Our results revealed that diffusion hindrance of small particles is controlled by the enhanced viscosity of the ECM gel in conformance with the Stokes-Einstein equation rather than by geometrical factors. Moreover, we provided a strong rationale that the enhanced ECM viscosity is largely contributed to by unassembled ECM macromolecules. We also reported that gels with the lowest D exhibited diffusion hindrance closest to the large physiologic hindrance of brain tissue, which has a typical pore size much smaller than ECM gels. Conversely, sparse gels (≤1mg/ml), which are extensively used in 3D cultures, failed to reproduce the hindered diffusion of tissues, thereby supporting that dense (but not sparse) ECM gels are suitable tissue surrogates in terms of macromolecular transport. Finally, the consequences of reduced diffusivity in terms of optimizing the design of 3D culture experiments were addressed in detail. Copyright © 2014 Elsevier B.V. All rights reserved.

48 CFR 401.371 - AGAR Advisories.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false AGAR Advisories. 401.371 Section 401.371 Federal Acquisition Regulations System DEPARTMENT OF AGRICULTURE GENERAL AGRICULTURE ACQUISITION REGULATION SYSTEM Agency Acquisition Regulations 401.371 AGAR Advisories. The SPE may issue AGAR...

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.

Demonstrating Diffusion

ERIC Educational Resources Information Center

Foy, Barry G.

1977-01-01

Two demonstrations are described. Materials and instructions for demonstrating movement of molecules into cytoplasm using agar blocks, phenolphthalein, and sodium hydroxide are given. A simple method for demonstrating that the rate of diffusion of a gas is inversely proportional to its molecular weight is also presented. (AJ)

Evaluation of genotoxic effects of lead in pottery-glaze workers using micronucleus assay, alkaline comet assay and DNA diffusion assay.

PubMed

Kašuba, V; Rozgaj, R; Milić, M; Zelježić, D; Kopjar, N; Pizent, A; Kljaković-Gašpić, Z; Jazbec, A

2012-10-01

We investigated genotoxic effects of occupational exposure to lead acetate in pottery-glaze ceramic workers. The study was carried out in 30 exposed workers and 30 matched controls, to whom several biochemical parameters-the blood lead (B-Pb; range: exposed, 41.68-404.77; controls, 12-52) and cadmium (B-Cd) level, the activity of delta-aminolevulinic acid dehydratase (ALAD), erythrocyte protoporphyrin (EP), the level of vitamin B(12) and folate in serum-were measured. The genotoxic effects were evaluated by the alkaline comet assay, the DNA diffusion assay and micronucleus test in peripheral blood lymphocytes. Subjects exposed to lead had significantly higher B-Pb level and, consequently, increased values of tail intensity (TI), frequency of apoptotic and necrotic cells, and frequency of micronuclei (MN). In contrast, their activity of ALAD, the level of vitamin B(12) and folate in serum were significantly lower compared to controls. Poisson regression analysis showed a significant correlation of profession, duration of exposure, smoking, level of cadmium in blood, ALAD and EP with primary DNA damage. A majority of primary damage repairs in a short period after exposure to a genotoxic agent. In addition, the influence of gender and level of vitamin B(12) and folate in serum MN frequency in exposed group was observed. In this study, DNA diffusion and micronucleus test showed higher influence of tested parameters to DNA damage. The results indicate a need for concomitant use of at least two different biomarkers of exposure when estimating a genetic risk of lead exposure.

Application of firefly luciferase assay for adenosine triphosphate (ATP) to antimicrobial drug sensitivity testing

NASA Technical Reports Server (NTRS)

Picciolo, G. L.; Tuttle, S. A.; Schrock, C. G.; Deming, J. W.; Barza, M. J.; Wienstein, L.; Chappelle, E. W.

1977-01-01

The development of a rapid method for determining microbial susceptibilities to antibiotics using the firefly luciferase assay for adenosine triphosphate (ATP) is documented. The reduction of bacterial ATP by an antimicrobial agent was determined to be a valid measure of drug effect in most cases. The effect of 12 antibiotics on 8 different bacterial species gave a 94 percent correlation with the standard Kirby-Buer-Agar disc diffusion method. A 93 percent correlation was obtained when the ATP assay method was applied directly to 50 urine specimens from patients with urinary tract infections. Urine samples were centrifuged first to that bacterial pellets could be suspended in broth. No primary isolation or subculturing was required. Mixed cultures in which one species was predominant gave accurate results for the most abundant organism. Since the method is based on an increase in bacterial ATP with time, the presence of leukocytes did not interfere with the interpretation of results. Both the incubation procedure and the ATP assays are compatible with automation.

Use of agar/glycerol and agar/glycerol/water as a translucent brain simulant for ballistic testing.

PubMed

Falland-Cheung, Lisa; Waddell, J Neil; Lazarjan, Milad Soltanipour; Jermy, Mark C; Winter, Taylor; Tong, Darryl; Brunton, Paul A

2017-01-01

The suitability of agar/glycerol/water and agar/glycerol mixtures as brain simulants was investigated. Test specimens (n=15) (50x27×37mm) were fabricated for these different mixtures and conditioned to 12°C, 22°C, and 26°C prior to testing. For comparison, fresh deer brain specimens (n=20) were sourced and prepared to the same dimensions as the agar/glycerol(/water) mixtures and conditioned to 12°C and 37°C. High impact tests were carried out with a 0.22-caliber air rifle pellet and a high-speed camera was used to record the projectile as it passed through the specimens, allowing for energy loss and vertical displacement velocity calculation. Although the agar/glycerol/water mixture presented with similar vertical expansion and contraction of the specimens to the warm and cold deer brains, a two-fold decrease of the vertical expansion and contraction was noticed with the agar/glycerol specimens. Also considerably less extrusion of this mixture out of the exit and entry sides after specimen penetration was observed. Of the simulants tested, agar/glycerol/water was the most suitable brain simulant for ballistic testing and impact studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

Comparison of chromogenic Biolog Rainbow agar Shigella/Aeromonas with xylose lysine desoxycholate agar for isolation and detection of Shigella spp. from foods.

PubMed

Zhang, Guodong; Lampel, Keith A

2010-08-01

Shigella outbreaks are widely reported throughout the world. However, it remains a challenge to isolate Shigella spp. from foods by using conventional microbiological media. The main objective of this study was to determine the effectiveness of a novel chromogenic medium, Rainbow agar Shigella/Aeromonas (Rainbow agar), for the isolation and detection of Shigella spp. in foods. All four Shigella species, S. sonnei, S. flexneri, S. dysenteriae, and S. boydii, were studied. Rainbow agar was compared with tryptic soy agar, xylose lysine desoxycholate agar (XLD), and Salmonella Shigella agar (SSA) for enumeration of Shigella spp. in pure culture. This chromogenic agar and XLD were also used to isolate Shigella spp. in artificially contaminated foods (4.8 log CFU/g of food), including lettuce, parsley, cilantro, spinach, potato salad, and shrimp. The inhibitory effect on Shigella growth by Rainbow agar was between that of XLD and SSA. All vegetables studied showed a moderately high background microflora on XLD and Rainbow agar. With artificially inoculated produce, Rainbow agar recovered about 1 to 2 log CFU more S. sonnei, S. dysenteriae, and S. boydii per g of food than did XLD. For potato salad and shrimp, which had low background microflora on Rainbow agar, Rainbow agar was slightly better in recovering Shigella spp. than XLD was in most cases. However, we found that the addition of streptomycin (6.25 mg/liter) to Rainbow agar could facilitate the isolation of Shigella in vegetables tested. In conclusion, Rainbow agar was a much more effective medium than was XLD for the isolation of Shigella spp. from foods.

Detection of group B streptococci in Lim broth by use of group B streptococcus peptide nucleic acid fluorescent in situ hybridization and selective and nonselective agars.

PubMed

Montague, Naomi S; Cleary, Timothy J; Martinez, Octavio V; Procop, Gary W

2008-10-01

The sensitivity, specificity, and positive and negative predictive values for the detection of group B streptococci from Lim enrichment broth with sheep blood agar (SBA), with selective Streptococcus agar (SSA), and by a peptide nucleic acid fluorescent in situ hybridization (PNA FISH) assay were as follows: for culture on SBA, 68.4%, 100%, 100%, and 87.9%, respectively; for culture on SSA, 85.5%, 100%, 100%, and 94.1%, respectively; and for the PNA FISH assay, 97.4%, 98.3%, 96.1%, and 98.9%, respectively.

21 CFR 866.4600 - Ouchterlony agar plate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Ouchterlony agar plate. 866.4600 Section 866.4600 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED....4600 Ouchterlony agar plate. (a) Identification. An ouchterlony agar plate for clinical use is a device...

21 CFR 866.4600 - Ouchterlony agar plate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Ouchterlony agar plate. 866.4600 Section 866.4600 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED....4600 Ouchterlony agar plate. (a) Identification. An ouchterlony agar plate for clinical use is a device...

Molecular Diffusion Coefficients: Experimental Determination and Demonstration.

ERIC Educational Resources Information Center

Fate, Gwendolyn; Lynn, David G.

1990-01-01

Presented are laboratory methods which allow the demonstration and determination of the diffusion coefficients of compounds ranging in size from water to small proteins. Included are the procedures involving the use of a spectrometer, UV cell, triterated agar, and oxygen diffusion. Results including quantification are described. (CW)

Antimicrobial activity of highly stable silver nanoparticles embedded in agar-agar matrix as a thin film.

PubMed

Ghosh, S; Kaushik, R; Nagalakshmi, K; Hoti, S L; Menezes, G A; Harish, B N; Vasan, H N

2010-10-13

Highly stable silver nanoparticles (Ag NPs) in agar-agar (Ag/agar) as inorganic-organic hybrid were obtained as free-standing film by in situ reduction of silver nitrate by ethanol. The antimicrobial activity of Ag/agar film on Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Candida albicans (C. albicans) was evaluated in a nutrient broth and also in saline solution. In particular, films were repeatedly tested for antimicrobial activity after recycling. UV-vis absorption and TEM studies were carried out on films at different stages and morphological studies on microbes were carried out by SEM. Results showed spherical Ag NPs of size 15-25 nm, having sharp surface plasmon resonance (SPR) band. The antimicrobial activity of Ag/agar film was found to be in the order, C. albicans>E. coli>S. aureus, and antimicrobial activity against C. albicans was almost maintained even after the third cycle. Whereas, in case of E. coli and S. aureus there was a sharp decline in antimicrobial activity after the second cycle. Agglomeration of Ag NPs in Ag/agar film on exposure to microbes was observed by TEM studies. Cytotoxic experiments carried out on HeLa cells showed a threshold Ag NPs concentration of 60 μg/mL, much higher than the minimum inhibition concentration of Ag NPs (25.8 μg/mL) for E. coli. The mechanical strength of the film determined by nanoindentation technique showed almost retention of the strength even after repeated cycle. Copyright © 2010 Elsevier Ltd. All rights reserved.

A fixed-time diffusion analysis method determines that the three cheV genes of Helicobacter pylori differentially affect motility

PubMed Central

Lowenthal, Andrew C.; Simon, Christopher; Fair, Amber S.; Mehmood, Khalid; Terry, Karianne; Anastasia, Stephanie; Ottemann, Karen M.

2009-01-01

Helicobacter pylori is a chemotactic bacterium that has three CheV proteins in its predicted chemotaxis signal transduction system. CheV proteins contain both CheW- and response-regulator-like domains. To determine the function of these proteins, we developed a fixed-time diffusion method that would quantify bacterial direction change without needing to define particular behaviours, to deal with the many behaviours that swimming H. pylori exhibit. We then analysed mutants that had each cheV gene deleted individually and found that the behaviour of each mutant differed substantially from wild-type and the other mutants. cheV1 and cheV2 mutants displayed smooth swimming behaviour, consistent with decreased cellular CheY-P, similar to a cheW mutant. In contrast, the cheV3 mutation had the opposite effect and the mutant cells appeared to change direction frequently. Additional analysis showed that the cheV mutants displayed aberrant behaviour as compared to the wild-type in the soft-agar chemotaxis assay. The soft-agar assay phenotype was less extreme compared to that seen in the fixed-time diffusion model, suggesting that the cheV mutants are able to partially compensate for their defects under some conditions. Each cheV mutant furthermore had defects in mouse colonization that ranged from severe to modest, consistent with a role in chemotaxis. These studies thus show that the H. pylori CheV proteins each differently affect swimming behaviour. PMID:19332820

Improving agar electrospinnability with choline-based deep eutectic solvents.

PubMed

Sousa, Ana M M; Souza, Hiléia K S; Uknalis, Joseph; Liu, Shih-Chuan; Gonçalves, Maria P; Liu, LinShu

2015-09-01

Very recently our group has produced novel agar-based fibers by an electrospinning technique using water as solvent and polyvinyl alcohol (PVA) as co-blending polymer. Here, we tested the deep eutectic solvent (DES), (2-hydroxyethyl)trimethylammonium chloride/urea prepared at 1:2 molar ratio, as an alternative solvent medium for agar electrospinning. The electrospun materials were collected with an ethanol bath adapted to a previous electrospinning set-up. One weight percent agar-in-DES showed improved viscoelasticity and hence, spinnability, when compared to 1 wt% agar-in-water and pure agar nanofibers were successfully electrospun if working above the temperature of sol-gel transition (∼80 °C). By changing the solvent medium we decreased the PVA concentration (5 wt% starting solution) and successfully produced composite fibers with high agar contents (50/50 agar/PVA). Best composite fibers were formed with the 50/50 and 30/70 agar/PVA solutions. These fibers were mechanically resistant, showed tailorable surface roughness and diverse size distributions, with most of the diameters falling in the sub-micron range. Both nano and micro forms of agar fibers (used separately or combined) may have potential for the design of new and highly functional agar-based materials. Published by Elsevier B.V.

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

Characterization of agar/soy protein biocomposite films: Effect of agar on the extruded pellets and compression moulded films.

PubMed

Garrido, T; Etxabide, A; Guerrero, P; de la Caba, K

2016-10-20

Agar/soy protein biocomposite films were successfully processed by extrusion and compression moulding, obtaining transparent and homogeneous films. The conformational changes occurred during the extrusion process and the effect of agar on the final properties were analyzed. As shown by differential scanning calorimetry (DSC) and specific mechanical energy (SME) values, during the extrusion process protein denatured and unfolded protein chains could interact with agar. These interactions were analyzed by Fourier transform infrared spectroscopy (FTIR) and the secondary structure was determined from the amide I band. Those interactions were supported by the decrease of film solubility. Furthermore, the good compatibility between agar and soy protein was confirmed by the images from scanning electron microscopy (SEM). Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

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.

A Novel Chromogenic Ester Agar Medium for Detection of Salmonellae

PubMed Central

Cooke, Venitia M.; Miles, R. J.; Price, R. G.; Richardson, A. C.

1999-01-01

A novel agar medium, chromogenic Salmonella esterase (CSE) agar, for the differentiation of salmonellae is described. The agar contains peptones and nutrient extracts together with the following (grams per liter unless otherwise specified): 4-[2-(4-octanoyloxy-3,5-dimethoxyphenyl)-vinyl]-quinolinium-1-(propan-3-yl carboxylic acid) bromide (SLPA-octanoate; bromide form), 0.3223; lactose, 14.65; trisodium citrate dihydrate, 0.5; Tween 20, 3.0; ethyl 4-dimethylaminobenzoate, 0.035% (wt/vol), novobiocin, 70 mg liter−1. The key component of the medium is SLPA-octanoate, a newly synthesized ester formed from a C8 fatty acid and a phenolic chromophore. In CSE agar, the ester is hydrolyzed by Salmonella spp. to yield a brightly colored phenol which remains tightly bound within colonies. After 24 h of incubation at 37 or 42°C, colonies of typical Salmonella spp. were burgundy colored on a transparent yellow background, whereas non-Salmonella spp. were white, cream, yellow or transparent. CSE agar was evaluated by using a panel of strains including a high proportion of Salmonella and non-Salmonella strains giving atypical reactions on other differential agars. The sensitivity (93.1%) of CSE agar for non-typhi salmonellae compared favorably with those of Rambach (82.8%), xylose-lysine-deoxycholate (XLD; 91.4%), Hektoen-enteric (89.7%), and SM ID (91.4%) agars. The specificity (93.9%) was also comparable to those of other Salmonella media (SM ID agar, 95.9%; Rambach agar, 91.8%; XLD agar, 91.8%; Hektoen-enteric agar, 87.8%). Strains of Citrobacter freundii and Proteus spp. giving false-positive reactions with other media gave a negative color reaction on CSE agar. CSE agar enabled the detection of >30 Salmonella serotypes, including agona, anatum, enteritidis, hadar, heidelberg, infantis, montevideo, thompson, typhimurium, and virchow, which accounted for 91.8% of the salmonella isolates recorded by the Public Health Laboratory Service (Colindale, London, England) for 1997

Borelli's lactritmel agar induces conidiation in rare-macroconidia producing dermatophytic fungi.

PubMed

Ilkit, Macit; Gümral, Ramazan; Döğen, Aylin

2012-10-01

Macroconidia are among the most important indicators used to identify dermatophytic fungi, but several do not usually sporulate and/or produce macroconidia on Sabouraud glucose agar. Specifically, Microsporum audouinii, M. ferrugineum, Trichophyton concentricum, T. schoenleinii, T. verrucosum, and T. violaceum (including T. soudanense and T. yaoundei) rarely form macroconidia and, therefore, cannot be easily identified. In this study, we investigated the production of macroconidia on nine common laboratory media, including Borelli's lactritmel agar (BLA), modified Borelli's lactritmel agar (MBLA), brain heart infusion agar (BHIA), Christensen's urease agar in Petri dishes (UPA), cornmeal dextrose agar (CMDA), Lowenstein-Jensen agar (LJA), malt extract agar (MEA), oatmeal agar (OA), and potato dextrose agar (PDA). The performance of these media was evaluated using 18 rare-macroconidia producing isolates, including representative of the six species mentioned above. All cultures in this study were incubated at 26°C on the bench, and conidia formation on each was investigated at 5, 10, 15, 20, 25, and 30 days of incubation. BLA apparently improved macroconidia production after 15 days and was the most useful nutrient agar medium to induce these phenotypic characters in daily practice, closely followed by OA, PDA, and MBLA.

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

Multi-chamber electroosmosis using textile reinforced agar membranes--A promising concept for the future of hemodialysis.

PubMed

Kofler, Markus; Lenninger, Margit; Mayer, Gert; Neuwirt, Hannes; Grimm, Michael; Bechtold, Thomas

2016-01-20

Renal replacement therapy options are limited to hemodialysis and peritoneal dialysis (70% of US patients) or renal transplantation. Diffusion processes are the main physico-chemical principle behind hemodialysis. An alternative way to achieve liquid flow through membranes bases on the electroosmotic flow which is observed as electrokinetic phenomenon in porous membranes which bear surface charges. Agar consists of the non-ionic agarose and the negatively charged agaropectine thus an electroosmotic flux is observed in analytical electrophoresis. In this study the potential electroosmosis on textile reinforced agar membranes as separation method was investigated. Using a five-chamber electrolysis cell and an agar membrane/cellulose fabric composite an intensive electroosmotic flow of 1-2 ml cm(2) h(-1) at 100 mA cell current could be observed. The movement of cations in the negatively charged agar structure led to an intensive electroosmotic flux, which also transported uncharged molecules such as urea, glucose through the membrane. Separation of uncharged low molecular weight molecules is determined by the membrane characteristic. The transport of ions (K(+), PO4(3-), creatinine) and uncharged molecules (urea, glucose) in electroosmotic separation experiments was monitored using a pH 5.5 phosphate electrolyte with the aim to assess the overall transport processes in the electrochemical cell. The results demonstrate the potential of the method for filtration of biological fluids in the absence of external pressure or high shear rates. Copyright © 2015 Elsevier Ltd. All rights reserved.

Green synthesis of gold nanoparticles of different sizes and shapes using agar-agar water solution and femtosecond pulse laser irradiation

NASA Astrophysics Data System (ADS)

Almeida de Matos, Ricardo; da Silva Cordeiro, Thiago; Elgul Samad, Ricardo; Dias Vieira, Nilson; Coronato Courrol, Lilia

2012-11-01

We report a method to create gold nanoparticles of different sizes and shapes using agar-agar water solution and irradiation with light from a xenon lamp, followed by ultrashort laser pulses. No additives, such as solvents, surfactants or reducing agents, were used in the procedure. Laser irradiation (laser ablation) was important to the reduction of the nanoparticles diameter and formation of another shapes. Distilled water was used as solvent and agar-agar (hydrophilic colloid extracted from certain seaweeds) was important for the stabilization of gold nanoparticles, avoiding their agglomeration. The formation of gold nanoparticles was confirmed with ultraviolet-visible absorption and TEM microscopy. The gold nanoparticles acquired spherical, prism, and rod shapes depending on the laser parameters. Variation of laser irradiation parameters as pulse energy, irradiation time and repetition rate was assessed. The relevant mechanisms contributing for the gold nanoparticles production are discussed.

Acanthamoeba on Sabouraud's agar from a patient with keratitis

PubMed Central

Baradkar, Vasant; Samal, Badhuli; Mali, Swapna A; Kulkarni, Ketaki; Shastri, Jayanthi

2011-01-01

A 25-year-old transgender patient came with complaints of watery discharge, red eye and photophobia in the left eye since 2 days. The patient had a history of wearing colored contact lenses since 4 years and cleaning the lens with tap water. Culture of lenses on Mac Conkey and blood agar yielded Klebsiella pneumoniae and Pseudomonas aeruginosa. Sabouroud's agar showed yeast cells and double-walled cysts of Acanthamoeba species. On further incubation of Sabouroud's agar, the cysts transformed to trophozoites. Parallel results were obtained on tap water agar. The previous therapy of moxifloxacin was changed to local Neosporin application. PMID:23508061

Antibacterial activity of Greek and Cypriot honeys against Staphylococcus aureus and Pseudomonas aeruginosa in comparison to manuka honey.

PubMed

Anthimidou, Eleni; Mossialos, Dimitris

2013-01-01

The antibacterial activity of 31 Greek and Cypriot honeys against Staphylococcus aureus and Pseudomonas aeruginosa was initially screened using an agar-well diffusion assay in comparison with manuka honey. The minimum inhibitory concentration (MIC) was determined in broth using a spectrophotometric-based assay. The MIC of treated honeys with catalase or proteinase K was determined and compared with those of untreated honeys. All tested honeys demonstrated antibacterial activity against S. aureus on agar-well diffusion assay. MICs of tested honeys were determined as 3.125-25% (v/v), compared with manuka honey at 6.25% (v/v). Similarly, 21 of 31 tested honeys demonstrated antibacterial activity on agar-well diffusion assay against P. aeruginosa. Their MICs ranged from 6.25% to 25% (v/v) compared with 12.5% (v/v) for manuka honey. Antibacterial activity of tested honeys could be largely attributed to hydrogen peroxide formation and in some cases to unidentified proteinaceous compounds. In conclusion, Greek and Cypriot honeys demonstrated significant but variable antibacterial activity against P. aeruginosa and especially S. aureus. To the best of our knowledge this is the first study that has thoroughly examined the antibacterial activity of Greek and Cypriot honeys compared with manuka honey. The high antibacterial activity exerted by some tested honeys warrants further investigation.

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

Relative diffusion of paramagnetic metal complexes of MRI contrast agents in an isotropic hydrogel medium.

PubMed

Weerakoon, Bimali Sanjeevani; Osuga, Toshiaki

2017-03-01

The observation of molecular diffusion by means of magnetic resonance imaging (MRI) is significant in the evaluation of the metabolic activity of living tissues. Series of MRI examinations were conducted on a diffusion model to study the behaviour of the diffusion process of different-molecular-weight (MW) paramagnetic MRI contrast agents in an isotropic agar hydrogel medium. The model consisted of a solidified 1 % agar gel with an initial concentration of 0.5 mmol/L contrast solution layered on top of the gel. The diffusion process was monitored at pre-determined time intervals of immediately, 1, 6, 9, 23, and 48 h after introduction of the contrast agents onto the agar gel with a T1-weighted spin-echo (SE) pulse sequence. Three types of paramagnetic contrast agents, Gd-DTPA with a MW of 547.57 g/mol, Prohance with a MW of 558.69 g/mol and MnCl 2 with a MW of 125.84 g/mol, resulted in an approximate average diffusional displacement ratio of 1:1:2 per hour, respectively, within 48 h of the experiment. Therefore, the results of this study supported the hypothesis that the rate of the diffusion process of MRI contrast agents in the agar hydrogel medium is inversely related to their MWs. However, more repetitions are necessary under various types of experimental conditions and also with various types of contrast media of different MWs for further confirmation and validation of these results.

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

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

Echinocandin Susceptibility Testing of Candida Species: Comparison of EUCAST EDef 7.1, CLSI M27-A3, Etest, Disk Diffusion, and Agar Dilution Methods with RPMI and IsoSensitest Media▿

PubMed Central

Arendrup, Maiken Cavling; Garcia-Effron, Guillermo; Lass-Flörl, Cornelia; Lopez, Alicia Gomez; Rodriguez-Tudela, Juan-Luis; Cuenca-Estrella, Manuel; Perlin, David S.

2010-01-01

This study compared nine susceptibility testing methods and 12 endpoints for anidulafungin, caspofungin, and micafungin with the same collection of blinded FKS hot spot mutant (n = 29) and wild-type isolates (n = 94). The susceptibility tests included EUCAST Edef 7.1, agar dilution, Etest, and disk diffusion with RPMI-1640 plus 2% glucose (2G) and IsoSensitest-2G media and CLSI M27A-3. Microdilution plates were read after 24 and 48 h. The following test parameters were evaluated: fks hot spot mutants overlapping the wild-type distribution, distance between the two populations, number of very major errors (VMEs; fks mutants misclassified as susceptible), and major errors (MEs; wild-type isolates classified as resistant) using a wild-type-upper-limit value (WT-UL) (two twofold-dilutions higher than the MIC50) as the susceptibility breakpoint. The methods with the lowest number of errors (given as VMEs/MEs) across the three echinocandins were CLSI (12%/1%), agar dilution with RPMI-2G medium (14%/0%), and Etest with RPMI-2G medium (8%/3%). The fewest errors overall were observed for anidulafungin (4%/1% for EUCAST, 4%/3% for CLSI, and 3%/9% for Etest with RPMI-2G). For micafungin, VME rates of 10 to 71% were observed. For caspofungin, agar dilution with either medium was superior (VMEs/MEs of 0%/1%), while CLSI, EUCAST with IsoSensitest-2G medium, and Etest were less optimal (VMEs of 7%, 10%, and 10%, respectively). Applying the CLSI breakpoint (S ≤ 2 μg/ml) for CLSI results, 89.2% fks hot spot mutants were classified as anidulafungin susceptible, 60.7% as caspofungin susceptible, and 92.9% as micafungin susceptible. In conclusion, no test was perfect, but anidulafungin susceptibility testing using the WT-UL to define susceptibility reliably identified fks hot spot mutants. PMID:19884370

Chocolate agar, a differential medium for gram-positive cocci.

PubMed Central

Gunn, B A

1984-01-01

Reactions incurred on chocolate agar by gram-positive cocci were correlated with species identity. Darkening and clearing of the medium was usually associated with the species Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus simulans, and Streptococcus faecalis. Yellowing of chocolate agar was associated with alpha-hemolytic species of Streptococcus. The study demonstrated that reactions occurring on chocolate agar are useful in identifying gram-positive cocci. PMID:6490866

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.

Comparison between Disk Diffusion and Microdilution Methods for Determining Susceptibility of Clinical Fungal Isolates to Caspofungin▿

PubMed Central

Milici, Maria Eleonora; Maida, Carmelo Massimo; Spreghini, Elisabetta; Ravazzolo, Barbara; Oliveri, Salvatore; Scalise, Giorgio; Barchiesi, Francesco

2007-01-01

We compared the caspofungin (CAS) susceptibility testing results generated by the disk diffusion (DD) assay with the results of the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BD) reference method for 106 yeast isolates. The isolates represented 11 different fungal species, including Candida albicans (n = 50), C. parapsilosis (n = 10), C. glabrata (n = 10), C. tropicalis (n = 10), C. guillermondii (n = 6), C. rugosa (n = 5), C. krusei (n = 5), C. kefyr (n = 2), C. pelliculosa (n = 2), Saccharomyces cerevisiae (n = 3), and Geotrichum candidum (n = 3). The DD assay was performed in supplemented Mueller-Hinton agar with CAS, which was tested at concentrations of 2, 10, and 25 μg per disk. MICs and inhibition zone diameters were evaluated at 24 and 48 h. In general, the results obtained by the DD assay correlated well with those obtained by the BD method. In particular, a significant correlation between methods was observed when CAS was used at concentration of 2 μg/disk at a reading time of either 24 or 48 h. PMID:17728477

diskImageR: quantification of resistance and tolerance to antimicrobial drugs using disk diffusion assays.

PubMed

Gerstein, Aleeza C; Rosenberg, Alexander; Hecht, Inbal; Berman, Judith

2016-07-01

Microbial pathogens represent an increasing threat to human health. Although many infections can be successfully treated and cleared, drug resistance is a widespread problem. The existence of subpopulations of 'tolerant' cells (where a fraction of the population is able to grow above the population resistance level) may increase the rate of treatment failure; yet, existing methods to measure subpopulation effects are cumbersome. Here we describe diskImageR, a computational pipeline that analyses photographs of disk diffusion assays to determine the degree of drug susceptibility [the radius of inhibition, (RAD)], and two aspects of subpopulation growth [the fraction of growth (FoG) within the zone of inhibition, (ZOI), and the rate of change in growth from no drug to inhibitory drug concentrations, (SLOPE)]. diskImageR was used to examine the response of the human fungal pathogen Candida albicans to the antifungal drug fluconazole across different strain backgrounds and growth conditions. Disk diffusion assays performed under Clinical and Laboratory Standards Institute (CLSI) conditions led to more susceptibility and less tolerance than assays performed using rich medium conditions. We also used diskImageR to quantify the effects of three drugs in combination with fluconazole, finding that all three combinations affected tolerance, with the effect of one drug (doxycycline) being very strain dependent. The three drugs had different effects on susceptibility, with doxycycline generally having no effect, chloroquine generally increasing susceptibility and pyrvinium pamoate generally reducing susceptibility. The ability to simultaneously quantitate different aspects of microbial drug responses will facilitate the study of mechanisms of subpopulation responses in the presence of antimicrobial drugs.

Factors Affecting Selectivity of Brilliant Green-Phenol Red Agar for Salmonellae

PubMed Central

Moats, W. A.; Kinner, J. A.

1974-01-01

Commercial brilliant green (BG)-sulfa agar was found to be nonselective toward a test series of Enterobacteriaceae. Various formulations of BG were prepared by using Trypticase soy agar (BBL) as a base. Results were more reproducible when BG dye was added after sterilization than before. Sulfonamides improved selectivity as compared with brilliant green alone. Sulfanilamide (SN) was slightly more selective for salmonellae than other sulfonamides tested. Bile salts and sodium dodecyl sulfate markedly reduced the toxicity of BG to all the test bacteria. Enterobacter strains were most difficult to inhibit. A combination of 5 mg of BG and 1 g of SN/liter prevented growth of Proteus mirabilis and Escherichia coli and retarded growth of Enterobacter strains. The BG-SN agars were superior in selectivity to a series of commercial agars tested, and numbers of salmonellae recovered on BG-SN agar and Trypticase soy agar (BBL) were the same. Brilliant green agars with various degrees of selectivity are described. PMID:4589120

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

Selection of Brain Metastasis-Initiating Breast Cancer Cells Determined by Growth on Hard Agar

PubMed Central

Guo, Lixia; Fan, Dominic; Zhang, Fahao; Price, Janet E.; Lee, Ju-Seog; Marchetti, Dario; Fidler, Isaiah J.; Langley, Robert R.

2011-01-01

An approach that facilitates rapid isolation and characterization of tumor cells with enhanced metastatic potential is highly desirable. Here, we demonstrate that plating GI-101A human breast cancer cells on hard (0.9%) agar selects for the subpopulation of metastasis-initiating cells. The agar-selected cells, designated GI-AGR, were homogeneous for CD44+ and CD133+ and five times more invasive than the parental GI-101A cells. Moreover, mice injected with GI-AGR cells had significantly more experimental brain metastases and shorter overall survival than did mice injected with GI-101A cells. Comparative gene expression analysis revealed that GI-AGR cells were markedly distinct from the parental cells but shared an overlapping pattern of gene expression with the GI-101A subline GI-BRN, which was generated by repeated in vivo recycling of GI-101A cells in an experimental brain metastasis model. Data mining on 216 genes shared between GI-AGR and GI-BRN breast cancer cells suggested that the molecular phenotype of these cells is consistent with that of cancer stem cells and the aggressive basal subtype of breast cancer. Collectively, these results demonstrate that analysis of cell growth in a hard agar assay is a powerful tool for selecting metastasis-initiating cells in a heterogeneous population of breast cancer cells, and that such selected cells have properties similar to those of tumor cells that are selected based on their potential to form metastases in mice. PMID:21514446

Light transfer in agar immobilized microalgae cell cultures

NASA Astrophysics Data System (ADS)

Kandilian, Razmig; Jesus, Bruno; Legrand, Jack; Pilon, Laurent; Pruvost, Jérémy

2017-09-01

This paper experimentally and theoretically investigates light transfer in agar-immobilized cell cultures. Certain biotechnological applications such as production of metabolites secreted by photosynthetic microorganisms require cells to be immobilized in biopolymers to minimize contamination and to facilitate metabolite recovery. In such applications, light absorption by cells is one of the most important parameters affecting cell growth or metabolite productivity. Modeling light transfer therein can aid design and optimize immobilized-cell reactors. In this study, Parachlorella kessleri cells with areal biomass concentrations ranging from 0.36 to 16.9 g/m2 were immobilized in 2.6 mm thick agar gels. The average absorption and scattering cross-sections as well as the scattering phase function of P. kessleri cells were measured. Then, the absorption and transport scattering coefficients of the agar gel were determined using an inverse method based on the modified two-flux approximation. The forward model was used to predict the normal-hemispherical transmittance and reflectance of the immobilized-cell films accounting for absorption and scattering by both microalgae and the agar gel. Good agreement was found between the measured and predicted normal-hemispherical transmittance and reflectance provided absorption and scattering by agar were taken into account. Moreover, good agreement was found between experimentally measured and predicted mean rate of photon absorption. Finally, optimal areal biomass concentration was determined to achieve complete absorption of the incident radiation.

[Evaluation of in vitro antimicrobial activity of cefazolin alone and in combination with cefmetazole or flomoxef using agar dilution method and disk diffusion method].

PubMed

Matsuo, K; Uete, T

1992-10-01

Antimicrobial activities of cefazolin (CEZ) against 251 strains of various clinical isolates obtained during 1989 and 1990 were determined using the Mueller-Hinton agar dilution method at an inoculum level 10(6) CFU/ml. The reliability of the disk susceptility test was also studied using Mueller-Hinton agar and various disks at inoculum levels of 10(3-4) CFU/cm2 in estimating approximate values of MICs. In addition, antimicrobial activities of CEZ and cefmetazole (CMZ) or flomoxef (FMOX) in combination were investigated against methicillin-sensitive and -resistant Staphylococcus aureus (MSSA and MRSA) using the checkerboard agar dilution MIC method and the disk diffusion test either with the disks contained CEZ, CMZ, and FMOX alone, or CEZ, and CMZ or FMOX in combination. In this study, the MICs of CEZ against S. aureus were distributed with the 3 peak values at 0.39 microgram/ml, 3.13 micrograms/ml and > 100 micrograms/ml. MICs against MSSA were 0.39 microgram/ml to 0.78 microgram/ml, whereas those against MRSA were greater than 0.78 microgram/ml. MICs against majority of strains of Enterococcus faecalis were 25 micrograms/ml. Over 90% of strains of Escherichia coli and Klebsiella pneumoniae were inhibited at the level of 3.13 micrograms/ml. About 60% of isolates of indole negative Proteus spp. were inhibited at the levels of less than 3.13 micrograms/ml and 100% at 6.25 micrograms/ml, but MICs against indole positive Proteus spp., Serratia spp. and Pseudomonas aeruginosa were over 100 micrograms/ml. The antimicrobial activities of CEZ against these clinical isolates were not significantly different compared to those reported about 15-20 years ago, except for S. aureus. Highly resistant strains of S. aureus to CEZ were more prevalent in this study. The inhibitory zones obtained with the disk test were compared with MICs. The results of CEZ disk susceptibility test with 30 micrograms disk (Showa) or 10 micrograms disk (prepared in this laboratory) were well

Antimicrobial Susceptibility of Flavobacterium psychrophilum from Chilean Salmon Farms and Their Epidemiological Cut-Off Values Using Agar Dilution and Disk Diffusion Methods.

PubMed

Miranda, Claudio D; Smith, Peter; Rojas, Rodrigo; Contreras-Lynch, Sergio; Vega, J M Alonso

2016-01-01

Flavobacterium psychrophilum is the most important bacterial pathogen for freshwater farmed salmonids in Chile. The aims of this study were to determine the susceptibility to antimicrobials used in fish farming of Chilean isolates and to calculate their epidemiological cut-off (CO WT ) values. A number of 125 Chilean isolates of F. psychrophilum were isolated from reared salmonids presenting clinical symptoms indicative of flavobacteriosis and their identities were confirmed by 16S rRNA polymerase chain reaction. Susceptibility to antibacterials was tested on diluted Mueller-Hinton by using an agar dilution MIC method and a disk diffusion method. The CO WT values calculated by Normalized Resistance Interpretation (NRI) analysis allow isolates to be categorized either as wild-type fully susceptible (WT) or as manifesting reduced susceptibility (NWT). When MIC data was used, NRI analysis calculated a CO WT of ≤0.125, ≤2, and ≤0.5 μg mL -1 for amoxicillin, florfenicol, and oxytetracycline, respectively. For the quinolones, the CO WT were ≤1, ≤0.5, and ≤0.125 μg mL -1 for oxolinic acid, flumequine, and enrofloxacin, respectively. The disk diffusion data sets obtained in this work were extremely diverse and were spread over a wide range. For the quinolones there was a close agreement between the frequencies of NWT isolates calculated using MIC and disk data. For oxolinic acid, flumequine, and enrofloxacin the frequencies were 45, 39, and 38% using MIC data, and 42, 41, and 44%, when disk data were used. There was less agreement with the other antimicrobials, because NWT frequencies obtained using MIC and disk data, respectively, were 24 and 10% for amoxicillin, 8 and 2% for florfenicol, and 70 and 64% for oxytetracycline. Considering that the MIC data was more precise than the disk diffusion data, MIC determination would be the preferred method for susceptibility testing for this species and the NWT frequencies derived from the MIC data sets should be

An in vitro assay for entry into cilia reveals unique properties of the soluble diffusion barrier

PubMed Central

Breslow, David K.; Koslover, Elena F.; Seydel, Federica; Spakowitz, Andrew J.

2013-01-01

Specific proteins are concentrated within primary cilia, whereas others remain excluded. To understand the mechanistic basis of entry into cilia, we developed an in vitro assay using cells in which the plasma membrane was permeabilized, but the ciliary membrane was left intact. Using a diffusion-to-capture system and quantitative analysis, we find that proteins >9 nm in diameter (∼100 kD) are restricted from entering cilia, and we confirm these findings in vivo. Interference with the nuclear pore complex (NPC) or the actin cytoskeleton in permeabilized cells demonstrated that the ciliary diffusion barrier is mechanistically distinct from those of the NPC or the axon initial segment. Moreover, applying a mass transport model to this system revealed diffusion coefficients for soluble and membrane proteins within cilia that are compatible with rapid exploration of the ciliary space in the absence of active transport. Our results indicate that large proteins require active transport for entry into cilia but not necessarily for movement inside cilia. PMID:24100294

Rheological and structural characterization of agar/whey proteins insoluble complexes.

PubMed

Rocha, Cristina M R; Souza, Hiléia K S; Magalhães, Natália F; Andrade, Cristina T; Gonçalves, Maria Pilar

2014-09-22

Complex coacervation between whey proteins and carboxylated or highly sulphated polysaccharides has been widely studied. The aim of this work was to characterise a slightly sulphated polysaccharide (agar) and whey protein insoluble complexes in terms of yield, composition and physicochemical properties as well as to study their rheological behaviour for better understanding their structure. Unlike other sulphated polysaccharides, complexation of agar and whey protein at pH 3 in the absence of a buffering agent resulted in a coacervate that was a gel at 20°C with rheological properties and structure similar to those of simple agar gels, reinforced by proteins electrostatically aggregated to the agar network. The behaviour towards heat treatment was similar to that of agar alone, with a high thermal hysteresis and almost full reversibility. In the presence of citrate buffer, the result was a "flocculated solid", with low water content (75-81%), whose properties were governed by protein behaviour. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

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

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.

Characterization of the species Malassezia pachydermatis and re-evaluation of its lipid dependence using a synthetic agar medium.

PubMed

Puig, Laura; Bragulat, M Rosa; Castellá, Gemma; Cabañes, F Javier

2017-01-01

The genus Malassezia includes lipophilic yeasts, which are part of the skin microbiota of various mammals and birds. Unlike the rest of Malassezia species, M. pachydermatis is described as non-lipid-dependent, as it is able to grow on Sabouraud glucose agar (SGA) without lipid supplementation. In this study we have examined the phenotypic variability within M. pachydermatis and confirmed its lipid-dependent nature using a synthetic agar medium. We used a selection of representative non-lipid-dependent strains from different animal species and three atypical lipid-dependent strains of this species, which were not able to grow after multiple passages on SGA. More than 400 lipid-dependent Malassezia isolates from animals were studied in order to detect the three lipid-dependent strains of M. pachydermatis. The identity of the atypical strains was confirmed by DNA sequencing. On the other hand, we have modified the Tween diffusion test, which is widely used in the characterization of these yeasts, by using a synthetic agar-based medium instead of SGA. This modification has proved to be useful for differentiation of M. pachydermatis strains, providing reproducible results and a straightforward interpretation. The finding of these peculiar lipid-dependent strains exemplifies the large variability within the species M. pachydermatis, which involves rare atypical strains with particular growth requirements.

New Chromogenic Agar Medium for the Identification of Candida spp.

PubMed Central

Cooke, Venitia M.; Miles, R. J.; Price, R. G.; Midgley, G.; Khamri, W.; Richardson, A. C.

2002-01-01

A new chromogenic agar medium (Candida diagnostic agar [CDA]) for differentiation of Candida spp. is described. This medium is based on Sabouraud dextrose agar (Oxoid CM41) and contains (per liter) 40.0 g of glucose, 10.0 g of mycological peptone, and 15.0 g of agar along with a novel chromogenic glucosaminidase substrate, ammonium 4-{2-[4-(2-acetamido-2-deoxy-β-d-glucopyranosyloxy)-3-methoxyphenyl]-vinyl}-1-(propan-3-yl-oate)-quinolium bromide (0.32 g liter−1). The glucosaminidase substrate in CDA was hydrolyzed by Candida albicans and Candida dubliniensis, yielding white colonies with deep-red spots on a yellow transparent background after 24 to 48 h of incubation at 37°C. Colonies of Candida tropicalis and Candida kefyr were uniformly pink, and colonies of other Candida spp., including Candida glabrata and Candida parapsilosis, were white. CDA was evaluated by using 115 test strains of Candida spp. and other clinically important yeasts and was compared with two commercially available chromogenic agars (Candida ID agar [bioMerieux] and CHROMagar Candida [CHROMagar Company Ltd.]). On all three agars, colonies of C. albicans were not distinguished from colonies of C. dubliniensis. However, for the group containing C. albicans plus C. dubliniensis, both the sensitivity and the specificity of detection when CDA was used were 100%, compared with values of 97.6 and 100%, respectively, with CHROMagar Candida and 100 and 96.8%, respectively, with Candida ID agar. In addition, for the group containing C. tropicalis plus C. kefyr, the sensitivity and specificity of detection when CDA was used were also 100%, compared with 72.7 and 98.1%, respectively, with CHROMagar Candida. Candida ID agar did not differentiate C. tropicalis and C. kefyr strains but did differentiate members of a broader group (C. tropicalis, C. kefyr, Candida lusitaniae plus Candida guilliermondii); the sensitivity and specificity of detection for members of this group were 94.7 and 93

Development of a nematode offspring counting assay for rapid and simple soil toxicity assessment.

PubMed

Kim, Shin Woong; Moon, Jongmin; Jeong, Seung-Woo; An, Youn-Joo

2018-05-01

Since the introduction of standardized nematode toxicity assays by the American Society for Testing and Materials (ASTM) and International Organization for Standardization (ISO), many studies have reported their use. Given that the currently used standardized nematode toxicity assays have certain limitations, in this study, we examined the use of a novel nematode offspring counting assay for evaluating soil ecotoxicity based on a previous soil-agar isolation method used to recover live adult nematodes. In this new assay, adult Caenorhabditis elegans were exposed to soil using a standardized toxicity assay procedure, and the resulting offspring in test soils attracted by a microbial food source in agar plates were counted. This method differs from previously used assays in terms of its endpoint, namely, the number of nematode offspring. The applicability of the bioassay was demonstrated using metal-spiked soils, which revealed metal concentration-dependent responses, and with 36 field soil samples characterized by different physicochemical properties and containing various metals. Principal component analysis revealed that texture fraction (clay, sand, and silt) and electrical conductivity values were the main factors influencing the nematode offspring counting assay, and these findings warrant further investigation. The nematode offspring counting assay is a rapid and simple process that can provide multi-directional toxicity assessment when used in conjunction with other standard methods. Copyright © 2018 Elsevier Ltd. 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.

Screening of Microbial Extracts for Anticancer Compounds Using Streptomyces Kinase Inhibitor Assay.

PubMed

Shanbhag, Prashant; Bhave, Sarita; Vartak, Ashwini; Kulkarni-Almeida, Asha; Mahajan, Girish; Villanueva, Ivan; Davies, Julian

2015-07-01

Eukaryotic kinases are known to play an important role in signal transduction pathways by phosphorylating their respective substrates. Abnormal phosphorylations by these kinases have resulted in diseases. Hence inhibitors of kinases are of considerable pharmaceutical interest for a wide variety of disease targets, especially cancers. A number of reports have been published which indicate that eukaryotic-like kinases may complement two-component kinase systems in several bacteria. In Streptomyces sp. such kinases have been found to have a role in formation of aerial hyphae, spores, pigmentation & even in antibiotic production in some strains. Eukaryotic kinase inhibitors are seen to inhibit formation of aerial mycelia in Streptomyces without inhibiting vegetative mycelia. This property has been used to design an assay to screen for eukaryotic kinase inhibitors. The assay involves testing of compounds against Streptomyces 85E ATCC 55824 using agar well diffusion method. Inhibitors of kinases give rise to "bald" colonies where aerial mycelia and sporulation inhibition is seen. The assay has been standardized using known eukaryotic protein kinase inhibiting anticancer agents like AG-490, AG-1295, AG-1478, Flavopiridol and Imatinib as positive controls, at a concentration ranging from 10 μg/well to 100 μg/well. Anti-infective compounds which are not reported to inhibit eukaryotic protein kinases were used as negative controls. A number of microbial cultures have been screened for novel eukaryotic protein kinase inhibitors. Further these microbial extracts were tested in various cancer cell lines like Panel, HCT116, Calul, ACHN and H460 at a concentration of 10 μg/mL/ well. The anticancer data was seen correlating well with the Streptomyces kinase assay thus validating the assay.

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 .

[Investigation on antibacterial activity of Forsythia suspense Vahl in vitro with Mueller-Hinton agar].

PubMed

Li, Z X; Wang, X H; Zhao, J H; Yang, J F; Wang, X

2000-12-01

To evaluate the antibacterial activity of Forsythia suspensa in vitro with different media. MIC determination of Forsythia suspensa against Staphylococci was performed by the agar dilution method. MIC90 of decoction of Forsythia suspensa against Staphylococcus epidermidis in M-H agar was 1:640, but in nutrient agar 1:40, the antibacterial activity with M-H agar being 16 fold higher than nutrient agar. The M-H agar should be recommended to replace nutrient agar as medium in the antibacterial experiment of Traditional Chinese medicine, and it is better to use multipoint inoculating device in the sensitivity test.

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 �

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.

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

Diagnostic Accuracy Assessment of Sensititre and Agar Disk Diffusion for Determining Antimicrobial Resistance Profiles of Bovine Clinical Mastitis Pathogens▿

PubMed Central

Saini, V.; Riekerink, R. G. M. Olde; McClure, J. T.; Barkema, H. W.

2011-01-01

Determining the accuracy and precision of a measuring instrument is pertinent in antimicrobial susceptibility testing. This study was conducted to predict the diagnostic accuracy of the Sensititre MIC mastitis panel (Sensititre) and agar disk diffusion (ADD) method with reference to the manual broth microdilution test method for antimicrobial resistance profiling of Escherichia coli (n = 156), Staphylococcus aureus (n = 154), streptococcal (n = 116), and enterococcal (n = 31) bovine clinical mastitis isolates. The activities of ampicillin, ceftiofur, cephalothin, erythromycin, oxacillin, penicillin, the penicillin-novobiocin combination, pirlimycin, and tetracycline were tested against the isolates. Diagnostic accuracy was determined by estimating the area under the receiver operating characteristic curve; intertest essential and categorical agreements were determined as well. Sensititre and the ADD method demonstrated moderate to highly accurate (71 to 99%) and moderate to perfect (71 to 100%) predictive accuracies for 74 and 76% of the isolate-antimicrobial MIC combinations, respectively. However, the diagnostic accuracy was low for S. aureus-ceftiofur/oxacillin combinations and other streptococcus-ampicillin combinations by either testing method. Essential agreement between Sensititre automatic MIC readings and MIC readings obtained by the broth microdilution test method was 87%. Essential agreement between Sensititre automatic and manual MIC reading methods was 97%. Furthermore, the ADD test method and Sensititre MIC method exhibited 92 and 91% categorical agreement (sensitive, intermediate, resistant) of results, respectively, compared with the reference method. However, both methods demonstrated lower agreement for E. coli-ampicillin/cephalothin combinations than for Gram-positive isolates. In conclusion, the Sensititre and ADD methods had moderate to high diagnostic accuracy and very good essential and categorical agreement for most udder pathogen

The effects of nutrient chemotaxis on bacterial aggregation patterns with non-linear degenerate cross diffusion

NASA Astrophysics Data System (ADS)

Leyva, J. Francisco; Málaga, Carlos; Plaza, Ramón G.

2013-11-01

This paper studies a reaction-diffusion-chemotaxis model for bacterial aggregation patterns on the surface of thin agar plates. It is based on the non-linear degenerate cross diffusion model proposed by Kawasaki et al. (1997) [5] and it includes a suitable nutrient chemotactic term compatible with such type of diffusion, as suggested by Ben-Jacob et al. (2000) [20]. An asymptotic estimation predicts the growth velocity of the colony envelope as a function of both the nutrient concentration and the chemotactic sensitivity. It is shown that the growth velocity is an increasing function of the chemotactic sensitivity. High resolution numerical simulations using Graphic Processing Units (GPUs), which include noise in the diffusion coefficient for the bacteria, are presented. The numerical results verify that the chemotactic term enhances the velocity of propagation of the colony envelope. In addition, the chemotaxis seems to stabilize the formation of branches in the soft-agar, low-nutrient regime.

Physicochemical and morphological properties of plasticized poly(vinyl alcohol)-agar biodegradable films.

PubMed

Madera-Santana, T J; Freile-Pelegrín, Y; Azamar-Barrios, J A

2014-08-01

The effects of the addition of glycerol (GLY) on the physicochemical and morphological properties of poly(vinyl alcohol) (PVA)-agar films were reported. PVA-agar films were prepared by solution cast method, and the addition of GLY in PVA-agar films altered the optical properties, resulting in a decrease in opacity values and in the color difference (ΔE) of the films. Structural characterization using Fourier transformation infrared (FTIR) spectroscopy and X-ray diffraction (XRD) indicated that the presence of GLY altered the intensity of the bands (from 1200 to 800cm(-1)) and crystallinity. The characterization of the thermal properties indicated that an increase in the agar content produces a decrease in the melting temperature and augments the heat of fusion. Similar tendencies were observed in plasticized films, but at different magnification. The formulation that demonstrated the lowest mechanical properties contained 25wt.% agar, whereas the formulation that contained 75wt.% agar demonstrated a significant improvement. The water vapor transmission rate (WVTR) and surface morphology analysis demonstrated that the structure of PVA-agar films is reorganized upon GLY addition. The physicochemical properties of PVA-agar films using GLY as a plasticizer provide information for the application of this formulation as packaging material for specific food applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Hichrom candida agar for identification of Candida species.

PubMed

Baradkar, V P; Mathur, M; Kumar, S

2010-01-01

Chromogenic media are frequently used in direct and rapid identification of yeasts because different Candida species produce unique colors on these media. We used 60 isolates of Candida species including 30 C. albicans, 10 C. parapsilosis, 11 C. glabrata, five C. tropicalis, and four C. dubliniensis, isolated from various clinical specimens, to evaluate the performance of HiChrome Candida agar. These strains had been identified by germ tube test, morphology on cornmeal agar, chlamydospore formation on tobacco agar and sugar assimilation tests. The sensitivity and specificity results were: C. albicans (96.55 and 96.42%); C. parapsilosis (80 and 98.03%), C. glabrata (90.90 and 88.23%), C. tropicalis (100 and 100%) and C. dubliniensis (60 and 96.55%) respectively. HiChrom Candida agaris medium has been useful and capable of presumptive, rapid identification of Candida species within 48 hours.

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.

Characteristics of thermoplastic sugar palm Starch/Agar blend: Thermal, tensile, and physical properties.

PubMed

Jumaidin, R; Sapuan, S M; Jawaid, M; Ishak, M R; Sahari, J

2016-08-01

The aim of this work is to study the behavior of biodegradable sugar palm starch (SPS) based thermoplastic containing agar in the range of 10-40wt%. The thermoplastics were melt-mixed and then hot pressed at 140°C for 10min. SEM investigation showed good miscibility between SPS and agar. FT-IR analysis confirmed that SPS and agar were compatible and inter-molecular hydrogen bonds existed between them. Incorporation of agar increased the thermoplastic starch tensile properties (Young's modulus and tensile strength). The thermal stability and moisture uptake increased with increasing agar content. The present work shows that starch-based thermoplastics with 30wt% agar content have the highest tensile strength. Higher content of agar (40wt%) resulted to more rough cleavage fracture and slight decrease in the tensile strength. In conclusion, the addition of agar improved the thermal and tensile properties of thermoplastic SPS which widened the potential application of this eco-friendly material. The most promising applications for this eco-friendly material are short-life products such as packaging, container, tray, etc. Copyright © 2016 Elsevier B.V. All rights reserved.

A Simple Experiment for Visualizing Diffusion

ERIC Educational Resources Information Center

Helseth, L. E.

2011-01-01

We propose a simple and fascinating experiment for studying diffusion in gels using a pH-sensitive dye. By doping agar with methyl red, we obtain a gel which rapidly reacts to changes in pH by changing its absorption spectrum. The pH gradients can be followed using a digital camera, and we demonstrate here that the pH-sensitive colour changes can…

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.

Characterization of the species Malassezia pachydermatis and re-evaluation of its lipid dependence using a synthetic agar medium

PubMed Central

Puig, Laura; Castellá, Gemma

2017-01-01

The genus Malassezia includes lipophilic yeasts, which are part of the skin microbiota of various mammals and birds. Unlike the rest of Malassezia species, M. pachydermatis is described as non-lipid-dependent, as it is able to grow on Sabouraud glucose agar (SGA) without lipid supplementation. In this study we have examined the phenotypic variability within M. pachydermatis and confirmed its lipid-dependent nature using a synthetic agar medium. We used a selection of representative non-lipid-dependent strains from different animal species and three atypical lipid-dependent strains of this species, which were not able to grow after multiple passages on SGA. More than 400 lipid-dependent Malassezia isolates from animals were studied in order to detect the three lipid-dependent strains of M. pachydermatis. The identity of the atypical strains was confirmed by DNA sequencing. On the other hand, we have modified the Tween diffusion test, which is widely used in the characterization of these yeasts, by using a synthetic agar-based medium instead of SGA. This modification has proved to be useful for differentiation of M. pachydermatis strains, providing reproducible results and a straightforward interpretation. The finding of these peculiar lipid-dependent strains exemplifies the large variability within the species M. pachydermatis, which involves rare atypical strains with particular growth requirements. PMID:28586389

New closed tube loop mediated isothermal amplification assay for prevention of product cross-contamination.

PubMed

Karthik, K; Rathore, Rajesh; Thomas, Prasad; Arun, T R; Viswas, K N; Dhama, Kuldeep; Agarwal, R K

2014-01-01

Loop mediated isothermal amplification (LAMP) assay, a promising diagnostic test, has been developed for detection of different pathogens of human as well as animals. Various positive points support its use as a field level test but the major problem is product cross contamination leading to false positive results. Different methods were adopted by various researchers to control this false positive amplification due to cross contamination but all have their own advantages and disadvantages. A new closed tube LAMP assay based on agar dye capsule was developed in the present study and this technique has some advantages over the other closed tube technique.•Agar at the concentration of 1.5% was used to sandwich SYBR green dye I with the aid of intradermal syringe. This agar dye capsule was placed over the LAMP reaction mixture before it was amplified.•To eliminate the hazardous nature of Ultra Violet (UV) light during result visualization of LAMP products, the present study demonstrates the use of Light Emitting Diode (LED) lights for result visualization.•LAMP was carried out for Brucella species detection using this modified techniques yielding good results without any cross contamination and LED showed similar fluorescence compared to UV.

[GROWTH OF MICROMYCETES FROM DIFFERENT ECOLOGICAL NICHES ON AGAR NUTRIENT MEDIA].

PubMed

Kurchenko, I M; Yurieva, E M; Voychuk, S I

2015-01-01

Radial growth rate of (K(r)) 153 strains 6 species of micromycetes from different ecological niches was studied on 7 agar media: three standard (malt extract agar, potato-dextrose agar, Czapek's agar), and on agar media with plant polymers (carboxymethylcellulose, xylan, soluble starch and apple pectin). Endophytic and plant pathogenic strains (biotrophs) of all studied species did not differ significantly in their ability to grow on nutrient media of different composition--average values of K(r) for these two groups were the same (0,200 and 0,199 mm/h, respectively). Soil micromycetes (saprophytes) characterized by the lowest average growth rate (0,169 mm/h) and significantly differed from the endophytic and plant pathogenic ones. Average of the radial growth rates of studied microscopic fungi were higher on standard nutrient media than with plant polymers ones. Growth parameters of endophytes and plant pathogens of all studied species on various agar media differed from the soil strains. High growth rate of endophytic and plant pathogenic strains of Fusarium poae, Alternaria alternata and Ceratocystis sp. provides them the rapid colonization of plants. Penicillium funiculosum strains equally can exist as saprophytes in soil and as endophytic plant symbionts. A wide range of K(r) variation of endophytic dark pigmented Mycelia sterilia indicates the presence in this group of different species of micromycetes, which have no sporulation.

Comparison of two antifungal susceptibility testing of Candida sp. isolates using agar diffusion method: Neo-sensitabs® tablets and Bio-rad® disks.

PubMed

Uwingabiye, J; Iken, M; Zohoun, A G; Boumhil, L; Lemkhente, Z; Naoui, H; Bouchrik, M; Lmimouni, B

2016-03-01

The aim of our study was to evaluate the concordance between the two antifungal susceptibility testing of Candida sp. isolates using agar diffusion method: Neo-Sensitabs(®) tablets and Bio-Rad(®) disks. This is a prospective study conducted in the Laboratory of Parasitology and Mycology of the Mohammed V military teaching hospital from February to August 2012. Upon receiving blood cultures and peripheral sites samples, the identification of Candida isolates performed using routine phenotypic standard tests and the realization of the antifungal susceptibility was carried out on Neo-sensitabs(®) tablets and Bio-Rad(®) disks. A total of 38 Candida strains were isolated: 15 C. albicans (39%), 13 C. glabrata (34%), 5 C. tropicalis (13%), 4 C. krusei (11%) and 1 C. dubliniensis (3%). There were no significant difference (P>0.05) in susceptibility rate between both methods for all antifungal agents tested except for 5-fluorocytosine. The concordance percentage between two methods was 100% for amphotericin B, 97.4% for fluconazole, 94.7% for voriconazole and 73% for 5-fluorocytosine. Both methods are easy to perform, rapid and cost effective. Our results showed the best agreement between the two methods for testing the susceptibility of Candida isolates to amphotericin B, fluconazole and voriconazole while for the 5-fluorocytosine, the concordance rate was low. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Proposal for agar disk diffusion interpretive criteria for susceptibility testing of bovine mastitis pathogens using cefoperazone 30μg disks.

PubMed

Feßler, Andrea T; Kaspar, Heike; Lindeman, Cynthia J; Peters, Thomas; Watts, Jeffrey L; Schwarz, Stefan

2017-02-01

Cefoperazone is a third generation cephalosporin which is commonly used for bovine mastitis therapy. Bacterial pathogens involved in bovine mastitis are frequently tested for their susceptibility to cefoperazone. So far, the cefoperazone susceptibility testing using 30μg disks has been hampered by the lack of quality control (QC) ranges as well as the lack of interpretive criteria. In 2014, QC ranges for 30 μg cefoperazone disks have been established for Staphylococcus aureus ATCC ® 25923 and Escherichia coli ATCC ® 25922. As a next step, interpretive criteria for the susceptibility testing of bovine mastitis pathogens should be developed. For this, 637 bovine mastitis pathogens (including 112 S. aureus, 121 coagulase-negative staphylococci (CoNS), 103 E. coli, 101 Streptococcus agalactiae, 100 Streptococcus dysgalactiae and 100 Streptococcus uberis) were investigated by agar disk diffusion according to the document Vet01-A4 of the Clinical and Laboratory Standards Institute (CLSI) using 30μg cefoperazone disks and the results were compared to the corresponding MIC values as determined by broth microdilution also according to the aforementioned CLSI document. Based on the results obtained and taking into account the achievable milk concentration of cefoperazone after regular dosing, the following interpretive criteria were proposed as a guidance for mastitis diagnostic laboratories: for staphylococci and E. coli ≥23mm (susceptible), 18-22mm (intermediate) and ≤17mm (resistant) and for streptococci ≥18mm (susceptible), and ≤17mm (non-susceptible). These proposed interpretive criteria shall contribute to a harmonization of cefoperazone susceptibility testing of bovine mastitis pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

The molecular bacterial load assay replaces solid culture for measuring early bactericidal response to antituberculosis treatment.

PubMed

Honeyborne, Isobella; Mtafya, Bariki; Phillips, Patrick P J; Hoelscher, Michael; Ntinginya, Elias N; Kohlenberg, Anke; Rachow, Andrea; Rojas-Ponce, Gabriel; McHugh, Timothy D; Heinrich, Norbert

2014-08-01

We evaluated the use of the molecular bacterial load (MBL) assay, for measuring viable Mycobacterium tuberculosis in sputum, in comparison with solid agar and liquid culture. The MBL assay provides early information on the rate of decline in bacterial load and has technical advantages over culture in either form. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Evaluation of a Method for Rapid Detection of Listeria monocytogenes in Dry-Cured Ham Based on Impedanciometry Combined with Chromogenic Agar.

PubMed

Labrador, Mirian; Rota, María C; Pérez, Consuelo; Herrera, Antonio; Bayarri, Susana

2018-05-01

The food industry is in need of rapid, reliable methodologies for the detection of Listeria monocytogenes in ready-to-eat products, as an alternative to the International Organization of Standardization (ISO) 11290-1 reference method. The aim of this study was to evaluate impedanciometry combined with chromogenic agar culture for the detection of L. monocytogenes in dry-cured ham. The experimental setup consisted in assaying four strains of L. monocytogenes and two strains of Listeria innocua in pure culture. The method was evaluated according to the ISO 16140:2003 standard through a comparative study with the ISO reference method with 119 samples of dry-cured ham. Significant determination coefficients ( R 2 of up to 0.99) for all strains assayed in pure culture were obtained. The comparative study results had 100% accuracy, 100% specificity, and 100% sensitivity. Impedanciometry followed by chromogenic agar culture was capable of detecting 1 CFU/25 g of food. L. monocytogenes was not detected in the 65 commercial samples tested. The method evaluated herein represents a promising alternative for the food industry in its efforts to control L. monocytogenes. Overall analysis time is shorter and the method permits a straightforward analysis of a large number of samples with reliable results.

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

Use of bile-esculin agar for rapid differentiation of Enterobacteriaceae.

PubMed Central

Lindell, S S; Quinn, P

1975-01-01

Bile-esculin agar has been used for several years for the presumptive identification of group D streptococci. All members of the Enterobacteriaceae family will also grow on this medium, but only certain ones can hydrolyze esculin to 6,7-dihydroxycoumarin, which reacts with iron to produce a characteristic blackening of the medium. One thousand and six cultures from clinical specimens representing 20 genera were isolated and identified. Heavy inocula from fresh pure culture isolates on heart infusion agar were placed on bile-esculin agar slants and incubated at 35 C. The slants were examined at 4 h and again at 18 h for esculin hydrolysis. Shigella, Salmonella, Arizona, Proteus mirabilis, Proteus morganii, Providencia alcalifaciens, and Providencia stuartii all produced negative results. Klebsiella pneumoniae, Enterobacter aerogenes, Serratia marcescens, and Serratia rubidaea produced a positive reaction in 4 h. The other remaining eight genera exhibited varying results. The use of this medium in conjunction with triple sugar iron-lysine iron agar has been of great value in differentiating the Klebsiella-Enterobacter-Serratia group from other Enterobacteriaceae. PMID:1176613

Current knowledge on agarolytic enzymes and the industrial potential of agar-derived sugars.

PubMed

Yun, Eun Ju; Yu, Sora; Kim, Kyoung Heon

2017-07-01

Agar is a major cell wall carbohydrate of red macroalgae (Rhodophyta). Sugars derived from agar, such as agarooligosaccharides (AOSs), neoagarooligosaccharides (NAOSs), neoagarobiose (NAB), and 3,6-anhydro-L-galactose (L-AHG), possess various physiological activities. These agar-derived sugars can be produced by hydrolysis using chemicals or agarolytic enzymes. Despite the industrial potential of agar-derived sugars, their application has been hampered mainly due to the absence of efficient processes for the liquefaction and saccharification of agar. In this review, we have focused on strategies for producing high value-added sugars from agarose via chemical or enzymatic liquefaction and enzymatic saccharification. The liquefaction of agarose is a key step for preventing gelling and increasing the solubility of agarose in water by prehydrolyzing agarose into AOSs or NAOSs. For the industrial use of agar-derived sugars, AOS, NAOS, NAB, and L-AHG can be used as functional biomaterials owing to their physiological activities such as antiinflammation, skin whitening, and moisturizing. Recently, it was reported that AHG could be considered as a new anticariogenic sugar to replace xylitol. This review provides a comprehensive overview of processes for the hydrolysis of agar or agarose to produce high value-added sugars and the industrial application of these sugars.

Non-invasive Measurement of Thermal Diffusivity Using High-Intensity Focused Ultrasound and Through-Transmission Ultrasonic Imaging.

PubMed

Yeshurun, Lilach; Azhari, Haim

2016-01-01

Thermal diffusivity at the site ablated by high-intensity focused ultrasound (HIFU) plays an important role in the final therapeutic outcome, as it influences the temperature's spatial and temporal distribution. Moreover, as tissue thermal diffusivity is different in tumors as compared with normal tissue, it could also potentially be used as a new source of imaging contrast. The aim of this study was to examine the feasibility of combining through-transmission ultrasonic imaging and HIFU to estimate thermal diffusivity non-invasively. The concept was initially evaluated using a computer simulation. Then it was experimentally tested on phantoms made of agar and ex vivo porcine fat. A computerized imaging system combined with a HIFU system was used to heat the phantoms to temperatures below 42°C to avoid irreversible damage. Through-transmission scanning provided the time-of-flight values in a region of interest during its cooling process. The time-of-flight values were consequently converted into mean values of speed of sound. Using the speed-of-sound profiles along with the developed model, we estimated the changes in temperature profiles over time. These changes in temperature profiles were then used to calculate the corresponding thermal diffusivity of the studied specimen. Thermal diffusivity for porcine fat was found to be lower by one order of magnitude than that obtained for agar (0.313×10(-7)m(2)/s vs. 4.83×10(-7)m(2)/s, respectively, p < 0.041). The fact that there is a substantial difference between agar and fat implies that non-invasive all-ultrasound thermal diffusivity mapping is feasible. The suggested method may particularly be suitable for breast scanning. Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Influence of the extraction process on the rheological and structural properties of agars.

PubMed

Sousa, Ana M M; Borges, João; Silva, A Fernando; Gonçalves, Maria P

2013-07-01

Agars obtained by traditional hot-water (TWE) and microwave-assisted (MAE) extractions were compared in terms of their rheological and physicochemical properties and molecular self-association in solutions of low (0.05%, w/w) and high (1.5%, w/w) polymer concentrations. At low concentration, thin gelled layers were imaged by AFM. Slow or rapid cooling of the solutions influenced structure formation. In each case, TWE and MAE agar structures were different and apparently larger for MAE. At high concentration, progressive structural reinforcement was seen; while TWE agar showed a more open and irregular 3D network, MAE agar gel imaged by cryoSEM was denser and fairly uniform. The rheological (higher thermal stability and consistency) and mechanical (higher gel strength) behaviors of MAE agar seemed consistent with a positive effect of molecular mass and 3,6-anhydro-α-l-galactose content. MAE produced non-degraded agar comparable with commercial ones and if properly monitored, could be a promising alternative to TWE. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

Diffusion of Antimicrobials Across Silicone Hydrogel Contact Lenses.

PubMed

Zambelli, Alison M; Brothers, Kimberly M; Hunt, Kristin M; Romanowski, Eric G; Nau, Amy C; Dhaliwal, Deepinder K; Shanks, Robert M Q

2015-09-01

To measure the diffusion of topical preparations of moxifloxacin, amphotericin B (AmB), and polyhexamethylene biguanide (PHMB) through silicone hydrogel (SH) contact lenses (CLs) in vitro. Using an in vitro model, the diffusion of three antimicrobials through SH CLs was measured. Diffused compounds were measured using a spectrophotometer at set time points over a period of 4 hr. The amount of each diffused antimicrobial was determined by comparing the experimental value with a standard curve. A biological assay was performed to validate the CL diffusion assay by testing antimicrobial activity of diffused material against lawns of susceptible bacteria (Staphylococcus epidermidis) and yeast (Saccharomyces cerevisiae). Experiments were repeated at least two times with a total of at least four independent replicates. Our data show detectable moxifloxacin and PHMB diffusion through SH CLs at 30 min, whereas AmB diffusion remained below the limit of detection within the 4-hr experimental period. In the biological assay, diffused moxifloxacin demonstrated microbial killing starting at 20 min on bacterial lawns, whereas PHMB and AmB failed to demonstrate killing on microbial lawns over the course of the 60-min experiment. In vitro diffusion assays demonstrate limited penetration of certain anti-infective agents through SH CLs. Further studies regarding the clinical benefit of using these agents along with bandage CL for corneal pathologic condition are warranted.

New closed tube loop mediated isothermal amplification assay for prevention of product cross-contamination

PubMed Central

Karthik, K.; Rathore, Rajesh; Thomas, Prasad; Arun, T.R.; Viswas, K.N.; Dhama, Kuldeep; Agarwal, R.K.

2014-01-01

Loop mediated isothermal amplification (LAMP) assay, a promising diagnostic test, has been developed for detection of different pathogens of human as well as animals. Various positive points support its use as a field level test but the major problem is product cross contamination leading to false positive results. Different methods were adopted by various researchers to control this false positive amplification due to cross contamination but all have their own advantages and disadvantages. A new closed tube LAMP assay based on agar dye capsule was developed in the present study and this technique has some advantages over the other closed tube technique.•Agar at the concentration of 1.5% was used to sandwich SYBR green dye I with the aid of intradermal syringe. This agar dye capsule was placed over the LAMP reaction mixture before it was amplified.•To eliminate the hazardous nature of Ultra Violet (UV) light during result visualization of LAMP products, the present study demonstrates the use of Light Emitting Diode (LED) lights for result visualization.•LAMP was carried out for Brucella species detection using this modified techniques yielding good results without any cross contamination and LED showed similar fluorescence compared to UV. PMID:26150945

Application of agar liquid-gel transition in cultivation and harvesting of microalgae for biodiesel production.

PubMed

Kumar, Vinod; Nanda, Manisha; Verma, Monu

2017-11-01

In order to increase microalgal biomass productivity efficient cultivation and harvesting methods are needed against the available traditional methods. The present study focuses on the same by harvesting microalgae using agar gel. Agar medium containing bold's basal medium (BBM) undergoes a thermoreversible gel transition. As compared to the traditional protocols, this gel is used to cultivate microalgae without even affecting the total productivity. To develop the gel for microalgae cultivation, agar was boiled in BBM. Then the agar was cooled to 35°C and microalgae culture was added to it. After seeding the microalgae the temperature of the agar was further decreased by 10°C to induce gelation. Instead of isolated cells microalgae were grown in clusters within the agar gel. Microalgal clusters gravimetrically settle at the bottom within 2h. In this method agar can be reused. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

Comparative evaluation of chromogenic agar CM1046 and mFC agar for detection of E. coli and thermotolerant coliform bacteria from water samples.

PubMed

Wohlsen, T D

2011-08-01

The equivalence of Oxoid (CM 1046) Brilliance((TM)) E. coli/coliform selective agar to mFC agar, as used in the Australian/New Zealand Standard Method to detect thermotolerant coliforms and Escherichia coli in water samples, was assessed. A total of 244 water samples were analysed in parallel over a 5-month period. Sewage effluent samples (n = 131, sites = 43), freshwater (n = 62, sites = 18) and marine/brackish water samples (n = 51, sites = 23) were analysed. The Wilcoxon matched-pairs signed-ranks test showed a varying degree of statistical difference between the two methods. All matrices had a higher recovery in the trial method. Enterococci faecalis, Aeromonas spp. and Vibrio spp. did not grow on the CM1046 agar, and Pseudomonas aeruginosa and Enterobacter aerogenes were inhibited. The use of CM 1046 for the detection and enumeration of E. coli and thermotolerant coliforms in water samples is a suitable alternative to the AS/NZS Standard Method. The use of CM1046 agar was less labour intensive and time consuming, as no secondary confirmation steps were required. Confirmed results could be reported within 24 h of sample analysis, as compared to 48 h with the reference method. Public health concerns can be addressed in a more efficient manner. © 2011 Unitywater. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

Films based on soy protein-agar blends for wound dressing: Effect of different biopolymer proportions on the drug release rate and the physical and antibacterial properties of the films.

PubMed

Rivadeneira, Josefina; Audisio, M C; Gorustovich, Alejandro

2018-04-01

No single material can provide all requirements for wound dressings. Here, we evaluated the influence of different soy protein isolate and agar proportions (3:1, 1:1, and 1:3) in blend films on some of their physical-chemical and antibacterial properties to elucidate their potential as wound dressings. The films were synthesized by the gel casting method and ciprofloxacin hydrochloride was incorporated into the films. Films were characterized based on their surface morphology, water uptake ability, and weight loss profile. Also, the ciprofloxacin hydrochloride release kinetics was quantified spectrophotometrically. The antibacterial effect was evaluated against Staphylococcus aureus and Pseudomonas aeruginosa strains. The soy protein isolate-agar ratio affected the water uptake of the films and the release profile of ciprofloxacin hydrochloride but not the weight loss profile. The amount of drug released decreased near 80% because of the decrease in agar content in the films. The release kinetics of ciprofloxacin hydrochloride data best fitted to the Korsmeyer-Peppas model, suggesting that the mechanism of drug release was mainly of the diffusion type. All ciprofloxacin hydrochloride-releasing soy protein isolate-agar films strongly inhibited the cell viability of the bacterial strains studied. We concluded that water uptake and ciprofloxacin hydrochloride release can be controlled by changing the soy protein isolate-agar proportion. The proportions did not lead to changes in the antibacterial strength of the films.

Diffusion of Antimicrobials Across Silicone Hydrogel Contact Lenses

PubMed Central

Zambelli, Alison M.; Brothers, Kimberly M.; Hunt, Kristin M.; Romanowski, Eric G.; Nau, Amy C.; Dhaliwal, Deepinder K.; Shanks, Robert M. Q.

2014-01-01

Objectives To measure the diffusion of topical preparations of moxifloxacin, amphotericin B (AmB), and polyhexamethylene biguanide (PHMB) through silicone hydrogel (SH) contact lenses in vitro. Methods Using an in vitro model, the diffusion of three antimicrobials through SH contact lenses was measured. Diffused compounds were measured using a spectrophotometer at set time points over a period of four hours. The amount of each diffused antimicrobial was determined by comparing the experimental value to a standard curve. A biological assay was performed to validate the contact lens diffusion assay by testing antimicrobial activity of diffused material against lawns of susceptible bacteria (Staphylococcus epidermidis) and yeast (Saccharomyces cerevisiae). Experiments were repeated at least two times with a total of at least 4 independent replicates. Results Our data show detectable moxifloxacin and PHMB diffusion through SH contact lenses at 30 minutes, while amphotericin B diffusion remained below the limit of detection within the 4 hour experimental period. In the biological assay, diffused moxifloxacin demonstrated microbial killing starting at 20 minutes on bacterial lawns, whereas PHMB and amphotericin B failed to demonstrate killing on microbial lawns over the course of the 60 minute experiment. Conclusions In vitro diffusion assays demonstrate limited penetration of certain anti-infective agents through silicone hydrogel contact lenses. Further studies regarding the clinical benefit of using these agents along with bandage contact lens use for corneal pathology are warranted. PMID:25806673

A novel agar formulation for isolation and direct enumeration of Vibrio vulnificus from oyster tissue.

PubMed

Griffitt, Kimberly J; Grimes, D Jay

2013-08-01

A new selective and differential medium, Vibrio vulnificus X-Gal (VVX), was developed for direct enumeration of V. vulnificus (Vv) from oyster samples. This agar utilizes cellobiose and lactose as carbon sources, and the antibiotics colistin and polymyxin B as selective agents. Hydrolysis of 5-bromo-4-chloro-3-indolyl- beta-d-galactopyranoside (x-gal), used in the agar as a lactose analog, produces an insoluble blue dye that makes lactose positive colonies easily distinguishable from any non-lactose fermenting bacteria. Various bacterial species were spot plated onto thiosulfate-citrate-bile salts-sucrose agar (TCBS), and CHROMagar Vibrio, two vibrio-specific selective agars, non-selective agar, and VVX to compare selectivity of VVX to other widely used media. A V. vulnificus pure culture was serially diluted on VVX and non-selective agar to determine the VVX percent recovery. Water and oyster samples were spread plated on VVX agar and allowed to incubate for 16-18 h at 33 °C. Blue and white colonies from VVX agar were picked and screened by end point PCR for the Vv hemolysin vvhA. VVX agar showed a significant improvement over TCBS and CHROMagar at preventing non-target growth. There was an 87.5% recovery compared to non-selective plating and a 98% positivity rate of blue colonies picked from oyster tissue plating. The findings suggest that this new agar is a fast, distinctive, and accurate method for enumeration of V. vulnificus from the environment. Copyright © 2013 Elsevier B.V. All rights reserved.

Photothermal characterization of the gelation process in Gelidium robustum Agar

NASA Astrophysics Data System (ADS)

Freile-Pelegrín, Y.; Bante, J.; Alvarado-Gil, J. J.; Yánez-Limón, J. M.

2005-06-01

Agar is a hydrophilic colloid formed by polysaccharides, whose ability to form reversible gels simply by cooling hot aqueous solutions is the most important property and can be regarded as the prototype and model for all gelling systems. In this paper the evolution of the gelation process of agar obtained from algae of the species Gelidium robustum, using the photopyroelectric technique is reported. It is shown that thermal effusivity increase when the agar is cooled, reaching a maximum value around 37°C. The increase in thermal effusivity can be related to the increasing of the bondings in the gel as temperature decreases, reaching the maximum at the gelation point. The decrease of the thermal effusivity at lower temperature could be due to the syneresis process involving a gradual release of water after gelation.

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.

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.

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.

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

Proton beam writing of microstructures in Agar gel for patterned cell growth

NASA Astrophysics Data System (ADS)

Larisch, Wolfgang; Koal, Torsten; Werner, Ronald; Hohlweg, Marcus; Reinert, Tilo; Butz, Tilman

2011-10-01

A rather useful prerequisite for many biological and biophysical studies, e.g., for cell-cell communication or neuronal networks, is confined cell growth on micro-structured surfaces. Solidified Agar layers have smooth surfaces which are electrically neutral and thus inhibit receptor binding and cell adhesion. For the first time, Agar microstructures have been manufactured using proton beam writing (PBW). In the irradiated Agar material the polysaccharides are split into oligosaccharides which can easily be washed off leaving Agar-free areas for cell adhesion. The beam diameter of 1 μm allows the fabrication of compartments accommodating single cells which are connected by micrometer-sized channels. Using the external beam the production process is very fast. Up to 50 Petri dishes can be produced per day which makes this technique very suitable for biological investigations which require large throughputs.

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.

Xanthan gum: an economical substitute for agar in plant tissue culture media.

PubMed

Jain, R; Babbar, S B

2006-03-01

Xanthan gum, a microbial desiccation-resistant polysaccharide prepared commercially by aerobic submerged fermentation from Xanthomonas campestris, has been successfully used as a solidifying agent for plant tissue culture media. Its suitability as a substitute to agar was demonstrated for in vitro seed germination, caulogenesis and rhizogenesis of Albizzia lebbeck, androgenesis in anther cultures of Datura innoxia, and somatic embryogenesis in callus cultures of Calliandra tweedii. Culture media used for eliciting these morphogenic responses were gelled with either 1% xanthan gum or 0.9% agar. Xanthan gum, like agar, supported all these responses.

Evaluating the Risk of Tumors Diseases Based on Measurement of Urinary and Serumal Antioxidants Using the New Agar Diffusion Methods

PubMed Central

Zhou, Ying; Chen, Jing; Wang, Zhen

2017-01-01

Objectives. To discuss the characteristics of the amount of urinary total antioxidants in tumor diseases and the possibility of utilizing the changing regulation of urinary antioxidants to diagnose tumor diseases. Method. Urine and serum specimens from 130 healthy people were used to investigate the variation of antioxidant capacity against age. Urine and serum specimens from 44 unselected patients with tumors and 44 healthy people with same age background were used to explore the significance of urinary antioxidant capacity in clinic to diagnose tumor diseases. Potassium permanganate agar method and iodine starch method were used to determine the amount of total antioxidants. Results. In healthy people, more antioxidants in urine were measured in older people, while the results were opposite in serum. More antioxidants were found in urine of tumor patients than in healthy people with same age-range. Conclusions. According to the results of 130 measurements, the amount of antioxidants in urine varies by age. By using agar methods to measure antioxidants, the effect of age is required to be considered. Antioxidants levels from tumor patients were significantly higher than healthy individuals in urine. The combination of urine and serum to determine total antioxidants can better diagnose tumor diseases based on iodine starch method, with area under the receiver operating characteristics curve at 0.787. PMID:28458777

Growth characteristics of Bacillus anthracis compared to other Bacillus spp. on the selective nutrient media Anthrax Blood Agar and Cereus Ident Agar.

PubMed

Tomaso, Herbert; Bartling, Carsten; Al Dahouk, Sascha; Hagen, Ralf M; Scholz, Holger C; Beyer, Wolfgang; Neubauer, Heinrich

2006-01-01

Anthrax Blood Agar (ABA) and Cereus Ident Agar (CEI) were evaluated as selective growth media for the isolation of Bacillus anthracis using 92 B. anthracis and 132 other Bacillus strains from 30 species. The positive predictive values for the identification of B. anthracis on ABA, CEI, and the combination of both were 72%, 71%, and 90%, respectively. Thus, less than 10% of all species were misidentified using both nutrient media. Species which might be misidentified as B. anthracis were B. cereus, B. mycoides, and B. thuringiensis. Particularly, 30% of B. weihenstephanensis strains were misidentified as B. anthracis.

Preparation, characterization, and in vitro gastrointestinal digestibility of oil-in-water emulsion-agar gels.

PubMed

Wang, Zheng; Neves, Marcos A; Kobayashi, Isao; Uemura, Kunihiko; Nakajima, Mitsutoshi

2013-01-01

Soybean oil-in-water (O/W) emulsion-agar gel samples were prepared and their digestibility evaluated by using an in vitro gastrointestinal digestion model. Emulsion-agar sols were obtained by mixing the prepared O/W emulsions with a 1.5 wt % agar solution at 60 °C, and their subsequent cooling at 5 °C for 1 h formed emulsion-agar gels. Their gel strength values increased with increasing degree of polymerization of the emulsifiers, and the relative gel strength increased in the case of droplets with an average diameter smaller than 700 nm. Flocculation and coalescence of the released emulsion droplets depended strongly on the emulsifier type; however, the emulsifier type hardly affected the ζ-potential of emulsion droplets released from the emulsion-agar gels during in vitro digestion. The total FFA content released from each emulsion towards the end of the digestion period was nearly twice that released from the emulsion-agar gel, indicating that gelation of the O/W emulsion may have delayed lipid hydrolysis.

Effect of lignin on water vapor barrier, mechanical, and structural properties of agar/lignin composite films.

PubMed

Shankar, Shiv; Reddy, Jeevan Prasad; Rhim, Jong-Whan

2015-11-01

Biodegradable composite films were prepared using two renewable resources based biopolymers, agar and lignin alkali. The lignin was used as a reinforcing material and agar as a biopolymer matrix. The effect of lignin concentration (1, 3, 5, and 10wt%) on the performance of the composite films was studied. In addition, the mechanical, water vapor barrier, UV light barrier properties, FE-SEM, and TGA of the films were analyzed. The agar/lignin films exhibited higher mechanical and UV barrier properties along with lower water vapor permeability compared to the neat agar film. The FTIR and SEM results showed the compatibility of lignin with agar polymer. The swelling ratio and moisture content of agar/lignin composite films were decreased with increase in lignin content. The thermostability and char content of agar/lignin composite films increased with increased lignin content. The results suggested that agar/lignin films have a potential to be used as a UV barrier food packaging material for maintaining food safety and extending the shelf-life of the packaged food. Copyright © 2015 Elsevier B.V. All rights reserved.

[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

Mupirocin-mucin agar for selective enumeration of Bifidobacterium bifidum.

PubMed

Pechar, Radko; Rada, Vojtech; Parafati, Lucia; Musilova, Sarka; Bunesova, Vera; Vlkova, Eva; Killer, Jiri; Mrazek, Jakub; Kmet, Vladimir; Svejstil, Roman

2014-11-17

Bifidobacterium bifidum is a bacterial species exclusively found in the human intestinal tract. This species is becoming increasingly popular as a probiotic organism added to lyophilized products. In this study, porcine mucin was used as the sole carbon source for the selective enumeration of B. bifidum in probiotic food additives. Thirty-six bifidobacterial strains were cultivated in broth with mucin. Only 13 strains of B. bifidum utilized the mucin to produce acids. B. bifidum was selectively enumerated in eight probiotic food supplements using agar (MM agar) containing mupirocin (100 mg/L) and mucin (20 g/L) as the sole carbon source. MM agar was fully selective if the B. bifidum species was presented together with Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, and Bifidobacterium longum subsp. longum species and with lactic acid bacteria (lactobacilli, streptococci). Isolated strains of B. bifidum were identified using biochemical, PCR, MALDI-TOF procedures and 16S rRNA gene sequencing. The novel selective medium was also suitable for the isolation of B. bifidum strains from human fecal samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of nutrient agar for the culture of Mycobacterium tuberculosis using the microcolony detection method.

PubMed

Satti, L; Abbasi, S; Faiz, U

2012-07-01

We evaluated nutrient agar using the microcolony detection method for the recovery of Mycobacterium tuberculosis on 37 acid-fast bacilli (AFB) positive sputum specimens, and compared it with conventional Löwenstein-Jensen (LJ) medium. Nutrient agar detected 35 isolates compared to 34 on LJ medium. The mean time to detection of mycobacteria on nutrient agar and LJ medium was respectively 9.6 and 21.4 days. The contamination rate on nutrient agar and LJ medium was respectively 5.4% and 2.7%. Nutrient agar detects M. tuberculosis more rapidly than LJ medium, and could be an economical, rapid culture method in resource-poor settings, provided our findings are confirmed by further studies.

Characterization of bionanocomposite films prepared with agar and paper-mulberry pulp nanocellulose.

PubMed

Reddy, Jeevan Prasad; Rhim, Jong-Whan

2014-09-22

Crystallized nanocellulose (CNC) was separated from paper-mulberry (Broussonetia kazinoki Siebold) bast pulp by sulfuric acid hydrolysis method and they were blended with agar to prepare bionanocomposite films. The effect of CNC content (1, 3, 5 and 10 wt% based on agar) on the mechanical, water vapor permeability (WVP), and thermal properties of the nanocomposites were studied. Changes of the cellulose fibers in structure, morphology, crystallinity, and thermal properties of the films were evaluated using FT-IR, TEM, SEM, XRD, and TGA analysis methods. The CNC was composed of fibrous and spherical or elliptic granules of nano-cellulose with sizes of 50-60 nm. Properties of agar film such as mechanical and water vapor barrier properties were improved significantly (p<0.05) by blending with the CNC. The tensile modulus and tensile strength of agar film increased by 40% and 25%, respectively, in the composite film with 5 wt% of CNC, and the WVP of agar film decreased by 25% after formation of nanocomposite with 3 wt% of CNC. The CNC obtained from the paper-mulberry bast pulp can be used as a reinforcing agent for the preparation of bio-nanocomposites, and they have a high potential for the development of completely biodegradable food packaging materials. Copyright © 2014 Elsevier Ltd. All rights reserved.

A Simple Assay to Screen Antimicrobial Compounds Potentiating the Activity of Current Antibiotics

PubMed Central

Iqbal, Junaid; Kazmi, Shahana Urooj; Khan, Naveed Ahmed

2013-01-01

Antibiotic resistance continues to pose a significant problem in the management of bacterial infections, despite advances in antimicrobial chemotherapy and supportive care. Here, we suggest a simple, inexpensive, and easy-to-perform assay to screen antimicrobial compounds from natural products or synthetic chemical libraries for their potential to work in tandem with the available antibiotics against multiple drug-resistant bacteria. The aqueous extract of Juglans regia tree bark was tested against representative multiple drug-resistant bacteria in the aforementioned assay to determine whether it potentiates the activity of selected antibiotics. The aqueous extract of J. regia bark was added to Mueller-Hinton agar, followed by a lawn of multiple drug-resistant bacteria, Salmonella typhi or enteropathogenic E. coli. Next, filter paper discs impregnated with different classes of antibiotics were placed on the agar surface. Bacteria incubated with extract or antibiotics alone were used as controls. The results showed a significant increase (>30%) in the zone of inhibition around the aztreonam, cefuroxime, and ampicillin discs compared with bacteria incubated with the antibiotics/extract alone. In conclusion, our assay is able to detect either synergistic or additive action of J. regia extract against multiple drug-resistant bacteria when tested with a range of antibiotics. PMID:23865073

Broth versus solid agar culture of swab samples of cadaveric allograft musculoskeletal tissue.

PubMed

Varettas, Kerry

2013-12-01

As part of the donor assessment protocol, bioburden assessment must be performed on allograft musculoskeletal tissue samples collected at the time of tissue retrieval. Swab samples of musculoskeletal tissue allografts from cadaveric donors are received at the microbiology department of the South Eastern Area Laboratory Services (Australia) to determine the presence of bacteria and fungi. This study will review the isolation rate of organisms from solid agar and broth culture of swab samples of cadaveric allograft musculoskeletal tissue over a 6-year period, 2006-2011. Swabs were inoculated onto horse blood agar (anaerobic, 35 °C) and chocolate agar (CO2, 35 °C) and then placed into a cooked meat broth (aerobic, 35 °C). A total of 1,912 swabs from 389 donors were received during the study period. 557 (29.1 %) swabs were culture positive with the isolation of 713 organisms, 249 (34.9 %) from solid agar culture and an additional 464 (65.1 %) from broth culture only. This study has shown that the broth culture of cadaveric allograft musculoskeletal swab samples recovered a greater amount of organisms than solid agar culture. Isolates such as Clostridium species and Staphylococcus aureus would not have been isolated from solid agar culture alone. Broth culture is an essential part of the bioburden assessment protocol of swab samples of cadaveric allograft musculoskeletal tissue in this laboratory.

Comparison of the BBL CHROMagar Staph aureus Agar Medium to Conventional Media for Detection of Staphylococcus aureus in Respiratory Samples

PubMed Central

Flayhart, Diane; Lema, Clara; Borek, Anita; Carroll, Karen C.

2004-01-01

Screening for Staphylococcus aureus has become routine in certain patient populations. This study is the first clinical evaluation of the BBL CHROMagar Staph aureus agar (CSA) medium (BD Diagnostics, Sparks, Md.) for detection of S. aureus in nasal surveillance cultures and in respiratory samples from cystic fibrosis (CF) patients. S. aureus colonies appear mauve on CSA. Other organisms are inhibited or produce a distinctly different colony color. S. aureus was identified from all media by slide coagulase, exogenous DNase, and mannitol fermentation assays. Susceptibility testing was performed using the agar dilution method. A total of 679 samples were evaluated. All samples were inoculated onto CSA. Nasal surveillance cultures were inoculated onto sheep blood agar (SBA) (BD Diagnostics), and samples from CF patients were inoculated onto mannitol salt agar (MSA) (BD Diagnostics). Of the 679 samples cultured, 200 organisms produced a mauve color on CSA (suspicious for S. aureus) and 180 were positive for S. aureus on SBA or MSA. Of 200 CSA-positive samples 191 were identified as S. aureus. Nine mauve colonies were slide coagulase negative and were subsequently identified as Staphylococcus lugdunensis (one), Staphylococcus epidermidis (three), Staphylococcus haemolyticus (one), and Corynebacterium species (four). CSA improved the ability to detect S. aureus by recovering 12 S. aureus isolates missed by conventional media. Of the 192 S. aureus isolates recovered, 122 were methicillin susceptible and 70 were methicillin resistant. Overall, the sensitivity and specificity of CSA in this study were 99.5 and 98%, respectively. There was no difference in the performance of the slide coagulase test or in susceptibility testing performed on S. aureus recovered from CSA compared to SBA or MSA. Our data support the use of CSA in place of standard culture media for detection of S. aureus in heavily contaminated respiratory samples. PMID:15297498

Physical-mechanical properties of agar/κ-carrageenan blend film and derived clay nanocomposite film.

PubMed

Rhim, Jong-Whan

2012-12-01

Binary blend films with different mixing ratio of agar and κ-carrageenan were prepared using a solution casting method with and without nanoclay and the effect of their composition on the mechanical, water vapor barrier, and water resistance properties was tested. The tensile strength (TS) of the κ-carrageenan film was greater than that of agar film. The water vapor permeability (WVP) of the agar film was lower than that of κ-carrageenan film, the swelling ratio (SR) and water solubility (WS) of κ-carrageenan film were higher than those of agar film. Each property of the binary blend films varied proportionately depending on the mixing ratio of each component. The XRD result indicated that the nanocomposite with agar/κ-carrageenan/clay (Cloisite(®) Na(+)) was intercalated. Consequently, the mechanical strength, water vapor barrier properties, and water contact angle (CA) were significantly (P < 0.05) improved through nanocomposite formation. © 2012 Institute of Food Technologists®

Detection of Agar, by Analysis of Sugar Markers, Associated with Bacillus Anthracis Spores, After Culture

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

Wunschel, David S.; Colburn, Heather A.; Fox, Alvin

2008-08-01

Detection of small quantities of agar associated with spores of Bacillus anthracis could provide key information regarding its source or growth characteristics. Agar, widely used in growth of bacteria on solid surfaces, consists primarily of repeating polysaccharide units of 3,6-anhydro-L-galactose (AGal) and galactose (Gal) with sulfated and O-methylated galactoses present as minor constituents. Two variants of the alditol acetate procedure were evaluated for detection of potential agar markers associated with spores. The first method employed a reductive hydrolysis step, to stabilize labile anhydrogalactose, by converting to anhydrogalactitol. The second eliminated the reductive hydrolysis step simplifying the procedure. Anhydrogalactitol, derived frommore » agar, was detected using both derivatization methods followed by gas chromatography-mass spectrometry (GC-MS) analysis. However, challenges with artefactual background (reductive hydrolysis) or marker destruction (hydrolysis) lead to the search for alternative sugar markers. A minor agar component, 6-O-methyl galactose (6-O-M gal), was readily detected in agar-grown but not broth-grown bacteria. Detection was optimized by the use of gas chromatography-tandem mass spectrometry (GC-MS-MS). With appropriate choice of sugar marker and analytical procedure, detection of sugar markers for agar has considerable potential in microbial forensics.« less

In vitro susceptibility of filamentous fungi to copper nanoparticles assessed by rapid XTT colorimetry and agar dilution method.

PubMed

Ghasemian, E; Naghoni, A; Tabaraie, B; Tabaraie, T

2012-12-01

Metal nanoparticles and their uses in various aspects have recently drawn a great deal of attention. One of the major applications is that it can be used as an antimicrobial agent. They can be considered in approaches targeted to decrease the harms caused by microorganisms, specifically fungi, threatening the medical and industrial areas. The aim of this study was to investigate the antifungal activity of synthesized copper nanoparticles (CuNPs) against four filamentous fungi including Alternaria alternata, Aspergillus flavus, Fusarium solani, and Penicillium chrysogenum. Zerovalent copper nanoparticles of mean size 8nm were synthesized by inert gas condensation (IGC) method. The antifungal activity of these synthesized copper nanoparticles was measured against selected fungi by using two different techniques including agar dilution method and XTT reduction assay. The minimal inhibitory concentrations (MICs) for copper nanoparticles by agar dilution method were less or equal to 40mg/L for P. chrysogenum, less or equal to 60mg/L for A. alternata, less or equal to 60mg/L for F. solani, and less or equal to 80mg/L for A. flavus. And also MICs obtained by XTT reduction assay ranged from 40 to 80mg/L. Our data demonstrated that the copper nanoparticles inhibited fungal growth, but the fungal sensitivity to copper nanoparticles varies depending on the fungal species. Therefore, it is advisable that the minimal inhibitory concentrations (MICs) be examined before using these compounds. It is hoped that, in future, copper nanoparticles could replace some antifungal agents, making them applicable to many different medical devices and antimicrobial control system. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

[Screening and identification of a bacterium capable of converting agar to neoagaro oligosaccharides].

PubMed

Han, Junping; Huang, Yayan; Ye, Jing; Xiao, Meitian

2015-09-04

To screen and identify a bacterium capable of converting agar to neoagaro oligosaccharides. We took samples of porphyra haitanensis and nearby seawater, and then used the medium containing 1 per thousand agar to enrich the target bacteria. The target isolates were obtained by dilution-plate method, of which crude enzymes were further obtained by liquid culture. We adopted DNS method to determine the target bacteria which can convert agar to neoagaro oligosaccharides. The phylogenetics was identified by analyzing 16S rDNA sequence and combining the strain's morphological and bacterial colonial physiological biochemical characteristics. We isolated a gram-negative bacterial strain HJPHYXJ-1 capable of transforming agar to neoagaro oligosaccharides. Basic Local Alignment Search Tool (BLAST) search of HJPHYXJ-1's 16S rDNA sequence on GenBank suggested that the similarity between this strain and Vibrio natriegens reached 99% . In addition, the morphological and physiological biochemical characteristics of HJPHYXJ-1 also showed highly similarity to Vibrio natriegens. So we identified HJPHYXJ-1 as Vibrio natriegens. The results of HPLC suggested that the metabolite of enzymatic degradation was neoagaro oligosaccharides. HJPHYXJ-1 or the new isolate of Vibrio natriegens was capable of converting agar to neoagaro oligosaccharides.

Cell-of-Origin in Diffuse Large B-Cell Lymphoma: Are the Assays Ready for the Clinic?

PubMed

Scott, David W

2015-01-01

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma worldwide and consists of a heterogeneous group of cancers classified together on the basis of shared morphology, immunophenotype, and aggressive clinical behavior. It is now recognized that this malignancy comprises at least two distinct molecular subtypes identified by gene expression profiling: the activated B-cell-like (ABC) and the germinal center B-cell-like (GCB) groups-the cell-of-origin (COO) classification. These two groups have different genetic mutation landscapes, pathobiology, and outcomes following treatment. Evidence is accumulating that novel agents have selective activity in one or the other COO group, making COO a predictive biomarker. Thus, there is now a pressing need for accurate and robust methods to assign COO, to support clinical trials, and ultimately guide treatment decisions for patients. The "gold standard" methods for COO are based on gene expression profiling (GEP) of RNA from fresh frozen tissue using microarray technology, which is an impractical solution when formalin-fixed paraffin-embedded tissue (FFPET) biopsies are the standard diagnostic material. This review outlines the history of the COO classification before examining the practical implementation of COO assays applicable to FFPET biopsies. The immunohistochemistry (IHC)-based algorithms and gene expression-based assays suitable for the highly degraded RNA from FFPET are discussed. Finally, the technical and practical challenges that still need to be addressed are outlined before robust gene expression-based assays are used in the routine management of patients with DLBCL.

Application of solid-phase extraction to agar-supported fermentation.

PubMed

Le Goff, Géraldine; Adelin, Emilie; Cortial, Sylvie; Servy, Claudine; Ouazzani, Jamal

2013-09-01

Agar-supported fermentation (Ag-SF), a variant of solid-state fermentation, has recently been improved by the development of a dedicated 2 m(2) scale pilot facility, Platotex. We investigated the application of solid-phase extraction (SPE) to Ag-SF in order to increase yields and minimize the contamination of the extracts with agar constituents. The selection of the appropriate resin was conducted on liquid-state fermentation and Diaion HP-20 exhibited the highest recovery yield and selectivity for the metabolites of the model fungal strains Phomopsis sp. and Fusarium sp. SPE applied to Ag-SF resulted in a particular compartmentalization of the culture. The mycelium that requires oxygen to grow migrates to the top layer and formed a thick biofilm. The resin beads intercalate between the agar surface and the mycelium layer, and trap directly the compounds secreted by the mycelium through a "solid-solid extraction" (SSE) process. The resin/mycelium layer is easily recovered by scraping the surface and the target metabolites extracted by methanol. Ag-SF associated to SSE represents an ideal compromise for the production of bioactive secondary metabolites with limited economic and environmental impact.

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...

Effect of impact stress on microbial recovery on an agar surface.

PubMed Central

Stewart, S L; Grinshpun, S A; Willeke, K; Terzieva, S; Ulevicius, V; Donnelly, J

1995-01-01

Microbial stress due to the impaction of microorganisms onto an agar collection surface was studied experimentally. The relative recovery rates of aerosolized Pseudomonas fluorescens and Micrococcus luteus were determined as a function of the impaction velocity by using a moving agar slide impactor operating over a flow rate range from 3.8 to 40 liters/min yielding impaction velocities from 24 to 250 m/s. As a reference, the sixth stage of the Andersen Six-Stage Viable Particle Sizing Sampler was used at its operating flow rate of 28.3 liters/min (24 m/s). At a collection efficiency of close to 100% for the agar slide impactor, an increase in sampling flow rate and, therefore, in impaction velocity produced a significant decline in the percentage of microorganisms recovered. Conversely, when the collection efficiency was less than 100%, greater recovery and lower injury rates occurred. The highest relative rate of recovery (approximately 51% for P. fluorescens and approximately 62% for M. luteus) was obtained on the complete (Trypticase soy agar) medium at 40 and 24 m/s (6.4 and 3.8 liters/min), respectively. M. luteus demonstrated less damage than P. fluorescens, suggesting the hardy nature of the gram-positive strain versus that of the gram-negative microorganism. Comparison of results from the agar slide and Andersen impactors at the same sampling velocity showed that recovery and injury due to collection depends not only on the magnitude of the impaction velocity but also on the degree to which the microorganisms may be embedded in the collection medium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7747946

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.

Influence of ethylenediamine-n,n’-disuccinic acid (EDDS) concentration on the bactericidal activity of fatty acids in vitro

USDA-ARS?s Scientific Manuscript database

The antibacterial activity of mixtures of ethylenediamine-N,N’-disuccinic acid (EDDS) and antibacterial fatty acids (FA) was examined using the agar diffusion assay. Solutions of caproic, caprylic, capric, and lauric acids dissolved in potassium hydroxide (KOH) were supplemented with 0, 5, or 10 mM ...

A vapour phase assay for evaluating the antimicrobial activities of essential oils against bovine respiratory bacterial pathogens.

PubMed

Amat, S; Baines, D; Alexander, T W

2017-12-01

The objectives of this study were to develop a new assay for the evaluation of the antimicrobial activities of essential oils (EOs) in vapour phase and to demonstrate the antimicrobial activities of commercial EOs against BRPs. To achieve the first objective, a microtube cap containing 100 μl of EO was embedded in an agar plate. An agar plug (diameter 13 mm) inoculated with a bacterial suspension containing10 8  CFU per ml was then placed over the cap and incubated at 37°C for 24 h. Subsequently, bacteria were recovered from the agar plug by immersion in 5 ml of broth for 10 min, followed by vortexing for 30 s, and the broths were then plated for enumeration. To demonstrate the usefulness of the assay, nine commercial EOs derived from the following specific plants: ajowan, carrot seed, cinnamon leaf, citronella, fennel, ginger grass, lavender, rosemary and thyme were first evaluated for their vapour phase antimicrobial activities against Mannheimia haemolytica serotype 1. Selected EOs were further tested against Pasteurella multocida and Histophilus somni. The EOs of ajowan, thyme and cinnamon leaf completely or partially inhibited BRPs growth. This new assay provided reproducible results on the vapour phase antimicrobial activities of EOs against BRPs. These results support further study of EOs as a potential mitigation strategy against BRPs. In this study, we present a new vapour phase assay for evaluating the antimicrobial activities of essential oils (EO) against bovine respiratory pathogens (BRPs). Using this assay, we identified EOs, such as ajowan, thyme and cinnamon leaf, that can effectively inhibit growth of the BRPs Mannheimia haemolytica serotype 1, Pasteurella multocida and Histophilus somni. This is the first study to demonstrate the vapour phase antimicrobial activity of EOs against BRPs. © 2017 Her Majesty the Queen in Right of Canada. © 2017 The Society for Applied Microbiology Reproduced with the permission of the Minister of the

Evaluation of modified dichloran 18% glycerol (DG18) agar for enumerating fungi in wheat flour: a collaborative study.

PubMed

Beuchat, L R; Hwang, C A

1996-04-01

Dichloran 18% glycerol agar base supplemented with 100 micrograms of chloramphenicol ml-1 (DG18 agar) was compared to DG18 agar supplemented with 100 micrograms of Triton X-301 ml-1 (DG18T) and DG18 agar supplemented with 1 microgram of iprodione [3-(3,5-dichlorophenyl)-N-(1-methyl-ethyl)-2,4-dioxo-1-imidazolidine- carboxamide] ml-1 (DG18I agar) for enumeration of fungi in ten brands of wheat flour. As the flours contained low fungal populations, all were inoculated with two to four strains of xerophilic fungi (Aspergillus candidus, A. penicillioides, Eurotium amstelodami, E. intermedium, E. repens, E. rubrum, E. tonophilum, E. umbrosum and Wallemia sebi), after which counts ranged from 3.87 to 6.37 log10 CFU g-1. Significantly higher populations (p < 0.05) were detected in four flours: three were on DG18T compared to DG18 and DG18I agar. A. candidus had been inoculated into all three flours. E. amstelodami, E. intermedium, E. repens or E. tonophilum had also been inoculated into at least one of the three flours showing significantly higher numbers of CFU on DG18T agar. Analysis of collapsed data from all samples showed that DG18T agar was significantly better than DG18 or DG18I agars at p < 0.10 but not at p < 0.05. Coefficients of variation for reproducibility (among-laboratory variation) were 8.4%, 7.5% and 8.6%, respectively, for DG18, DG18T and DG18I agars. DG18I agar restricted colony development most, especially for Eurotium species. Naturally occurring Penicillium species grew equally well on DG18 and DG18T agars, whereas W. sebi grew well on all three media. DG18T agar was judged to be superior to DG18 and DG18I agars for enumerating fungi in wheat flours.

Amino acid mediated synthesis of silver nanoparticles and preparation of antimicrobial agar/silver nanoparticles composite films.

PubMed

Shankar, Shiv; Rhim, Jong-Whan

2015-10-05

Silver nanoparticles (AgNPs) were synthesized using amino acids (tyrosine and tryptophan) as reducing and capping agents, and they were incorporated into the agar to prepare antimicrobial composite films. The AgNPs solutions exhibited characteristic absorption peak at 420 nm that showed a red shift to ∼434 nm after forming composite with agar. XRD data demonstrated the crystalline structure of AgNPs with dominant (111) facet. Apparent surface color and transmittance of agar films were greatly influenced by the AgNPs. The incorporation of AgNPs into agar did not exhibit any change in chemical structure, thermal stability, moisture content, and water vapor permeability. The water contact angle, tensile strength, and modulus decreased slightly, but elongation at break increased after AgNPs incorporation. The agar/AgNPs nanocomposite films possessed strong antibacterial activity against Listeria monocytogenes and Escherichia coli. The agar/AgNPs film could be applied to the active food packaging by controlling the food-borne pathogens. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparison of performance of the novel chromogenic spectra VRE agar to that of bile esculin azide and Campylobacter agars for detection of vancomycin-resistant enterococci in fecal samples.

PubMed

Jenkins, S G; Raskoshina, L; Schuetz, A N

2011-11-01

A total of 142 stool specimens were evaluated for vancomycin-resistant enterococcus (VRE). Twenty-four-hour sensitivities and specificities, respectively, were 98% and 95% for Spectra VRE chromogenic agar (Remel, Lenexa, KS), 86% and 92% for bile esculin azide with vancomycin (BEAV; Remel), and 96.5% and 92% for Campylobacter agar (CAMPY; Remel). Spectra VRE and CAMPY are significantly more sensitive at 24 h than BEAV.

A novel DTI-QA tool: Automated metric extraction exploiting the sphericity of an agar filled phantom.

PubMed

Chavez, Sofia; Viviano, Joseph; Zamyadi, Mojdeh; Kingsley, Peter B; Kochunov, Peter; Strother, Stephen; Voineskos, Aristotle

2018-02-01

To develop a quality assurance (QA) tool (acquisition guidelines and automated processing) for diffusion tensor imaging (DTI) data using a common agar-based phantom used for fMRI QA. The goal is to produce a comprehensive set of automated, sensitive and robust QA metrics. A readily available agar phantom was scanned with and without parallel imaging reconstruction. Other scanning parameters were matched to the human scans. A central slab made up of either a thick slice or an average of a few slices, was extracted and all processing was performed on that image. The proposed QA relies on the creation of two ROIs for processing: (i) a preset central circular region of interest (ccROI) and (ii) a signal mask for all images in the dataset. The ccROI enables computation of average signal for SNR calculations as well as average FA values. The production of the signal masks enables automated measurements of eddy current and B0 inhomogeneity induced distortions by exploiting the sphericity of the phantom. Also, the signal masks allow automated background localization to assess levels of Nyquist ghosting. The proposed DTI-QA was shown to produce eleven metrics which are robust yet sensitive to image quality changes within site and differences across sites. It can be performed in a reasonable amount of scan time (~15min) and the code for automated processing has been made publicly available. A novel DTI-QA tool has been proposed. It has been applied successfully on data from several scanners/platforms. The novelty lies in the exploitation of the sphericity of the phantom for distortion measurements. Other novel contributions are: the computation of an SNR value per gradient direction for the diffusion weighted images (DWIs) and an SNR value per non-DWI, an automated background detection for the Nyquist ghosting measurement and an error metric reflecting the contribution of EPI instability to the eddy current induced shape changes observed for DWIs. Copyright © 2017 Elsevier

Mineralized agar-based nanocomposite films: Potential food packaging materials with antimicrobial properties.

PubMed

Malagurski, Ivana; Levic, Steva; Nesic, Aleksandra; Mitric, Miodrag; Pavlovic, Vladimir; Dimitrijevic-Brankovic, Suzana

2017-11-01

New mineralized, agar-based nanocomposite films (Zn-carbonate and Zn-phosphate/agar) were produced by a combination of in situ precipitation and a casting method. The presence of minerals significantly influenced the morphology, properties and functionality of the obtained nanocomposites. Reinforcement with the Zn-mineral phase improved the mechanical properties of the carbonate-mineralized films, but had a negligible effect on the phosphate-mineralized samples. Both nanocomposites showed improved optical and thermal properties, better Zn(II) release potential in a slightly acidic environment and exhibited antimicrobial activity against S. aureus. These results suggest that Zn-mineralized agar nanocomposite films could be potentially used as affordable, eco-friendly and active food packaging materials. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

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.

Equilibrium and kinetic modelling of Cd(II) biosorption by algae Gelidium and agar extraction algal waste.

PubMed

Vilar, Vítor J P; Botelho, Cidália M S; Boaventura, Rui A R

2006-01-01

In this study an industrial algal waste from agar extraction has been used as an inexpensive and effective biosorbent for cadmium (II) removal from aqueous solutions. This biosorbent was compared with the algae Gelidium itself, which is the raw material for agar extraction. Equilibrium data follow both Langmuir and Redlich-Peterson models. The parameters of Langmuir equilibrium model are q(max)=18.0 mgg(-1), b=0.19 mgl(-1) and q(max)=9.7 mgg(-1), b=0.16 mgl(-1), respectively for Gelidium and the algal waste. Kinetic experiments were conducted at initial Cd(II) concentrations in the range 6-91 mgl(-1). Data were fitted to pseudo-first- and second-order Lagergren models. For an initial Cd(II) concentration of 91 mgl(-1) the parameters of the pseudo-first-order Lagergren model are k(1,ads)=0.17 and 0.87 min(-1); q(eq)=16.3 and 8.7 mgg(-1), respectively, for Gelidium and algal waste. Kinetic constants vary with the initial metal concentration. The adsorptive behaviour of biosorbent particles was modelled using a batch reactor mass transfer kinetic model. The model successfully predicts Cd(II) concentration profiles and provides significant insights on the biosorbents performance. The homogeneous diffusivity, D(h), is in the range 0.5-2.2 x10(-8) and 2.1-10.4 x10(-8)cm(2)s(-1), respectively, for Gelidium and algal waste.

Cytotoxic effects of denture adhesives on primary human oral keratinocytes, fibroblasts and permanent L929 cell lines.

PubMed

Chen, Fengying; Wu, Tianfu; Cheng, Xiangrong

2014-03-01

To date, there have been very little data on the cytotoxic responses of different cell lines to denture adhesives. To determine the cytotoxicity of three denture adhesives on primary human oral keratinocytes (HOKs), fibroblasts (HOFs) and permanent mouse fibroblasts cell lines (L929). Three commercial denture adhesives (two creams and one powder) were prepared for indirect contact using the agar diffusion test, as well as extracts in MTT assay. The results of the MTT assay were statistically analysed by one-way anova and Tukey's test (p < 0.05). All of the tested denture adhesives showed mild to moderate cytotoxicity to primary HOKs (p < 0.001), whereas none of three was toxic to L929 cells (p > 0.05) in both assays. For primary HOFs cultures, slight cytotoxicity was observed for one of the products from the agar diffusion test and undiluted eluates of all tested adhesives with MTT assay (p < 0.01). Denture adhesives are toxic to the primary HOKs and HOFs cultures, whereas non-toxic to L929 cells. The results suggest that primary human oral mucosal cells may provide more valuable information in toxicity screening of denture adhesives. © 2012 John Wiley & Sons A/S and The Gerodontology Association. Published by John Wiley & Sons Ltd.

Modification of kanamycin-esculin-azide agar to improve selectivity in the enumeration of fecal streptococci from water samples.

PubMed

Audicana, A; Perales, I; Borrego, J J

1995-12-01

Kanamycin-esculin-azide agar was modified by increasing the concentration of sodium azide to 0.4 g liter-1 and replacing kanamycin sulfate with 5 mg of oxolinic acid liter-1. The modification, named oxolinic acid-esculin-azide (OAA) agar, was compared with Slanetz-Bartley and KF agars by using drinking water and seawater samples. The OAA agar showed higher specificity, selectivity, and recovery efficiencies than those obtained by using the other media. In addition, no confirmation of typical colonies was needed when OAA agar was used, which significantly shortens the time of sample processing and increases the accuracy of the method.

Comparison of Performance of the Novel Chromogenic Spectra VRE Agar to That of Bile Esculin Azide and Campylobacter Agars for Detection of Vancomycin-Resistant Enterococci in Fecal Samples ▿

PubMed Central

Jenkins, S. G.; Raskoshina, L.; Schuetz, A. N.

2011-01-01

A total of 142 stool specimens were evaluated for vancomycin-resistant enterococcus (VRE). Twenty-four-hour sensitivities and specificities, respectively, were 98% and 95% for Spectra VRE chromogenic agar (Remel, Lenexa, KS), 86% and 92% for bile esculin azide with vancomycin (BEAV; Remel), and 96.5% and 92% for Campylobacter agar (CAMPY; Remel). Spectra VRE and CAMPY are significantly more sensitive at 24 h than BEAV. PMID:21880967

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.

Fusion of agarase and neoagarobiose hydrolase for mono-sugar production from agar.

PubMed

Alkotaini, Bassam; Han, Nam Soo; Kim, Beom Soo

2017-02-01

In enzymatic saccharification of agar, endo- and exo-agarases together with neoagarobiose hydrolase (NABH) are important key enzymes for the sequential hydrolysis reactions. In this study, a bifunctional endo/exo-agarase was fused with NABH for production of mono-sugars (D-galactose and 3,6-anhydro-L-galactose) from agar using only one fusion enzyme. Two fusion enzymes with either bifunctional agarase (Sco3476) or NABH (Zg4663) at the N-terminus, Sco3476-Zg4663 (SZ) and Zg4663-Sco3476 (ZS), were constructed. Both fusion enzymes exhibited their optimal agarase and NABH activities at 40 and 35 °C, respectively. Fusions SZ and ZS enhanced the thermostability of the NABH activity, while only fusion SZ showed a slight enhancement in the NABH catalytic efficiency (K cat /K M ) from 14.8 (mg/mL) -1  s -1 to 15.8 (mg/mL) -1  s -1 . Saccharification of agar using fusion SZ resulted in 2-fold higher mono-sugar production and 3-fold lower neoagarobiose accumulation when compared to the physical mixture of Sco3476 and Zg4663. Therefore, this fusion has the potential to reduce enzyme production cost, decrease intermediate accumulation, and increase mono-sugar yield in agar saccharification.

An extension of the Coconut Cream Agar method to screen Penicillium citrinum isolates for citrinin production.

PubMed

Mohamed, S; Flint, S; Palmer, J; Fletcher, G C; Pitt, J I

2013-09-01

A simple and rapid screening method was developed for the detection of citrinin in fungal cultures using Coconut Cream Agar (CCA) described previously for detecting aflatoxin and ochratoxin A. Fifteen isolates of Penicillium citrinum were inoculated onto CCA and incubated at 25 and 30°C for 10 days. All isolates produced a distinct yellow green fluorescence on CCA when the reverse side of the agar plates were viewed under long wavelength UV light. Detection was optimal at 25°C after four to 5 days of incubation. Isolates positive by the CCA method also tested positive for citrinin production by the TLC agar plug method after growth on CCA, Czapek yeast extract agar and yeast extract sucrose agar. Control cultures were negative by both methods, indicating that the CCA Petri dish method was suitable for screening cultures for citrinin production. © 2013 The Society for Applied Microbiology.

Serological purification of polysaccharide antigens from Streptococcus mutans serotypes a and d: characterization of multiple antigenic determinants.

PubMed

Linzer, R; Mukasa, H; Slade, H D

1975-10-01

The polysaccharide antigen preparations from serotype a and serotype d strains of Streptococcus mutans contained both a serotype-specific antigenic determinant and a common a-d antigenic determinant, as demonstrated by agar gel diffusion studies and a quantitative cross-precipitin assay. The chromatographically purified antigens were isolated by a method which depended on their serological specificity to determine if these two antigenic determinants were located on the same molecule. The a and d polysaccharides were recovered from specific antigen-antibody complexes and characterized with respect to their immunological specificity and chemical composition. Agar gel diffusion tests demonstrated that, in both the a and d preparations, the serotype-specific antigenic determinant and the common a-d antigenic determinant were present in one molecule.

Diffusion processes in tumors: A nuclear medicine approach

NASA Astrophysics Data System (ADS)

Amaya, Helman

2016-07-01

The number of counts used in nuclear medicine imaging techniques, only provides physical information about the desintegration of the nucleus present in the the radiotracer molecules that were uptaken in a particular anatomical region, but that information is not a real metabolic information. For this reason a mathematical method was used to find a correlation between number of counts and 18F-FDG mass concentration. This correlation allows a better interpretation of the results obtained in the study of diffusive processes in an agar phantom, and based on it, an image from the PETCETIX DICOM sample image set from OsiriX-viewer software was processed. PET-CT gradient magnitude and Laplacian images could show direct information on diffusive processes for radiopharmaceuticals that enter into the cells by simple diffusion. In the case of the radiopharmaceutical 18F-FDG is necessary to include pharmacokinetic models, to make a correct interpretation of the gradient magnitude and Laplacian of counts images.

Diffusion processes in tumors: A nuclear medicine approach

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

Amaya, Helman, E-mail: haamayae@unal.edu.co

The number of counts used in nuclear medicine imaging techniques, only provides physical information about the desintegration of the nucleus present in the the radiotracer molecules that were uptaken in a particular anatomical region, but that information is not a real metabolic information. For this reason a mathematical method was used to find a correlation between number of counts and {sup 18}F-FDG mass concentration. This correlation allows a better interpretation of the results obtained in the study of diffusive processes in an agar phantom, and based on it, an image from the PETCETIX DICOM sample image set from OsiriX-viewer softwaremore » was processed. PET-CT gradient magnitude and Laplacian images could show direct information on diffusive processes for radiopharmaceuticals that enter into the cells by simple diffusion. In the case of the radiopharmaceutical {sup 18}F-FDG is necessary to include pharmacokinetic models, to make a correct interpretation of the gradient magnitude and Laplacian of counts images.« less

Modification of kanamycin-esculin-azide agar to improve selectivity in the enumeration of fecal streptococci from water samples.

PubMed Central

Audicana, A; Perales, I; Borrego, J J

1995-01-01

Kanamycin-esculin-azide agar was modified by increasing the concentration of sodium azide to 0.4 g liter-1 and replacing kanamycin sulfate with 5 mg of oxolinic acid liter-1. The modification, named oxolinic acid-esculin-azide (OAA) agar, was compared with Slanetz-Bartley and KF agars by using drinking water and seawater samples. The OAA agar showed higher specificity, selectivity, and recovery efficiencies than those obtained by using the other media. In addition, no confirmation of typical colonies was needed when OAA agar was used, which significantly shortens the time of sample processing and increases the accuracy of the method. PMID:8534085

Agar/gelatin bilayer gel matrix fabricated by simple thermo-responsive sol-gel transition method.

PubMed

Wang, Yifeng; Dong, Meng; Guo, Mengmeng; Wang, Xia; Zhou, Jing; Lei, Jian; Guo, Chuanhang; Qin, Chaoran

2017-08-01

We present a simple and environmentally-friendly method to generate an agar/gelatin bilayer gel matrix for further biomedical applications. In this method, the thermally responsive sol-gel transitions of agar and gelatin combined with the different transition temperatures are exquisitely employed to fabricate the agar/gelatin bilayer gel matrix and achieve separate loading for various materials (e.g., drugs, fluorescent materials, and nanoparticles). Importantly, the resulting bilayer gel matrix provides two different biopolymer environments (a polysaccharide environment vs a protein environment) with a well-defined border, which allows the loaded materials in different layers to retain their original properties (e.g., magnetism and fluorescence) and reduce mutual interference. In addition, the loaded materials in the bilayer gel matrix exhibit an interesting release behavior under the control of thermal stimuli. Consequently, the resulting agar/gelatin bilayer gel matrix is a promising candidate for biomedical applications in drug delivery, controlled release, fluorescence labeling, and bio-imaging. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Strategies to improve the mechanical strength and water resistance of agar films for food packaging applications.

PubMed

Sousa, Ana M M; Gonçalves, Maria P

2015-11-05

Agar films possess several properties adequate for food packaging applications. However, their high cost-production and quality variations caused by physiological and environmental factors affecting wild seaweeds make them less attractive for industries. In this work, native (NA) and alkali-modified (AA) agars obtained from sustainably grown seaweeds (integrated multi-trophic aquaculture) were mixed with locust bean gum (LBG) to make 'knife-coated' films with fixed final concentration (1 wt%) and variable agar/LBG ratios. Agar films were easier to process upon LBG addition (viscosity increase and gelling character decrease of the film-forming solutions observed by dynamic oscillatory and steady shear measurements). The mechanical properties and water resistance were optimal for films with 50 and/or 75% LBG contents and best in the case of NA (cheaper to extract). These findings can help reduce the cost-production of agar packaging films. Moreover, the controlled cultivation of seaweeds can provide continuous and reliable feedstock for transformation industries. Copyright © 2015 Elsevier Ltd. All rights reserved.

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®

A comparison of in vitro cytotoxicity assays in medical device regulatory studies.

PubMed

Liu, Xuemei; Rodeheaver, Denise P; White, Jeffrey C; Wright, Ann M; Walker, Lisa M; Zhang, Fan; Shannon, Stephen

2018-06-06

Medical device biocompatibility testing is used to evaluate the risk of adverse effects on tissues from exposure to leachates/extracts. A battery of tests is typically recommended in accordance with regulatory standards to determine if the device is biocompatible. In vitro cytotoxicity, a key element of the standards, is a required endpoint for all types of medical devices. Each validated cytotoxicity method has different methodology and acceptance criteria that could influence the selection of a specific test. In addition, some guidances are more specific than others as to the recommended test methods. For example, the International Organization for Standardization (ISO 1 ) cites preference for quantitative methods (e.g., tetrazolium (MTT/XTT), neutral red (NR), or colony formation assays (CFA)) over qualitative methods (e.g., elution, agar overlay/diffusion, or direct), while a recent ISO standard for contact lens/lens care solutions specifically requires a qualitative direct test. Qualitative methods are described in United States Pharmacopeia (USP) while quantitative CFAs are listed in Japan guidance. The aim of this review is to compare the methodologies such as test article preparation, test conditions, and criteria for six cytotoxicity methods recommended in regulatory standards in order to inform decisions on which method(s) to select during the medical device safety evaluation. Copyright © 2018. Published by Elsevier Inc.

Quantitative SIMS Imaging of Agar-Based Microbial Communities.

PubMed

Dunham, Sage J B; Ellis, Joseph F; Baig, Nameera F; Morales-Soto, Nydia; Cao, Tianyuan; Shrout, Joshua D; Bohn, Paul W; Sweedler, Jonathan V

2018-05-01

After several decades of widespread use for mapping elemental ions and small molecular fragments in surface science, secondary ion mass spectrometry (SIMS) has emerged as a powerful analytical tool for molecular imaging in biology. Biomolecular SIMS imaging has primarily been used as a qualitative technique; although the distribution of a single analyte can be accurately determined, it is difficult to map the absolute quantity of a compound or even to compare the relative abundance of one molecular species to that of another. We describe a method for quantitative SIMS imaging of small molecules in agar-based microbial communities. The microbes are cultivated on a thin film of agar, dried under nitrogen, and imaged directly with SIMS. By use of optical microscopy, we show that the area of the agar is reduced by 26 ± 2% (standard deviation) during dehydration, but the overall biofilm morphology and analyte distribution are largely retained. We detail a quantitative imaging methodology, in which the ion intensity of each analyte is (1) normalized to an external quadratic regression curve, (2) corrected for isomeric interference, and (3) filtered for sample-specific noise and lower and upper limits of quantitation. The end result is a two-dimensional surface density image for each analyte. The sample preparation and quantitation methods are validated by quantitatively imaging four alkyl-quinolone and alkyl-quinoline N-oxide signaling molecules (including Pseudomonas quinolone signal) in Pseudomonas aeruginosa colony biofilms. We show that the relative surface densities of the target biomolecules are substantially different from values inferred through direct intensity comparison and that the developed methodologies can be used to quantitatively compare as many ions as there are available standards.

Bactericidal activity of alkaline salts of fatty acids towards bacteria associated with poultry processing

USDA-ARS?s Scientific Manuscript database

Antibacterial activity of alkaline salts of caproic, caprylic, capric, lauric, and myristic acids were determined using the agar diffusion assay. A 0.5M concentration of each fatty acid (FA) was dissolved in 1.0 M potassium hydroxide (KOH), and pH of the mixtures was adjusted to 10.5 with citric aci...

Effect of pH alkaline salts of fatty acids on the inhibition of bacteria associated with poultry processing

USDA-ARS?s Scientific Manuscript database

The agar diffusion assay was used to examine the effect of pH on the ability of alkaline salts of three fatty acids (FA) to inhibit growth of bacteria associated with poultry processing. FA solutions were prepared by dissolving 0.5 M concentrations of caprylic, capric, or lauric acid in separate ali...

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

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

Effects of shape and size of agar gels on heating uniformity during pulsed microwave treatment.

PubMed

Soto-Reyes, Nohemí; Temis-Pérez, Ana L; López-Malo, Aurelio; Rojas-Laguna, Roberto; Sosa-Morales, María Elena

2015-05-01

Model gel systems with different shape (sphere, cylinder, and slab) and size (180 and 290 g) were prepared with agar (5%) and sucrose (5%). Dielectric constant (ε'), loss factor (ε"), thermophysical properties, and temperature distribution of the model system were measured. Each agar model system was immersed and suspended in water, and then, heated in a microwave oven with intermittent heating until the core temperature reached 50 °C. The ε' and ε" of agar gels decreased when frequency increased. The density and thermal conductivity values of the agar gels were 1033 kg/m(3) and 0.55 W/m °C, respectively. The temperature distribution of sphere, cylinder, and slab was different when similar power doses were applied. The slab reached 50 °C in less time (10 min) and showed a more uniform heating than spheres and cylinders in both sizes. Agar model systems of 180 g heated faster than those of 290 g. The coldest point was the center of the model systems in all studied cases. Shape and size are critical food factors that affect the heating uniformity during microwave heating processes. © 2015 Institute of Food Technologists®

Rapid direct testing of susceptibility of Mycobacterium tuberculosis to isoniazid and rifampin on nutrient and blood agar in resource-starved settings.

PubMed

Satti, Luqman; Ikram, Aamer; Coban, Ahmet Yilmaz; Martin, Anandi

2012-05-01

In this study, we evaluated the performance of blood agar (by macroscopic growth) and nutrient agar (by a microcolony detection method) for drug susceptibility testing of Mycobacterium tuberculosis against rifampin (RIF) and isoniazid (INH), using 67 smear-positive sputum specimens. The direct proportion method on Lowenstein-Jensen (LJ) medium was used as the "gold standard." Compared with LJ medium, results for both media were in 100% agreement for RIF, while for INH the agreement levels for blood agar and nutrient agar were 98% and 95%, respectively. Within 2 weeks, 100% of specimens yielded results on blood agar, while 96.8% of specimens yielded results on nutrient agar. Our study showed that blood agar and nutrient agar can be used as alternative media for direct susceptibility testing of RIF and INH, especially in resource-poor settings.

Formation of Ramified Colony of Fungus Aspergillus Oryzae on Agar Media

NASA Astrophysics Data System (ADS)

Matsuura, Shu; Miyazima, Sasuke

Ramified colonies of fungus Aspergillus oryzae have been found to grow at a low growth rate on "liquid-like" agar media with low concentrations of agar and glucose. Box-counting fractal dimensions of the individual colony branches have been found to decrease with the time of incubation. Addition of glucose solution in the interior of branched colonies has brought about the production of the hyphal filaments almost only at the apical region of the colony branches. Active growth of the ramified colonies is localized in the peripheral zone, and this growth manner implies that the fungus is exhibiting a positive exploitation.

Disk Diffusion Testing Using Candida sp. Colonies Taken Directly from CHROMagar Candida Medium May Decrease Time Required To Obtain Results

PubMed Central

Klevay, Michael; Ebinger, Alex; Diekema, Daniel; Messer, Shawn; Hollis, Richard; Pfaller, Michael

2005-01-01

We compared results of disk diffusion antifungal susceptibility testing from Candida sp. strains passaged on CHROMagar and on potato dextrose agar. The overall categorical agreements for fluconazole and voriconazole disk testing were 95% and 98% with 0% and 0.5% very major errors, respectively. Disk diffusion testing by the CLSI (formerly NCCLS) M44-A method can be performed accurately by taking inocula directly from CHROMagar. PMID:16000489

A fresh liver agar substrate for rearing small numbers of forensically important blow flies (Diptera: Calliphoridae).

PubMed

Gruner, Susan V; Slone, Daniel H

2014-05-01

Forensically important calliphorids can be reared on a mixture of beef liver and agar. Small pieces of meat, especially fresh or frozen beef liver, will desiccate in 2-6 h, but this simple-to-make feeding substrate remains moist for at least 12 h at 25 and 30 degrees C without desiccation, even in small (5 g) amounts. We determined the survivorship of small numbers of Chrysomya megacephala (F.) (first-instar larvae to adult eclosion) raised on 5 g of liver agar and fresh beef liver. We found that all larvae raised on 5 g of liver died due to desiccation, but survivorship on 5 g of liver agar was equivalent to that on larger (50 g) pieces of either liver agar or beef liver.

Rapid Direct Testing of Susceptibility of Mycobacterium tuberculosis to Isoniazid and Rifampin on Nutrient and Blood Agar in Resource-Starved Settings

PubMed Central

Ikram, Aamer; Coban, Ahmet Yilmaz; Martin, Anandi

2012-01-01

In this study, we evaluated the performance of blood agar (by macroscopic growth) and nutrient agar (by a microcolony detection method) for drug susceptibility testing of Mycobacterium tuberculosis against rifampin (RIF) and isoniazid (INH), using 67 smear-positive sputum specimens. The direct proportion method on Lowenstein-Jensen (LJ) medium was used as the “gold standard.” Compared with LJ medium, results for both media were in 100% agreement for RIF, while for INH the agreement levels for blood agar and nutrient agar were 98% and 95%, respectively. Within 2 weeks, 100% of specimens yielded results on blood agar, while 96.8% of specimens yielded results on nutrient agar. Our study showed that blood agar and nutrient agar can be used as alternative media for direct susceptibility testing of RIF and INH, especially in resource-poor settings. PMID:22357498

Simple fluorescence-based high throughput cell viability assay for filamentous fungi.

PubMed

Chadha, S; Kale, S P

2015-09-01

Filamentous fungi are important model organisms to understand the eukaryotic process and have been frequently exploited in research and industry. These fungi are also causative agents of serious diseases in plants and humans. Disease management strategies include in vitro susceptibility testing of the fungal pathogens to environmental conditions and antifungal agents. Conventional methods used for antifungal susceptibilities are cumbersome, time-consuming and are not suitable for a large-scale analysis. Here, we report a rapid, high throughput microplate-based fluorescence method for investigating the toxicity of antifungal and stress (osmotic, salt and oxidative) agents on Magnaporthe oryzae and compared it with agar dilution method. This bioassay is optimized for the resazurin reduction to fluorescent resorufin by the fungal hyphae. Resazurin bioassay showed inhibitory rates and IC50 values comparable to the agar dilution method and to previously reported IC50 or MICs for M. oryzae and other fungi. The present method can screen range of test agents from different chemical classes with different modes of action for antifungal activities in a simple, sensitive, time and cost effective manner. A simple fluorescence-based high throughput method is developed to test the effects of stress and antifungal agents on viability of filamentous fungus Magnaporthe oryzae. This resazurin fluorescence assay can detect inhibitory effects comparable to those obtained using the growth inhibition assay with added advantages of simplicity, time and cost effectiveness. This high throughput viability assay has a great potential in large-scale screening of the chemical libraries of antifungal agents, for evaluating the effects of environmental conditions and hyphal kinetic studies in mutant and natural populations of filamentous fungi. © 2015 The Society for Applied Microbiology.

Homogeneous Matrix Deposition on Dried Agar for MALDI Imaging Mass Spectrometry of Microbial Cultures

NASA Astrophysics Data System (ADS)

Hoffmann, Thomas; Dorrestein, Pieter C.

2015-11-01

Matrix deposition on agar-based microbial colonies for MALDI imaging mass spectrometry is often complicated by the complex media on which microbes are grown. This Application Note demonstrates how consecutive short spray pulses of a matrix solution can form an evenly closed matrix layer on dried agar. Compared with sieving dry matrix onto wet agar, this method supports analyte cocrystallization, which results in significantly more signals, higher signal-to-noise ratios, and improved ionization efficiency. The even matrix layer improves spot-to-spot precision of measured m/z values when using TOF mass spectrometers. With this technique, we established reproducible imaging mass spectrometry of myxobacterial cultures on nutrient-rich cultivation media, which was not possible with the sieving technique.

Roughness-controlled self-assembly of mannitol/LB agar microparticles by polymorphic transformation for pulmonary drug delivery.

PubMed

Zhang, Fengying; Ngoc, Nguyen Thi Quynh; Tay, Bao Hui; Mendyk, Aleksander; Shao, Yu-Hsuan; Lau, Raymond

2015-01-05

Novel roughness-controlled mannitol/LB Agar microparticles were synthesized by polymorphic transformation and self-assembly method using hexane as the polymorphic transformation reagent and spray-dried mannitol/LB Agar microparticles as the starting material. As-prepared microparticles were characterized by Fourier transform infrared spectra (FTIR), X-ray diffraction spectra (XRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), thermal gravimetric analysis (TGA), and Andersen Cascade Impactor (ACI). The XRD and DSC results indicate that after immersing spray-dried mannitol/LB Agar microparticles in hexane, β-mannitol was completely transformed to α-mannitol in 1 h, and all the δ-mannitol was transformed to α form after 14 days. SEM shows that during the transformation the nanobelts on the spray-dried mannitol/LB Agar microparticles become more dispersed and the contour of the individual nanobelts becomes more noticeable. Afterward, the nanobelts self-assemble to nanorods and result in rod-covered mannitol/LB Agar microparticles. FTIR indicates new hydrogen bonds were formed among mannitol, LB Agar, and hexane. SEM images coupled with image analysis software reveal that different surface morphology of the microparticles have different drug adhesion mechanisms. Comparison of ACI results and image analysis of SEM images shows that an increase in the particle surface roughness can increase the fine particle fractions (FPFs) using the rod-covered mannitol microparticles as drug carriers. Transformed microparticles show higher FPFs than commercially available lactose carriers. An FPF of 28.6 ± 2.4% was achieved by microparticles transformed from spray-dried microparticles using 2% mannitol(w/v)/LB Agar as feed solution. It is comparable to the highest FPF reported in the literature using lactose and spray-dried mannitol as carriers.

Long-term biological hydrogen production by agar immobilized Rhodobacter capsulatus in a sequential batch photobioreactor.

PubMed

Elkahlout, Kamal; Alipour, Siamak; Eroglu, Inci; Gunduz, Ufuk; Yucel, Meral

2017-04-01

In this study, agar immobilization technique was employed for biological hydrogen production using Rhodobacter capsulatus DSM 1710 (wild type) and YO3 (hup-mutant) strains in sequential batch process. Different agar and glutamate concentrations were tested with defined nutrient medium. Agar concentration 4% (w/v) and 4 mM glutamate were selected for bacterial immobilization in terms of rate and longevity of hydrogen production. Acetate concentration was increased from 40 to 60-100 and 60 mM gave best results with both bacterial strains immobilized in 4% (w/v) agar. Cell concentration was increased from 2.5 to 5 mg dcw mL -1 agar and it was found that increasing cell concentration of wild-type strain caused decrease in yield and productivity while these parameters improved by increasing cell concentration of mutant strain. Also, the hydrogen production time has extended from 17 days up to 60 days according to the process conditions and parameters. Hydrogen production by immobilized photosynthetic bacteria is a convenient technology for hydrogen production as it enables to produce hydrogen with high organic acid concentrations comparing to suspended cultures. Besides, immobilization increases the stability of the system and allowed sequential batch operation for long-term application.

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)

The effect of agar jelly on energy expenditure, appetite, gastric emptying and glycaemic response.

PubMed

Clegg, Miriam E; Shafat, Amir

2014-01-01

Agar contains a high amount of soluble fibre and has been shown to delay gastric emptying (GE) without impacting on glycaemic response (GR). The current study aimed to further the limited data on the effect of agar on metabolism by assessing the effects on GE and GR as well as appetite- and diet-induced thermogenesis (DIT). In this randomized control trial, eleven healthy volunteers were tested on two occasions following an overnight fast. Following baseline and resting measurements, volunteers were either fed a fruit-flavoured drink (liquid) or consumed a fruit-flavoured jelly (jelly). The two were exactly the same in composition except the jelly contained 4 g of agar crystals. Both contained 50 g of available carbohydrate. DIT was measured using indirect calorimetry, GE using the (13)C sodium acetate breath test, appetite using visual analogue scale and GR using finger prick blood samples. The jelly significantly delayed GE across all time points-latency phase (p = 0.07), lag phase (p = 0.04), half-time (p < 0.0001), ascension time (p = 0.025). The jelly also increased all appetite parameters-hunger (p = 0.006), fullness (p = 0.035), desire to eat (p = 0.03) and prospective consumption (p = 0.011). However, there were no significant differences in either GR or postprandial DIT between the liquid and jelly. Agar delays GE and increases appetite but does not change GR or DIT most probably due to the increase in viscosity caused by the agar jelly.

Antimicrobial and physical-mechanical properties of agar-based films incorporated with grapefruit seed extract.

PubMed

Kanmani, Paulraj; Rhim, Jong-Whan

2014-02-15

The use of synthetic petroleum based packaging films caused serious environmental problems due to their difficulty in recycling and poor biodegradability. Therefore, present study was aimed to develop natural biopolymer-based antimicrobial packaging films as an alternative for the synthetic packaging films. As a natural antimicrobial agent, grapefruit seed extract (GSE) has been incorporated into agar to prepare antimicrobial packaging film. The films with different concentrations of GSE were prepared by a solvent casting method and the resulting composite films were examined physically and mechanically. In addition, the films were characterized by FE-SEM, XRD, FT-IR and TGA. The incorporation of GSE caused increase in color, UV barrier, moisture content, water solubility and water vapor permeability, while decrease in surface hydrophobicity, tensile strength and elastic modulus of the films. As the concentration of GSE increased from 0.6 to 13.3 μg/mL, the physical and mechanical properties of the films were affected significantly. The addition of GSE changed film microstructure of the film, but did not influence the crystallinity of agar and thermal stability of the agar-based films. The agar/GSE films exhibited distinctive antimicrobial activity against three test food pathogens, such as Listeria monocytogenes, Bacillus cereus and Escherichia coli. These results suggest that agar/GSE films have potential to be used in an active food packaging systems for maintaining food safety and extending the shelf-life of the packaged food. Copyright © 2013 Elsevier Ltd. All rights reserved.

A fresh liver agar substrate for rearing small numbers of forensically important blow flies (Diptera: Calliphoridae)

USGS Publications Warehouse

Gruner, Susan V.; Slone, Daniel H.

2014-01-01

Forensically important calliphorids can be reared on a mixture of beef liver and agar. Small pieces of meat, especially fresh or frozen beef liver, will desiccate in 2–6 h, but this simple-to-make feeding substrate remains moist for at least 12 h at 25 and 30°C without desiccation, even in small (5 g) amounts. We determined the survivorship of small numbers of Chrysomya megacephala (F.) (first-instar larvae to adult eclosion) raised on 5 g of liver agar and fresh beef liver. We found that all larvae raised on 5 g of liver died due to desiccation, but survivorship on 5 g of liver agar was equivalent to that on larger (50 g) pieces of either liver agar or beef liver.

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.

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.

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.

Homogeneous matrix deposition on dried agar for MALDI imaging mass spectrometry of microbial cultures.

PubMed

Hoffmann, Thomas; Dorrestein, Pieter C

2015-11-01

Matrix deposition on agar-based microbial colonies for MALDI imaging mass spectrometry is often complicated by the complex media on which microbes are grown. This Application Note demonstrates how consecutive short spray pulses of a matrix solution can form an evenly closed matrix layer on dried agar. Compared with sieving dry matrix onto wet agar, this method supports analyte cocrystallization, which results in significantly more signals, higher signal-to-noise ratios, and improved ionization efficiency. The even matrix layer improves spot-to-spot precision of measured m/z values when using TOF mass spectrometers. With this technique, we established reproducible imaging mass spectrometry of myxobacterial cultures on nutrient-rich cultivation media, which was not possible with the sieving technique. Graphical Abstract ᅟ.

Substrate Diffusion Heterogeneity Controls Bacterial Competition and Coexistence

NASA Astrophysics Data System (ADS)

Dechesne, A.; Or, D.; Smets, B. F.

2005-12-01

Diffusion has long been recognized as a key process affecting bacterial physiological functions ranging from nutrient uptake to removal of metabolic waste products. In the vadose zone, significant convective flows are limited and bacteria rely primarily on diffusion for nutrient supply. Even under relatively "wet" conditions (e.g. matric potentials -20 J/kg), soil water is fragmented and exists as thin liquid films or held in crevices imposing constraints on substrate diffusion. Our objective was to investigate the role of diffusion on soil microbial diversity, by focusing on one of the processes that shapes the structure of bacterial communities: competitive interactions. We used a simplified setup, in which the substrate (citrate) fluxes were controlled by different agar gels thicknesses and spatially heterogeneous diffusive pathways were created by an impermeable film with prescribed hole sizes and patterns. Our competition experiments involved two soil bacteria: Burkholderia xenovorans LB400 and Pseudomonas putida KT2440, which were tagged with different constitutive fluorescent markers, allowing for their on line microscopic detection. The growth parameters on citrate of these strains were thoroughly assessed. B. xenovorans LB400 is the weaker competitor. As a result, this strain was outcompeted by KT2440 under high substrate diffusivity and homogeneous conditions. Conversely, the disadvantage of the weakest competitor was not so marked under low substrate diffusivity condition. These results suggest that dry conditions in soil would provide conditions allowing the sustaining of weak bacterial competitors, resulting in the maintenance of high bacterial diversity.

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

Optimization of Phenotyping Assays for the Model Monocot Setaria viridis

PubMed Central

Acharya, Biswa R.; Roy Choudhury, Swarup; Estelle, Aiden B.; Vijayakumar, Anitha; Zhu, Chuanmei; Hovis, Laryssa; Pandey, Sona

2017-01-01

Setaria viridis (green foxtail) is an important model plant for the study of C4 photosynthesis in panicoid grasses, and is fast emerging as a system of choice for the study of plant development, domestication, abiotic stress responses and evolution. Basic research findings in Setaria are expected to advance research not only in this species and its close relative S. italica (foxtail millet), but also in other panicoid grasses, many of which are important food or bioenergy crops. Here we report on the standardization of multiple growth and development assays for S. viridis under controlled conditions, and in response to several phytohormones and abiotic stresses. We optimized these assays at three different stages of the plant’s life: seed germination and post-germination growth using agar plate-based assays, early seedling growth and development using germination pouch-based assays, and adult plant growth and development under environmentally controlled growth chambers and greenhouses. These assays will be useful for the community to perform large scale phenotyping analyses, mutant screens, comparative physiological analysis, and functional characterization of novel genes of Setaria or other related agricultural crops. Precise description of various growth conditions, effective treatment conditions and description of the resultant phenotypes will help expand the use of S. viridis as an effective model system. PMID:29312412

Optimization of Phenotyping Assays for the Model Monocot Setaria viridis.

PubMed

Acharya, Biswa R; Roy Choudhury, Swarup; Estelle, Aiden B; Vijayakumar, Anitha; Zhu, Chuanmei; Hovis, Laryssa; Pandey, Sona

2017-01-01

Setaria viridis (green foxtail) is an important model plant for the study of C4 photosynthesis in panicoid grasses, and is fast emerging as a system of choice for the study of plant development, domestication, abiotic stress responses and evolution. Basic research findings in Setaria are expected to advance research not only in this species and its close relative S. italica (foxtail millet), but also in other panicoid grasses, many of which are important food or bioenergy crops. Here we report on the standardization of multiple growth and development assays for S. viridis under controlled conditions, and in response to several phytohormones and abiotic stresses. We optimized these assays at three different stages of the plant's life: seed germination and post-germination growth using agar plate-based assays, early seedling growth and development using germination pouch-based assays, and adult plant growth and development under environmentally controlled growth chambers and greenhouses. These assays will be useful for the community to perform large scale phenotyping analyses, mutant screens, comparative physiological analysis, and functional characterization of novel genes of Setaria or other related agricultural crops. Precise description of various growth conditions, effective treatment conditions and description of the resultant phenotypes will help expand the use of S. viridis as an effective model system.

The routine use of modified Borelli's lactritmel agar (MBLA).

PubMed

Kaminski, G W

1985-07-01

The original formula of Borelli's lactritmel agar (BLA)(3) which contains wheat flour, milk and honey, has been modified by replacing the wheat flour with dehydrated Bacto Corn Meal Agar (Difco) and by slightly altering the concentrations of the milk and honey. The modified medium (MBLA) is less turbid, less particulate, and easier to prepare than BLA. Although Trichophyton rubrum usually produces a wine-red pigment with BLA, most strains initially produce a yellow pigment, with the red pigment developing later. The corn meal in MBLA reduces this tendency and stimulates the early formation of deep wine red pigment, MBLA enhances sporulation of dermatophytes and various fungi which fail to sporulate on other media, and maintains characteristic growth without developing pleomorphic degeneration. It has been used routinely since 1972 as a reliable aid to the differentiation of T. rubrum and T. mentagrophytes. Since 1975 selective MBLA has been used as a routine primary isolation medium for dermatophytes, and has proved to be most useful.

Preparation and characterization of bio-nanocomposite films of agar and silver nanoparticles: laser ablation method.

PubMed

Rhim, Jong-Whan; Wang, Long-Feng; Lee, Yonghoon; Hong, Seok-In

2014-03-15

Silver nanoparticles (AgNPs) were prepared by a laser ablation method and composite films with the AgNPs and agar were prepared by solvent casting method. UV-vis absorbance test and transmission electron microscopy (TEM) analysis results revealed that non-agglomerated spherical AgNPs were formed by the laser ablation method. The surface color of the resulting agar/AgNPs films exhibited the characteristic plasmonic effect of the AgNPs with the maximum absorption peaks of 400-407 nm. X-ray diffraction (XRD) test results also exhibited characteristic AgNPs crystals with diffraction peaks observed at 2θ values of 38.39°, 44.49°, and 64.45°, which were corresponding to (111), (200), and (220) crystallographic planes of face-centered cubic (fcc) silver crystals, respectively. Thermogravimetric analysis (TGA) results showed that thermal stability of the agar/AgNPs composite films was increased by the inclusion of metallic silver. Water vapor barrier properties and surface hydrophobicity of the agar/AgNPs films increased slightly with the increase in AgNPs content but they were not statistically significant (p>0.05), while mechanical strength and stiffness of the composite films decreased slightly (p<0.05). The agar/AgNPs films exhibited distinctive antimicrobial activity against both Gram-positive (Listeria monocytogenes) and Gram-negative (Escherichia coli O157:H7) bacterial pathogens. Copyright © 2014 Elsevier Ltd. All rights reserved.

Improving agar electrospinnability with choline-based deep eutectic solvents

USDA-ARS?s Scientific Manuscript database

One percent agar (% wt) was dissolved in the deep eutectic solvent (DES), (2-hydroxyethyl) trimethylammonium chloride/urea at a 1:2 molar ratio, and successfully electrospun into nanofibers. An existing electrospinning set-up, operated at 50 deg C, was adapted for use with an ethanol bath to collect...

Enzymatic desulfation of the red seaweeds agar by Marinomonas arylsulfatase.

PubMed

Wang, Xueyan; Duan, Delin; Fu, Xiaoting

2016-12-01

Agar and sulfated galactans were isolated from the red seaweeds Gracilariopsis lemaneiformis and Gelidium amansii. A previously purified arylsulfatase from Marinomonas sp. FW-1 was used to remove sulfate groups in agar and sulfated galactans. After enzymatic desulfation, the sulfate content decreased to about 0.16% and gel strength increased about two folds. Moreover, there was no difference between the DNA electrophoresis spectrum on the gel of the arylsulfatase-treated agar and that of the commercial agarose. In order to reveal the desulfation ratio and site, chemical and structural identification of sulfated galactan were carried out. G. amansii sulfated galactan with 7.4% sulfated content was composed of galactose and 3,6-anhydro-l-galactose. Meanwhile, G. lemaneiformis sulfated galactan with 8.5% sulfated content was composed of galactose, 3,6-anhydro-l-galactose, 2-O-methyl-3,6-anhydro-l-galactose and xylose. Data from 13 C NMR, FT-IR, GC-MS provided evidence of sulfate groups at C-4 and C-6 of d-galactose and C-6 of l-galactose both in GRAP and GEAP. Data from GC-MS revealed that desulfation was carried out by the arylsulfatase at the sulfate bonds at C-4 and C-6 of d-galactose and C-6 of l-galactose, with a desulfation ratio of 83.4% and 86.0% against GEAP and GRAP, respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

Engineering rheology of electrolytes using agar for improving the performance of bioelectrochemical systems.

PubMed

Rathinam, Navanietha Krishnaraj; Tripathi, Abhilash K; Smirnova, Alevtina; Beyenal, Haluk; Sani, Rajesh K

2018-04-24

The present study is focused on enhancing the rheological properties of the electrolyte and eliminating sedimentation of microorganisms/flocs without affecting the electron transfer kinetics for improved bioelectricity generation. Agar derived from polysaccharide agarose (0.05-0.2%, w/v) was chosen as a rheology modifying agent. Electroanalytical investigations showed that electrolytes modified with 0.15% agar display a nine-fold increase in current density (1.2 mA/cm 2 ) by a thermophilic strain (Geobacillus sp. 44C, 60 °C) when compared with the control. Sodium phosphate buffer (0.1 M, pH 7) electrolyte with riboflavin (0.1 mM) was used as the control. Electrolytes modified with 0.15% agar significantly improved chemical oxygen demand removal rates. This developed electrolyte will aid in improving bioelectricity generation in Bioelectrochemical Systems (BES). The developed strategy avoids the use of peristaltic pumps and magnetic stirrers, thereby improving the energy efficiency of the process. Copyright © 2018 Elsevier Ltd. All rights reserved.

Negligible fractionation of Kr and Xe isotopes by molecular diffusion in water

NASA Astrophysics Data System (ADS)

Tyroller, Lina; Brennwald, Matthias S.; Busemann, Henner; Maden, Colin; Baur, Heinrich; Kipfer, Rolf

2018-06-01

Molecular diffusion is a key transport process for noble gases in water. Such diffusive transport is often thought to cause a mass-dependent fractionation of noble gas isotopes that is inversely proportional to the square root of the ratio of their atomic mass, referred to as the square root relation. Previous studies, challenged the commonly held assumption that the square root relation adequately describes the behaviour of noble gas isotopes diffusing through water. However, the effect of diffusion on noble gas isotopes has only been determined experimentally for He, Ne and Ar to date, whereas the extent of fractionation of Kr and Xe has not been measured. In the present study the fractionation of Kr and Xe isotopes diffusing through water immobilised by adding agar was quantified through measuring the respective isotope ratio after diffusing through the immobilised water. No fractionation of Kr and Xe isotopes was observed, even using high-precision noble gas analytics. These results complement our current understanding on isotopic fractionation of noble gases diffusing through water. Therefore this complete data set builds a robust basis to describe molecular diffusion of noble gases in water in a physical sound manner which is fundamental to assess the physical aspects of gas dynamics in aquatic systems.

Comparative studies on the conformational change and aggregation behavior of irradiated carrageenans and agar by dynamic light scattering.

PubMed

Abad, Lucille; Okabe, Satoshi; Shibayama, Mitsuhiro; Kudo, Hisaaki; Saiki, Seiichi; Aranilla, Charito; Relleve, Lorna; de la Rosa, Alumanda

2008-01-01

The conformational associative properties of kappa-, iota-, and lambda-carrageenan and agar with irradiation dose were studied by dynamic light scattering. The random scission of the carrageenans and agar by gamma irradiation resulted in the formation of polydispersed lower molecular weight fragments. At high doses, the system moves towards uniformity. Conformational change from coil to helix was observed in all carrageenans and agar at doses up to 100 kGy. The conformational change in lambda-carrageenan may be due to the irregular and hybrid structure of this polysaccharide. Only agar and lambda-carrageenan still undergo conformational transition at a high dose of 200 kGy. Gelation is observed for kappa-, iota-carrageenan up to a dose of 50 kGy while gelation is still observed at 100 kGy for agar. Increase in the hydrodynamic radius with decreasing temperatures for the non-irradiated carrageenans follows this order: lambda-carrageenan>kappa-carrageenan>iota-carrageenan. Slight increases in hydrodynamic radius were observed with irradiation.

Nutrient agar with sodium chloride supplementation for presumptive detection of Moraxella catarrhalis in clinical specimens.

PubMed

Nishiyama, Hiroyuki; Saito, Ryoichi; Chida, Toshio; Sano, Kazumitsu; Tsuchiya, Tatsuyuki; Okamura, Noboru

2012-04-01

We previously reported that Nissui nutrient agar (N medium) promoted the growth of Moraxella catarrhalis but not commensal Neisseria spp. In the present study, we examined which constituent of N medium was responsible for the selective growth of M. catarrhalis using 209 M. catarrhalis and 100 commensal Neisseria spp. clinical strains. We found that peptone, but not meat extract or agar of N medium, had growth-promoting or growth-inhibiting ability with respect to M. catarrhalis and commensal Neisseria spp. Thus, we investigated the amino acid content of N peptone and found it had higher concentrations of amino acids than other commercial peptone products. On varying the sodium chloride concentration of reconstituted N medium, we noted that the concentration was an important factor in bacterial growth differences. Varying the sodium chloride concentration of other commercial nutrient agars achieved similar results to those for N medium. This is, to our knowledge, the first study observing that sodium chloride concentration is responsible for difference in growth between the two organisms. We also successfully isolated colonies of M. catarrhalis from respiratory specimens on N medium, whereas the growth of commensal Neisseria spp. was inhibited, and by adding bovine hematin and β-NAD we were able to isolate Haemophilus influenzae colonies as efficiently as with a chocolate agar. In conclusion, nutrient agar can be used as a medium for the preferential isolation of M. catarrhalis from upper respiratory tract specimens.

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.

Production of microbial medium from defatted brebra (Milletia ferruginea) seed flour to substitute commercial peptone agar

PubMed Central

Andualem, Berhanu; Gessesse, Amare

2013-01-01

Objective To investigate and optimize microbial media that substitute peptone agar using brebra seed defatted flour. Methods 'Defatted process, inoculums preparation, evaluation of bacterial growth, preparation of cooked and hydrolyzed media and growth turbidity of tested bacteria were determined. Results Two percent defatted flour was found to be suitable concentration for the growth of pathogenic bacteria: Escherichia coli (ATCC 25922) (E. coli), Pseudomonas aeruginosa (ATCC 27853), Salmonella (NCTC 8385) and Shigella flexneri (ATCC 12022) (S. flexneri), while 3% defatted flour was suitable for Staphylococcus aureus (ATCC 25923) (S. aureus). E. coli (93±1) and S. flexneri (524±1) colony count were significantly (P≤0.05) greater in defatted flour without supplement than in supplemented medium. E. coli [(3.72×109±2) CFU/mL], S. aureus [(7.4×109±2) CFU/mL], S. flexneri [(4.03×109±2) CFU/mL] and Salmonella [(2.37×109±1) CFU/mL] in non-hydrolyzed sample were statistically (P≤0.05) greater than hydrolyzed one and commercial peptone agar. Colony count of Salmonella [(4.55×109±3) CFU/mL], S. flexneri [(5.40×109±3) CFU/mL] and Lyesria moncytogenes (ATCC 19116) [(5.4×109±3) CFU/mL] on raw defatted flour agar was significantly (P≤0.05) greater than cooked defatted flour and commercial peptone agar. Biomass of E. coli, S. aureus, Salmonella and Enterococcus faecalis in non-hydrolyzed defatted flour is highly increased over hydrolyzed defatted flour and commercial peptone broth. Conclusions The defatted flour agar was found to be better microbial media or comparable with peptone agar. The substances in it can serve as sources of carbon, nitrogen, vitamins and minerals that are essential to support the growth of microorganisms without any supplements. Currently, all supplements of peptone agar are very expensive in the market. PMID:24075344

Production of microbial medium from defatted brebra (Milletia ferruginea) seed flour to substitute commercial peptone agar.

PubMed

Andualem, Berhanu; Gessesse, Amare

2013-10-01

To investigate and optimize microbial media that substitute peptone agar using brebra seed defatted flour. Defatted process, inoculums preparation, evaluation of bacterial growth, preparation of cooked and hydrolyzed media and growth turbidity of tested bacteria were determined. Two percent defatted flour was found to be suitable concentration for the growth of pathogenic bacteria: Escherichia coli (ATCC 25922) (E. coli), Pseudomonas aeruginosa (ATCC 27853), Salmonella (NCTC 8385) and Shigella flexneri (ATCC 12022) (S. flexneri), while 3% defatted flour was suitable for Staphylococcus aureus (ATCC 25923) (S. aureus). E. coli (93±1) and S. flexneri (524±1) colony count were significantly (P≤0.05) greater in defatted flour without supplement than in supplemented medium. E. coli [(3.72×10(9)±2) CFU/mL], S. aureus [(7.4×10(9)±2) CFU/mL], S. flexneri [(4.03×10(9)±2) CFU/mL] and Salmonella [(2.37×10(9)±1) CFU/mL] in non-hydrolyzed sample were statistically (P≤0.05) greater than hydrolyzed one and commercial peptone agar. Colony count of Salmonella [(4.55×10(9)±3) CFU/mL], S. flexneri [(5.40×10(9)±3) CFU/mL] and Lyesria moncytogenes (ATCC 19116) [(5.4×10(9)±3) CFU/mL] on raw defatted flour agar was significantly (P≤0.05) greater than cooked defatted flour and commercial peptone agar. Biomass of E. coli, S. aureus, Salmonella and Enterococcus faecalis in non-hydrolyzed defatted flour is highly increased over hydrolyzed defatted flour and commercial peptone broth. The defatted flour agar was found to be better microbial media or comparable with peptone agar. The substances in it can serve as sources of carbon, nitrogen, vitamins and minerals that are essential to support the growth of microorganisms without any supplements. Currently, all supplements of peptone agar are very expensive in the market. Copyright © 2013 Asian Pacific Tropical Biomedical Magazine. Published by Elsevier B.V. All rights reserved.

Characterization and immobilization of arylsulfatase on modified magnetic nanoparticles for desulfation of agar.

PubMed

Xiao, Qiong; Yin, Qin; Ni, Hui; Cai, Huinong; Wu, Changzheng; Xiao, Anfeng

2017-01-01

Carboxyl functioned magnetic nanoparticles (CMNPs) were prepared by a simple co-precipitation method and characterized by Fourier transform infrared spedtroscopy and scanning electron microscope. The prepared CMNPs were used for covalent immobilization of the arylsulfatase which could be applied in desulfation of agar. The optimal immobilizaion conditions were obtained as follows: glutaraldehyde concentration 1.0% (v/v), cross-linking time 3h, immobilization time 3h, immobilization temperature 5°C and enzyme dose 0.62U. Increase in properties of the arylsulfatase such as optimum temperature and pH was observed after immobilization. Immobilization led to increased tolerance of enzyme to some metal ions, inhibitors and detergents. The K m and k cat of the immobilized enzyme for hydrolysis of p-NPS at pH 7.5 and at 50°C were determined to be 0.89mmol/L and 256.91s -1 , respectively. The relative desulfuration rates of immobilized arylsulfatase maintained 61.7% of its initial desulfuration rates after seven cycles. After the reaction of agar with immobilized arylsulfatase for 90min at 50°C, 46% of the sulfate in the agar was removed. These results showed that the immobilization of arylsulfatase onto CMNPs is an efficient and simple way for preparation of stable arylsulfatase and have a great potential for application in enzymatic desulfation of agar. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Antifungal susceptibility testing of Malassezia yeast: comparison of two different methodologies.

PubMed

Rojas, Florencia D; Córdoba, Susana B; de Los Ángeles Sosa, María; Zalazar, Laura C; Fernández, Mariana S; Cattana, María E; Alegre, Liliana R; Carrillo-Muñoz, Alfonso J; Giusiano, Gustavo E

2017-02-01

All Malassezia species are lipophilic; thus, modifications are required in susceptibility testing methods to ensure their growth. Antifungal susceptibility of Malassezia species using agar and broth dilution methods has been studied. Currently, few tests using disc diffusion methods are being performed. The aim was to evaluate the in vitro susceptibility of Malassezia yeast against antifungal agents using broth microdilution and disc diffusion methods, then to compare both methodologies. Fifty Malassezia isolates were studied. Microdilution method was performed as described in reference document and agar diffusion test was performed using antifungal tablets and discs. To support growth, culture media were supplemented. To correlate methods, linear regression analysis and categorical agreement was determined. The strongest linear association was observed for fluconazole and miconazole. The highest agreement between both methods was observed for itraconazole and voriconazole and the lowest for amphotericin B and fluconazole. Although modifications made to disc diffusion method allowed to obtain susceptibility data for Malassezia yeast, variables cannot be associated through a linear correlation model, indicating that inhibition zone values cannot predict MIC value. According to the results, disc diffusion assay may not represent an alternative to determine antifungal susceptibility of Malassezia yeast. © 2016 Blackwell Verlag GmbH.

Recovery of Oesophagostomum dentatum from pigs by isolation of parasites migrating from large intestinal contents embedded in agar-gel.

PubMed

Slotved, H C; Barnes, E H; Bjørn, H; Christensen, C M; Eriksen, L; Roepstorff, A; Nansen, P

1996-06-01

Four groups with three pigs in each group were inoculated with Oesophagostomum dentatum larvae (L3 larvae). Groups 1 and 3 were inoculated with 20,000 larvae, and Groups 2 and 4 with 200,000 larvae. On Days 11 and 34, respectively, Groups 1 and 2 and Groups 3 and 4 were slaughtered, and the contents from the large intestines collected. Subsamples of intestinal contents were mixed with agar to a final concentration of 1% agar and allowed to set. The worms were allowed to migrate from the agar-gel into 38 degrees C 0.9% saline overnight. Then the worms were collected on a sieve (38 microns mesh) and counted. The worms retained in the agar-gel were counted after pouring the melted agar through a sieve (38 microns mesh). The results showed that more than 95% of the worms migrated out of the agar-gel, and subsequently were available for counting in an almost clean suspension. Additionally the method yielded a high worm recovery; all stages were recovered. The recovery percentage was not significantly affected by either the dose of parasites or the time interval from slaughtering to start of incubation (37-128 min).

In vivo and in vitro control activity of plant essential oils against three strains of Aspergillus niger.

PubMed

Kumar, Peeyush; Mishra, Sapna; Kumar, Atul; Kumar, Sanjeev; Prasad, Chandra Shekhar

2017-09-01

Contamination of environment and food from the prevalent spores and mycotoxins of Aspergillus niger has led to several diseases in humans and other animals. The present study investigated the control activity of plant essential oils against three strains of A. niger. In the elaborate assays done through microdilution plate assay and agar disk diffusion assay in the lab condition and in vivo assay on the stored wheat grains, the essential oil of Thymus vulgaris depicted overall superior efficacy. In microdilution plate assay, the oil of Anethum graveolens showed best fungistatic activity, while best fungicidal activity was depicted by Syzygium aromaticum oil. The oil of T. vulgaris showed moderate control efficacy against A. niger strains with its antifungal activity resulting mainly due to killing of microorganism rather than growth inhibition. In agar disk diffusion assay, T. vulgaris oil with a zone of inhibition (ZOI) of 23.3-61.1% was the most effective fungicide. The in vivo assay to evaluate the protection efficacy of oils for stored wheat grains against A. niger (AN1) revealed T. vulgaris (90.5-100%) to be the best control agent, followed by the oil of S. aromaticum (61.9-100%). The GC-MS analysis of T. vulgaris oil indicated the presence of thymol (39.11%), γ-terpinene (19.73%), o-cymene (17.21%), and β-pinene (5.38%) as major oil components. Phytotoxic effects of the oils on wheat seeds showed no significant phytotoxic effect of oils in terms of seed germination or seedling growth. The results of the study demonstrated control potentiality of essential oils for the protection of stored wheat against A. niger with prospect for development of eco-friendly antifungal products.

Using the overlay assay to qualitatively measure bacterial production of and sensitivity to pneumococcal bacteriocins.

PubMed

Maricic, Natalie; Dawid, Suzanne

2014-09-30

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Preparation of bioactive neoagaroligosaccharides through hydrolysis of Gracilaria lemaneiformis agar: A comparative study.

PubMed

Xu, Xin-Qi; Su, Bing-Mei; Xie, Jin-Sheng; Li, Ren-Kuan; Yang, Jie; Lin, Juan; Ye, Xiu-Yun

2018-02-01

Hydrolysis of Gracilaria lemaneiformis agar by β-agarase was compared with HCl hydrolysis. The results showed that optimum catalysis conditions for the β-agarase were pH 7.0 at 45°C. Mass spectroscopy, thin-layer chromatography and GPC results showed that the polymerization degrees of the hydrolysis products by the β-agarase were mainly four, six and eight (more specific than the hydrolysate by HCl). The enzymatic degradation products of agar were distinctly different from those of HCl hydrolysis in the ratios among galactose and 3,6-anhydro-galactose and sulfate group contents. The NMR spectrometry proved that the products of β-agarase were neoagaroligosaccharides, which was not found in the agarolytic products by HCl. The neoagarotetraose inhibited tyrosinase activity competitively with the K I value of 16.0mg/ml. Hydroxyl radical-scavenging ability of neoagaroligosaccharides was much greater than that of agar HCl hydrolysate. This work suggests that neoagaroligosaccharide products produced by our β-agarase could be more effective in function than products from acid hydrolysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

A microfluidics-based turning assay reveals complex growth cone responses to integrated gradients of substrate-bound ECM molecules and diffusible guidance cues.

PubMed

Joanne Wang, C; Li, Xiong; Lin, Benjamin; Shim, Sangwoo; Ming, Guo-Li; Levchenko, Andre

2008-02-01

Neuronal growth cones contain sophisticated molecular machinery precisely regulating their migration in response to complex combinatorial gradients of diverse external cues. The details of this regulation are still largely unknown, in part due to limitations of the currently available experimental techniques. Microfluidic devices have been shown to be capable of generating complex, stable and precisely controlled chemical gradients, but their use in studying growth cone migration has been limited in part due to the effects of shear stress. Here we describe a microfluidics-based turning-assay chip designed to overcome this issue. In addition to generating precise gradients of soluble guidance cues, the chip can also fabricate complex composite gradients of diffusible and surface-bound guidance cues that mimic the conditions the growth cones realistically counter in vivo. Applying this assay to Xenopus embryonic spinal neurons, we demonstrate that the presence of a surface-bound laminin gradient can finely tune the polarity of growth cone responses (repulsion or attraction) to gradients of brain-derived neurotrophic factor (BDNF), with the guidance outcome dependent on the mean BDNF concentration. The flexibility inherent in this assay holds significant potential for refinement of our understanding of nervous system development and regeneration, and can be extended to elucidate other cellular processes involving chemotaxis of shear sensitive cells.

Optimization of the Agar-gel Method for Isolation of Migrating Ascaris suum Larvae From the Liver and Lungs of Pigs

PubMed Central

Saeed, I; Roepstorff, A; Rasmussen, T; Høg, M; Jungersen, G

2001-01-01

Experiments on use of an agar-gel method for recovery of migrating Ascaris suum larvae from the liver and lungs of pigs were conducted to obtain fast standardized methods. Subsamples of blended tissues of pig liver and lungs were mixed with agar to a final concentration of 1% agar and the larvae allowed to migrate out of the agar-gel into 0.9% NaCl at 38°C. The results showed that within 3 h more than 88% of the recoverable larvae migrated out of the liver agar-gel and more than 83% of the obtained larvae migrated out of the lung agar-gel. The larvae were subsequently available in a very clean suspension which reduced the sample counting time. Blending the liver for 60 sec in a commercial blender showed significantly higher larvae recovery than blending for 30 sec. Addition of gentamycin to reduce bacterial growth during incubation, glucose to increase larval motility during migration or ice to increase sedimentation of migrated larvae did not influence larvae recovery significantly. PMID:11503373

[Rapid bioluminescent antibiotic susceptibility assay].

PubMed

Frundzhian, V G; Ugarova, N N; Blatun, L A; Terekhova, R P; Rusanova, E V

2009-01-01

Rapid testing of pathogen susceptibility to antibiotics is of great practical value for rational chemotherapy of pyoinflammatory deseases and postoperative complications of microbial etiology. The standard microbiological methods, i.e., the disk diffusion method and the method of serial dilutions are labour- and time-consuming (not less than 18-36 hours). The method of the authors is based on measuring bioluminescence resulting from interaction of adenosine-5'-triphosphate (ATP) and ATP reagent, a standard reaction mixture of firefly luciferase (an enzyme) and luciferin. The bioluminescence intensity is proportional to the ATP concentration in the reaction mixture and the ATP concentration is proportional to the number of the pathogen viable cells in the sample. The bioluminescence intensity value in the pathogen suspension aliquots with and without (control) the antibiotic were compared after the incubation for 5 hours and the coefficient of the microbial cell growth inhibition was calculated. Satisfactory correlation (R2 > 88%) of the results of the bioluminescent assay and the assay with the disk diffusion method and the method of serial dilutions was observed.

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.

Broad-range (pan) Salmonella and Salmonella serotype typhi-specific real-time PCR assays: potential tools for the clinical microbiologist.

PubMed

Farrell, John J; Doyle, Laura J; Addison, Rachel M; Reller, L Barth; Hall, Geraldine S; Procop, Gary W

2005-03-01

We describe broad-range salmonellae (ie, Salmonella) and Salmonella serotype Typhi-specific LightCycler (Roche Diagnostics, Indianapolis, IN) real-time polymerase chain reaction assays. We validated these with a battery of 280 bacteria, 108 of which were salmonellae representing 20 serotypes. In addition, 298 isolates from 170 clinical specimens that were suspected to possibly represent Salmonella were tested with the pan- Salmonella assay. Finally, the pan-Salmonella assay also was used to test DNA extracts from 101 archived, frozen stool specimens, 55 of which were culture-positive for salmonellae. Both assays were 100% sensitive and specific when cultured isolates of the battery were tested. The pan- Salmonella assay also characterized correctly all salmonellae on the primary isolation agar and was 96% sensitive (53/55) and 96% specific (49/51) when nucleic acid extracts from direct stool specimens were tested. These assays represent potential tools the clinical microbiologist could use to screen suspect isolates or stool specimens for Salmonella.

Intelligent pH indicator film composed of agar/potato starch and anthocyanin extracts from purple sweet potato.

PubMed

Choi, Inyoung; Lee, Jun Young; Lacroix, Monique; Han, Jaejoon

2017-03-01

A new colorimetric pH indicator film was developed using agar, potato starch, and natural dyes extracted from purple sweet potato, Ipomoea batatas. Both agar and potato starch are solid matrices used to immobilize natural dyes, anthocyanins. The ultraviolet-visible (UV-vis) spectrum of anthocyanin extract solutions and agar/potato starch films with anthocyanins showed color variations to different pH values (pH 2.0-10.0). Fourier transform infrared (FT-IR) and UV-vis region spectra showed compatibility between agar, starch, and anthocyanin extracts. Color variations of pH indicator films were measured by a colorimeter after immersion in different pH buffers. An application test was conducted for potential use as a meat spoilage sensor. The pH indicator films showed pH changes and spoilage point of pork samples, changing from red to green. Therefore, the developed pH indicator films could be used as a diagnostic tool for the detection of food spoilage. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

Use of an agar-gel technique for large scale application to recover Ascaris suum larvae from intestinal contents of pigs.

PubMed

Slotved, H C; Barnes, E H; Eriksen, L; Roepstorff, A; Nansen, P; Bjørn, H

1997-01-01

Four groups each of 3 pigs were inoculated with Ascaris suum eggs. Pigs in groups 1 and 3 were inoculated with 1000 eggs, and pigs in groups 2 and 4 with 10,000 eggs. On day 10 and 21 post-inoculation (p.i.), respectively, groups 1 + 2 and 3 + 4 were slaughtered, and the contents from the small intestines collected. The contents were mixed with agar to a final concentration of 1% agar and allowed to sediment. The larvae were allowed to migrate from the agar-gel into 38 degrees C 0.9% saline overnight, and were then collected on a sieve (20 microns mesh) and counted. The larvae retained in the agar-gel were counted after pouring the melted agar through a sieve (20 microns mesh). The results showed that more than 97% of the larvae migrated out of the agar-gel and were available for counting in an almost clean suspension. The inoculation dose level did not significantly affect the recovery percentage, neither did the larval stage (10 or 21 days old larvae). The variation in the time interval from slaughtering to start of incubation (interval 57-155 min) did not significantly affect the recovery percentage.

Comparison of the Cellient(™) automated cell block system and agar cell block method.

PubMed

Kruger, A M; Stevens, M W; Kerley, K J; Carter, C D

2014-12-01

To compare the Cellient(TM) automated cell block system with the agar cell block method in terms of quantity and quality of diagnostic material and morphological, histochemical and immunocytochemical features. Cell blocks were prepared from 100 effusion samples using the agar method and Cellient system, and routinely sectioned and stained for haematoxylin and eosin and periodic acid-Schiff with diastase (PASD). A preliminary immunocytochemical study was performed on selected cases (27/100 cases). Sections were evaluated using a three-point grading system to compare a set of morphological parameters. Statistical analysis was performed using Fisher's exact test. Parameters assessing cellularity, presence of single cells and definition of nuclear membrane, nucleoli, chromatin and cytoplasm showed a statistically significant improvement on Cellient cell blocks compared with agar cell blocks (P < 0.05). No significant difference was seen for definition of cell groups, PASD staining or the intensity or clarity of immunocytochemical staining. A discrepant immunocytochemistry (ICC) result was seen in 21% (13/63) of immunostains. The Cellient technique is comparable with the agar method, with statistically significant results achieved for important morphological features. It demonstrates potential as an alternative cell block preparation method which is relevant for the rapid processing of fine needle aspiration samples, malignant effusions and low-cellularity specimens, where optimal cell morphology and architecture are essential. Further investigation is required to optimize immunocytochemical staining using the Cellient method. © 2014 John Wiley & Sons Ltd.

Anti-Quorum Sensing Potential of Crude Kigelia africana Fruit Extracts

PubMed Central

Chenia, Hafizah Y.

2013-01-01

The increasing incidence of multidrug-resistant pathogens has stimulated the search for novel anti-virulence compounds. Although many phytochemicals show promising antimicrobial activity, their power lies in their anti-virulence properties. Thus the quorum sensing (QS) inhibitory activity of four crude Kigelia africana fruit extracts was assessed qualitatively and quantitatively using the Chromobacterium violaceum and Agrobacterium tumefaciens biosensor systems. Inhibition of QS-controlled violacein production in C. violaceum was assayed using the qualitative agar diffusion assay as well as by quantifying violacein inhibition using K. africana extracts ranging from 0.31–8.2 mg/mL. Qualitative modulation of QS activity was investigated using the agar diffusion double ring assay. All four extracts showed varying levels of anti-QS activity with zones of violacein inhibition ranging from 9–10 mm. The effect on violacein inhibition was significant in the following order: hexane > dichloromethane > ethyl acetate > methanol. Inhibition was concentration-dependent, with the ≥90% inhibition being obtained with ≥1.3 mg/mL of the hexane extract. Both LuxI and LuxR activity were affected by crude extracts suggesting that the phytochemicals target both QS signal and receptor. K. africana extracts with their anti-QS activity, have the potential to be novel therapeutic agents, which might be important in reducing virulence and pathogenicity of drug-resistant bacteria in vivo. PMID:23447012

Carbohydrate hydrogels with stabilized phage particles for bacterial biosensing: bacterium diffusion studies.

PubMed

Balcão, Victor M; Barreira, Sérgio V P; Nunes, Thiago M; Chaud, Marco V; Tubino, Matthieu; Vila, Marta M D C

2014-02-01

Bacteriophage particles have been reported as potentially useful in the development of diagnosis tools for pathogenic bacteria as they specifically recognize and lyse bacterial isolates thus confirming the presence of viable cells. One of the most representative microorganisms associated with health care services is the bacterium Pseudomonas aeruginosa, which alone is responsible for nearly 15% of all nosocomial infections. In this context, structural and functional stabilization of phage particles within biopolymeric hydrogels, aiming at producing cheap (chromogenic) bacterial biosensing devices, has been the goal of a previous research effort. For this, a detailed knowledge of the bacterial diffusion profile into the hydrogel core, where the phage particles lie, is of utmost importance. In the present research effort, the bacterial diffusion process into the biopolymeric hydrogel core was mathematically described and the theoretical simulations duly compared with experimental results, allowing determination of the effective diffusion coefficients of P. aeruginosa in the agar and calcium alginate hydrogels tested.

EFFECT OF IMPACT STRESS ON MICROBIAL RECOVERY ON AN AGAR SURFACE

EPA Science Inventory

Microbial stress due to the impaction of microorganisms onto an agar collection surface was studied experimentally. he relative recovery rates of aerosolized Pseudomonas fluorescens and Micrococcus luteus were determined as a function of the impaction velocity by using a moving a...

Development of a real-time TaqMan assay to detect mendocina sublineage Pseudomonas species in contaminated metalworking fluids.

PubMed

Saha, Ratul; Donofrio, Robert S; Bagley, Susan T

2010-08-01

A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but are also time-consuming. The primer-probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 10(1) colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer-probe pair was successful in determining concentrations from used MWF samples, indicating levels between 2.9 x 10(3) and 3.9 x 10(6) CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 x 10(1) to 1.4 x 10(5) CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer-probe pair can be efficiently used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs.

Efficacy of the thin agar layer method for the recovery of stressed Cronobacter spp. (Enterobacter sakazakii).

PubMed

Osaili, Tareq M; Al-Nabulsi, Anas A; Shaker, Reyad R; Al-Holy, Murad M; Al-Haddaq, Mohammed S; Olaimat, Amin N; Ayyash, Mutamed M; Al Ta'ani, Mahmoud K; Forsythe, Stephen J

2010-10-01

Cronobacter spp. (Enterobacter sakazakii) are emerging opportunistic pathogens for all age groups, and are of particular concern when it comes to infants. Prior to contaminating food, the organism may be exposed to a variety of stresses, leading to a generation of sublethally injured cells that may not be detected by selective media unless a protracted recovery period is included in the isolation procedure. This study evaluated the efficacy of the thin agar layer (TAL) method for the recovery of Cronobacter cells that had been exposed to various stress conditions. Five strains of C. sakazakii and C. muytjensii were exposed to starvation, heat, cold, acid, alkaline, chlorine, or ethanol, with or without further exposure to desiccation stress. The recovery of the stressed cells was determined on tryptone soy agar (TSA; nonselective control medium), violet red bile glucose agar (VRBGA; selective agar), Druggan-Forsythe-Iversen (DFI; selective agar), and TAL media (viz., VRBGA overlaid with TSA, and DFI overlaid with TSA). Regardless of stress type, there were no significant differences among the recoveries of stressed desiccated Cronobacter spp. cultures on TSA, DFI+TSA, and VRBGA+TSA, but there was significantly less recovery on VRBGA. The recovery of prestressed desiccated Cronobacter spp. on DFI+TSA was similar to that on TSA, whereas the recovery on VRBGA+TSA was lower. DFI+TSA performed better than VRBGA+TSA did in differentiating Cronobacter spp. within mixed bacterial cultures. The results of this study suggest the use of the TAL method DFI+TSA as an improved method for the direct recovery of stressed Cronobacter spp.

Agar Sediment Test for Assessing the Suitability of Organic Waste Streams for Recovering Nutrients by the Aquatic Worm Lumbriculus variegatus.

PubMed

Laarhoven, Bob; Elissen, H J H; Temmink, H; Buisman, C J N

2016-01-01

An agar sediment test was developed to evaluate the suitability of organic waste streams from the food industry for recovering nutrients by the aquatic worm Lumbriculus variegatus (Lv). The effects of agar gel, sand, and food quantities in the sediment test on worm growth, reproduction, and water quality were studied. Agar gel addition ameliorated growth conditions by reducing food hydrolysis and altering sediment structure. Best results for combined reproduction and growth were obtained with 0.6% agar-gel (20 ml), 10 g. fine sand, 40 g. coarse sand, and 105 mg fish food (Tetramin). With agar gel, ingestion and growth is more the result of addition of food in its original quality. Final tests with secondary potato starch sludge and wheat bran demonstrated that this test is appropriate for the comparison of solid feedstuffs and suspended organic waste streams. This test method is expected to be suitable for organic waste studies using other sediment dwelling invertebrates.

Agar Sediment Test for Assessing the Suitability of Organic Waste Streams for Recovering Nutrients by the Aquatic Worm Lumbriculus variegatus

PubMed Central

Laarhoven, Bob; Elissen, H. J. H.; Temmink, H.; Buisman, C. J. N.

2016-01-01

An agar sediment test was developed to evaluate the suitability of organic waste streams from the food industry for recovering nutrients by the aquatic worm Lumbriculus variegatus (Lv). The effects of agar gel, sand, and food quantities in the sediment test on worm growth, reproduction, and water quality were studied. Agar gel addition ameliorated growth conditions by reducing food hydrolysis and altering sediment structure. Best results for combined reproduction and growth were obtained with 0.6% agar-gel (20 ml), 10 g. fine sand, 40 g. coarse sand, and 105 mg fish food (Tetramin). With agar gel, ingestion and growth is more the result of addition of food in its original quality. Final tests with secondary potato starch sludge and wheat bran demonstrated that this test is appropriate for the comparison of solid feedstuffs and suspended organic waste streams. This test method is expected to be suitable for organic waste studies using other sediment dwelling invertebrates. PMID:26937632

A novel bicomponent hemolysin from Bacillus cereus.

PubMed Central

Beecher, D J; MacMillan, J D

1990-01-01

A procedure combining isoelectric focusing (Sephadex IEF) and fast protein liquid chromatography (Superose 12; Mono-Q) removed hemolytic activity (presumably a contaminant) from partially purified preparations of the multicomponent diarrheal enterotoxin produced by Bacillus cereus. However, when the separated fractions were recombined, hemolytic activity was restored, suggesting that hemolysis is a property of the enterotoxin components. Combined fractions exhibited a unique ring pattern in gel diffusion assays in blood agar. During diffusion of the hemolysin from an agar well, the erythrocytes closest to the well were not lysed initially. After diffusion, hemolysis was observed as a sharp ring beginning several millimeters away from the edge of the well. With time the cells closer to the well were also lysed. This novel hemolysin consists of a protein (component B) which binds to or alters cells, allowing subsequent lysis by a second protein (component L). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and Western blot analysis showed that hemolysin BL has properties similar to those described previously for the enterotoxin and that both components are distinct from cereolysin and cereolysin AB. Images PMID:2114359

Qualitative and Quantitative Assays of Transposition and Homologous Recombination of the Retrotransposon Tf1 in Schizosaccharomyces pombe.

PubMed

Sangesland, Maya; Atwood-Moore, Angela; Rai, Sudhir K; Levin, Henry L

2016-01-01

Transposition and homologous recombination assays are valuable genetic tools to measure the production and integration of cDNA from the long terminal repeat (LTR) retrotransposon Tf1 in the fission yeast (Schizosaccharomyces pombe). Here we describe two genetic assays, one that measures the transposition activity of Tf1 by monitoring the mobility of a drug resistance marked Tf1 element expressed from a multi-copy plasmid and another assay that measures homologous recombination between Tf1 cDNA and the expression plasmid. While the transposition assay measures insertion of full-length Tf1 cDNA mediated by the transposon integrase, the homologous recombination assay measures levels of cDNA present in the nucleus and is independent of integrase activity. Combined, these assays can be used to systematically screen large collections of strains to identify mutations that specifically inhibit the integration step in the retroelement life cycle. Such mutations can be identified because they reduce transposition activity but nevertheless have wild-type frequencies of homologous recombination. Qualitative assays of yeast patches on agar plates detect large defects in integration and recombination, while the quantitative approach provides a precise method of determining integration and recombination frequencies.

Properties and characterization of agar/CuNP bionanocomposite films prepared with different copper salts and reducing agents.

PubMed

Shankar, Shiv; Teng, Xinnan; Rhim, Jong-Whan

2014-12-19

Various types of agar-based bio-nanocomposite (BNC) films were prepared by blending agar and six different copper nanoparticles (CuNPs) with different shapes and sizes obtained from three different sources of copper salts and two different reducing agents. The BNC films were characterized by UV-visible, FE-SEM, FT-IR, and XRD. The thermogravimetric study showed that the melting point of BNC films was increased when ascorbic acid was used as a reducing agent for CuNPs synthesis. Apparent surface color and transmittance of agar film was greatly influenced by the reinforcement of CuNPs. However, mechanical and water vapor barrier properties did not change significantly (p>0.05) by blending with CuNPs. Tensile modulus and tensile strength decreased slightly for all types of CuNPs reinforced while elongation at break slightly increased when CuNPs produced by ascorbic acid were blended. The agar bio-nanocomposite films showed profound antibacterial activity against both Gram-positive and Gram-negative food-borne pathogenic bacteria. Copyright © 2014 Elsevier Ltd. All rights reserved.

Rapid diagnosis of acanthamoeba keratitis using non-nutrient agar with a lawn of E. coli.

PubMed

Borin, Samuel; Feldman, Ilan; Ken-Dror, Shifra; Briscoe, Daniel

2013-02-27

A patient presented with a corneal foreign body in his only eye. He was treated with prophylactic antibiotics and sent home, but deteriorated. He returned to the hospital 5 days later, and on slit-lamp examination, there was ciliary injection, corneal oedema and a 1 mm × 1 mm corneal abscess with mild anterior uveitis. Corneal scrapings were taken for culture on a non-nutrient agar with a lawn of Escherichia coli, on chocolate agar and on blood agar. He was treated with fortified gentamicin and cefazolin drops. He improved and was discharged 4 days after admission. On day 5, the culture results showed acanthamoeba. He was brought back to the hospital and treated with hourly chlorhexidine drops, ofloxacin six times daily and neomycin/dexamethasone drops once daily. On day 7, he was discharged to continue treatment at home, at which time his visual acuity in that eye was 6/9, and slit-lamp examination showed punctate keratitis and a stromal opacity with mild peripheral infiltration. Culture on non-nutrient agar with a lawn of E. coli is a rapid, reliable and less invasive alternative to corneal biopsy for the diagnosis of acanthamoeba infection. We suggest using this method where acanthamoeba is suspected. Owing to the risk of corneal abscess, orthokeratology should be avoided in an amblyopic patient or an only eye. Acanthamoeba infection may be masked by other eye diseases.

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.

Comparison of Six Chromogenic Agar Media for the Isolation of a Broad Variety of Non-O157 Shigatoxin-Producing Escherichia coli (STEC) Serogroups

PubMed Central

Verhaegen, Bavo; De Reu, Koen; Heyndrickx, Marc; De Zutter, Lieven

2015-01-01

The isolation of non-O157 STEC from food samples has proved to be challenging. The selection of a suitable selective isolation agar remains problematic. The purpose of this study was to qualitatively and quantitatively evaluate six chromogenic agar media for the isolation of STEC: Tryptone Bile X-glucuronide agar (TBX), Rainbow® Agar O157 (RB), Rapid E. coli O157:H7 (RE), Modified MacConkey Agar (mMac), CHROMagarTM STEC (Chr ST) and chromIDTM EHEC (Chr ID). During this study, 45 E. coli strains were used, including 39 STEC strains belonging to 16 different O serogroups and 6 non-STEC E. coli. All E. coli strains were able to grow on TBX and RB, whereas one STEC strain was unable to grow on Chr ID and a number of other STEC strains did not grow on mMac, CHROMagar STEC and Rapid E. coli O157:H7. However, only the latter three agars were selective enough to completely inhibit the growth of the non-STEC E. coli. Our conclusion was that paired use of a more selective agar such as CHROMagar STEC together with a less selective agar like TBX or Chr ID might be the best solution for isolating non-O157 STEC from food. PMID:26090610

Comparison of Six Chromogenic Agar Media for the Isolation of a Broad Variety of Non-O157 Shigatoxin-Producing Escherichia coli (STEC) Serogroups.

PubMed

Verhaegen, Bavo; De Reu, Koen; Heyndrickx, Marc; De Zutter, Lieven

2015-06-17

The isolation of non-O157 STEC from food samples has proved to be challenging. The selection of a suitable selective isolation agar remains problematic. The purpose of this study was to qualitatively and quantitatively evaluate six chromogenic agar media for the isolation of STEC: Tryptone Bile X-glucuronide agar (TBX), Rainbow® Agar O157 (RB), Rapid E. coli O157:H7 (RE), Modified MacConkey Agar (mMac), CHROMagarTM STEC (Chr ST) and chromIDTM EHEC (Chr ID). During this study, 45 E. coli strains were used, including 39 STEC strains belonging to 16 different O serogroups and 6 non-STEC E. coli. All E. coli strains were able to grow on TBX and RB, whereas one STEC strain was unable to grow on Chr ID and a number of other STEC strains did not grow on mMac, CHROMagar STEC and Rapid E. coli O157:H7. However, only the latter three agars were selective enough to completely inhibit the growth of the non-STEC E. coli. Our conclusion was that paired use of a more selective agar such as CHROMagar STEC together with a less selective agar like TBX or Chr ID might be the best solution for isolating non-O157 STEC from food.

Spore-to-spore agar culture of the myxomycete Physarum globuliferum.

PubMed

Liu, Pu; Wang, Qi; Li, Yu

2010-02-01

The ontogeny of the myxomycete Physarum globuliferum was observed on corn meal agar and hanging drop cultures without adding sterile oat flakes, bacteria or other microorganisms. Its complete life cycle including spore germination, myxamoebae, swarm cells, plasmodial development, and maturity of fructifications was demonstrated. Details of spore-to-spore development are described and illustrated.

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

Simple genetic transformation assay for rapid diagnosis of Moraxella osloensis.

PubMed

Juni, E

1974-01-01

A genetic transformation assay for unequivocal identification of strains of Moraxella osloensis is described. In this assay a stable tryptophan auxotroph is transformed to prototrophy by deoxyribonucleic acid (DNA) samples from other strains of M. osloensis but not by DNA samples from unrelated bacteria. The test is simple to perform and definitive results can be obtained in less than 24 h. The procedure, which is suitable for routine diagnosis in a clinical laboratory, involves a rapid method for preparation of crude transforming DNA from small quantities of bacterial cells and permits simultaneous examination of large numbers of isolated cultures. The assay was shown to correctly identify 27 strains previously classified as M. osloensis. Forty-five other gram-negative, oxidase-positive, nonmotile coccobacilli, which might be confused with M. osloensis unless subject to more extensive testing, were shown to be unrelated genetically to M. osloensis. The transformation assay clearly distinguishes M. osloensis from Acinetobacter. Although most strains of M. osloensis are nonfastidious, being able to grow in a mineral medium supplemented with a single organic carbon source, one of the strains tested was only able to grow on fairly complex media and could not be transformed to grow on simple media. Inability to alkalize Simmons citrate agar was shown not to be characteristic of all strains of M. osloensis.

Simple Genetic Transformation Assay for Rapid Diagnosis of Moraxella osloensis

PubMed Central

Juni, Elliot

1974-01-01

A genetic transformation assay for unequivocal identification of strains of Moraxella osloensis is described. In this assay a stable tryptophan auxotroph is transformed to prototrophy by deoxyribonucleic acid (DNA) samples from other strains of M. osloensis but not by DNA samples from unrelated bacteria. The test is simple to perform and definitive results can be obtained in less than 24 h. The procedure, which is suitable for routine diagnosis in a clinical laboratory, involves a rapid method for preparation of crude transforming DNA from small quantities of bacterial cells and permits simultaneous examination of large numbers of isolated cultures. The assay was shown to correctly identify 27 strains previously classified as M. osloensis. Forty-five other gram-negative, oxidase-positive, nonmotile coccobacilli, which might be confused with M. osloensis unless subject to more extensive testing, were shown to be unrelated genetically to M. osloensis. The transformation assay clearly distinguishes M. osloensis from Acinetobacter. Although most strains of M. osloensis are nonfastidious, being able to grow in a mineral medium supplemented with a single organic carbon source, one of the strains tested was only able to grow on fairly complex media and could not be transformed to grow on simple media. Inability to alkalize Simmons citrate agar was shown not to be characteristic of all strains of M. osloensis. Images PMID:4589126

Effect of time on migration of Oesophagostomum spp. and Hyostrongylus rubidus out of agar-gel.

PubMed

Nosal, P; Christensen, C M; Nansen, P

1998-01-01

The agar-gel migration technique has previously been described, however, aspects regarding the effect of timing on worm migration needed further scrutiny. In the first experiment, pigs inoculated with Oesophagostomum dentatum were slaughtered simultaneously and their intestines stored at 21-23 degrees C until processed pairwise 2, 4, 6, 8, 12 and 18 h after slaughter. More than 95% of the worms migrated out of the agar if processed within 6 h. In the second experiment, intestines were treated immediately after slaughter and the migratory speed of adult worms or 4th-stage larvae of O. dentatum or O. quadrispinulatum, or adult Hyostrongylus rubidus were studied. For both Oesophagostomum species, more than 90% of the worms were recovered within 1 h. H. rubidus was significantly slower; however, approximately 98% of the worms had migrated out of the agar-gel by 20 h. This information is essential in planning experiments where recovery of live worms is of value.

[Presumptive identification of Candida spp. and other clinically important yeasts: usefulness of Brilliance Candida Agar].

PubMed

Alfonso, Claudia; López, Mónica; Arechavala, Alicia; Perrone, María Del Carmen; Guelfand, Liliana; Bianchi, Mario

2010-06-30

Fungal infections caused by yeasts have increased during the last decades and invasive forms represent a serious problem for human health. Candida albicans is the species most frequently isolated from clinical samples. However, other emerging yeast pathogens are increasingly responsible for mycotic infections, and some of them are resistant to some antifungal drugs. Consequently, it is necessary to have methods that can provide a rapid presumptive identification at species level. Numerous chromogenic agar media have been shown to be of value as diagnostic tools. We have compared a chromogenic medium, Brilliance Candida Agar, with CHROMagar Candida, the chromogenic medium most used in our country. A multicentre study was conducted in 16 Hospitals belonging to the Mycology Net of Buenos Aires City Government. A total of 240 yeast isolates were included in this research. The new chromogenic agar showed results very similar to those obtained with CHROMagar Candida. Copyright 2009 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

Extracellular enzyme production and cheating in Pseudomonas fluorescens depend on diffusion rates

PubMed Central

Allison, Steven D.; Lu, Lucy; Kent, Alyssa G.; Martiny, Adam C.

2014-01-01

Bacteria produce extracellular enzymes to obtain resources from complex chemical substrates, but this strategy is vulnerable to cheating by cells that take up reaction products without paying the cost of enzyme production. We hypothesized that cheating would suppress enzyme production in co-cultures of cheater and producer bacteria, particularly under well-mixed conditions. To test this hypothesis, we monitored protease expression and frequencies of Pseudomonas fluorescens producer and cheater genotypes over time in mixed liquid cultures and on agar plates. In mixed culture inoculated with equal frequencies of cheaters and producers, enzyme concentration declined to zero after 20 days, consistent with our hypothesis. We observed a similar decline in cultures inoculated with producers only, suggesting that cheater mutants arose de novo and swept the population. DNA sequencing showed that genetic changes most likely occurred outside the protease operon. In one experimental replicate, the population regained the ability to produce protease, likely due to further genetic changes or population dynamics. Under spatially structured conditions on agar plates, cheaters did not sweep the population. Instead, we observed a significant increase in the variation of enzyme activity levels expressed by clones isolated from the population. Together these results suggest that restricted diffusion favors a diversity of enzyme production strategies. In contrast, well-mixed conditions favor population sweeps by cheater strains, consistent with theoretical predictions. Cheater and producer strategies likely coexist in natural environments with the frequency of cheating increasing with diffusion rate. PMID:24782855

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

Detection of Salmonella by indicator agar media and PCR as affected by alfalfa seed homogenates and native bacteria.

PubMed

Liao, C-H; Shollenberger, L M

2003-01-01

To investigate and prevent the undesirable effect of native bacteria and alfalfa seed homogenates on detection of Salmonella in alfalfa seeds by indicator agar media and polymerase chain reaction (PCR). The relative sensitivity of five indicator agar media, including modified semisolid RV (MSRV), xylose-lysine-Tergitol 4 (XLT4), Hektoen enteric agar (HEA), brilliant green agar (BGA) and bismuth sulphite agar (BSA), for detection of Salmonella in the presence of a large number of native bacteria from alfalfa seeds was examined. The detection limit as measured by the ratio between the numbers of native bacteria and Salmonella was estimated to be 10(6) to 1 for MSRV and 10(3) to 1 for XLT4, HEA, BGA or BSA. Presence of alfalfa seed homogenates markedly reduced the sensitivity of Salmonella detection by PCR. The minimal number of Salmonella detectable by PCR was determined to be 1-10 and 100-1000 CFU in the absence and presence of seed homogenate, respectively. Application of anti-Salmonella immunomagnetic beads permitted detection of 2-5 CFU of heat-injured cells in 25 g of seeds within 24 h by PCR. The MSRV medium is more sensitive than other indicator agars for detecting a small number of motile Salmonella in samples containing a large number of native bacteria. Application of immunomagnetic beads eliminates the PCR-inhibitory activity of seed homogenates and improves the detection of Salmonella in inoculated seeds. The results generated from this study will aid the seed distributors, sprout growers and public health officials to identify and recall the Salmonella-contaminated seed lots to be used for sprout production.

Comparison of different agar diffusion methods for the detection of residues in the kidneys of pigs treated with antimicrobial drugs.

PubMed

Korkeala, H; Sorvettula, O; Mäki-Petäys, O; Hirn, J

1983-01-01

Residue analyses of the kidneys of twenty-six pigs treated with various antimicrobial drugs 20 h before slaughter and of eleven untreated pigs were performed. The effects of storage temperature of the kidneys, and of sampling location, on the residue analysis were also studied. No method alone was sufficient for the detection of residues. Oxytetracycline residues could be detected at pH 6, dihydrostreptomycin residues at pH 8, and sulphonamide residues if trimethoprim was present in the medium. Chloramphenicol, penicillin G procaine, tylosin and lincomycin residues were not detectable with the methods used. The concentration of ampicillin decreased during the storage of samples at +4°C. Most methods also yielded zones of inhibition for the frozen kidneys from untreated pigs. It seems necessary to use agar media of two different pH values: the addition of trimethoprim to the medium is also needed. The use of fresh pig kidneys, and samples containing both kidney medulla and kidney cortex, is recommended in residue analysis. Copyright © 1983. Published by Elsevier Ltd.

Agar Block Smear Preparation: a Novel Method of Slide Preparation for Preservation of Native Fungal Structures for Microscopic Examination and Long-Term Storage▿

PubMed Central

Woo, Patrick C. Y.; Ngan, Antonio H. Y.; Chui, Hon-Kit; Lau, Susanna K. P.; Yuen, Kwok-Yung

2010-01-01

We describe a novel method of fungal slide preparation named “agar block smear preparation.” A total of 510 agar block smears of 25 fungal strains obtained from culture collections, 90 QC fungal strains, and 82 clinical fungal strains from our clinical microbiology laboratory, which included a total of 137 species of yeasts, molds, and thermal dimorphic fungi, were prepared and examined. In contrast to adhesive tape preparation, agar block smears preserved the native fungal structures, such as intact conidiophores of Aspergillus species and arrangements of conidia in Scopulariopsis brevicaulis. Furthermore, agar block smears allowed examination of fungal structures embedded in the agar, such as the ascomata with ascomal hairs in Chaetomium funicola; pycnidium of Phoma glomerata; the intercalary ovoidal chlamydospores arranged in chains of Fusarium dimerum; and the lateral, spherical chlamydospores arranged in pairs of Fusarium solani. After 1 year of storage, morphological integrity was found to have been maintained in 459 (90%) of the 510 agar block smears. After 3 years of storage, morphological integrity was found to have been maintained in 72 (71%) of the 102 smears prepared in 2006. Agar block smear preparation preserves the native fungal structures and allows long-term storage and examination of fungal structures embedded in the agar, hence overcoming the major drawbacks of adhesive tape preparation. The major roles of agar block smear should be diagnosis for difficult cases, accurate identification of fungal species for clinical management of patients and epidemiological studies, and long-term storage for transportation of slides and education purposes. PMID:20660221

Agar block smear preparation: a novel method of slide preparation for preservation of native fungal structures for microscopic examination and long-term storage.

PubMed

Woo, Patrick C Y; Ngan, Antonio H Y; Chui, Hon-Kit; Lau, Susanna K P; Yuen, Kwok-Yung

2010-09-01

We describe a novel method of fungal slide preparation named "agar block smear preparation." A total of 510 agar block smears of 25 fungal strains obtained from culture collections, 90 QC fungal strains, and 82 clinical fungal strains from our clinical microbiology laboratory, which included a total of 137 species of yeasts, molds, and thermal dimorphic fungi, were prepared and examined. In contrast to adhesive tape preparation, agar block smears preserved the native fungal structures, such as intact conidiophores of Aspergillus species and arrangements of conidia in Scopulariopsis brevicaulis. Furthermore, agar block smears allowed examination of fungal structures embedded in the agar, such as the ascomata with ascomal hairs in Chaetomium funicola; pycnidium of Phoma glomerata; the intercalary ovoidal chlamydospores arranged in chains of Fusarium dimerum; and the lateral, spherical chlamydospores arranged in pairs of Fusarium solani. After 1 year of storage, morphological integrity was found to have been maintained in 459 (90%) of the 510 agar block smears. After 3 years of storage, morphological integrity was found to have been maintained in 72 (71%) of the 102 smears prepared in 2006. Agar block smear preparation preserves the native fungal structures and allows long-term storage and examination of fungal structures embedded in the agar, hence overcoming the major drawbacks of adhesive tape preparation. The major roles of agar block smear should be diagnosis for difficult cases, accurate identification of fungal species for clinical management of patients and epidemiological studies, and long-term storage for transportation of slides and education purposes.

The in-vitro antimicrobial activities of some medicinal plants from Cameroon.

PubMed

Gangoué-Piéboji, J; Pegnyemb, D E; Niyitegeka, D; Nsangou, A; Eze, N; Minyem, C; Mbing, J Ngo; Ngassam, P; Tih, R Ghogomu; Sodengam, B L; Bodo, B

2006-04-01

The antimicrobial activities of 10 plant species (Voacanga africana, Crepis cameroonica, Plagiostyles africana, Crotalaria retusa, Mammea africana, Lophira lanceolata, Ochna afzelii, Ouratea elongata, Ou. flava and Ou. sulcata), each of which is currently used in the traditional medicine of Cameroon, were investigated in vitro. The activities of a methanol extract of each plant were tested, in disc-diffusion assays, against 37 reference or laboratory strains of seven species of microorganism (Staphylococcus aureus, S. epidermidis, Enterococcus hirae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Candida albicans). The minimal inhibitory concentrations of each extract were then estimated, against each of the more susceptible microorganisms (i.e. those giving an inhibition zone measuring at least 9 mm in diameter in the disc-diffusion assays), by agar dilution. Although, in the disc-diffusion assays, each of the 10 methanol extracts investigated displayed some degree of antimicrobial activity against at least one species of microorganism, no activity against the Gram-negative bacteria (Es. coli, K. pneumoniae and Ps. aeruginosa) was observed. The extract with the greatest antimicrobial activity was that of Pl. africana (Euphorbiaceae).

A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate.

PubMed

Levoye, Angélique; Zwier, Jurriaan M; Jaracz-Ros, Agnieszka; Klipfel, Laurence; Cottet, Martin; Maurel, Damien; Bdioui, Sara; Balabanian, Karl; Prézeau, Laurent; Trinquet, Eric; Durroux, Thierry; Bachelerie, Françoise

2015-01-01

Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET) between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z'-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS) of compounds that may modulate GPCRs internalization.

Investigation of dental alginate and agar impression materials as a brain simulant for ballistic testing.

PubMed

Falland-Cheung, Lisa; Piccione, Neil; Zhao, Tianqi; Lazarjan, Milad Soltanipour; Hanlin, Suzanne; Jermy, Mark; Waddell, J Neil

2016-06-01

Routine forensic research into in vitro skin/skull/brain ballistic blood backspatter behavior has traditionally used gelatin at a 1:10 Water:Powder (W:P) ratio by volume as a brain simulant. A limitation of gelatin is its high elasticity compared to brain tissue. Therefore this study investigated the use of dental alginate and agar impression materials as a brain simulant for ballistic testing. Fresh deer brain, alginate (W:P ratio 91.5:8.5) and agar (W:P ratio 81:19) specimens (n=10) (11×22×33mm) were placed in transparent Perspex boxes of the same internal dimensions prior to shooting with a 0.22inch caliber high velocity air gun. Quantitative analysis to establish kinetic energy loss, vertical displacement elastic behavior and qualitative analysis to establish elasticity behavior was done via high-speed camera footage (SA5, Photron, Japan) using Photron Fastcam Viewer software (Version 3.5.1, Photron, Japan) and visual observation. Damage mechanisms and behavior were qualitatively established by observation of the materials during and after shooting. The qualitative analysis found that of the two simulant materials tested, agar behaved more like brain in terms of damage and showed similar mechanical response to brain during the passage of the projectile, in terms of energy absorption and vertical velocity displacement. In conclusion agar showed a mechanical and subsequent damage response that was similar to brain compared to alginate. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species.

PubMed

Yeung, Marie; Thorsen, Trevor

2016-11-08

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 °C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V

Preparation of amine-impregnated silica foams using agar as the gelling agent

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

Jardim, Iara M., E-mail: iaramj01@yahoo.com.br

In this work we successfully prepared amine-impregnated gel-cast silica foams using agar and atmospheric air as the gelling agent and heat treatment atmosphere, respectively. The concentration of 3,6-anhydrogalactose in agar was evaluated by ultraviolet–visible spectroscopy (UV–Vis). The obtained foams were examined by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TG) coupled to mass spectrometry (TG-MS), scanning electron microscopy (SEM), X-ray microtomography (micro-CT), and Archimedes method. The cold crushing strength of the materials prepared in this work was assessed using a mechanical testing stage available in the micro-CT system. The obtained foams exhibited a highly interconnected pore network, with an expressivemore » presence of open pores. Samples heat-treated at 1300 °C for 2 h showed both an expressive porosity (≈ 77%) and a significant cold crushing strength (≈ 1.4 MPa). It was observed that the calcination of the prepared materials at 1200 °C for times as long as 16 h may lead to the rupture of pore walls. FTIR and TG-MS revealed that amine groups were properly incorporated into the foams structure. - Highlights: •Successful preparation of amine-impregnated gel-cast silica foams •Agar used as the gelling agent •Samples with expressive porosity and cold crushing strength •Sintering times as long as 16 h led to the rupture of the pore network.« less

Enhanced diffusion weighting generated by selective adiabatic pulse trains

NASA Astrophysics Data System (ADS)

Sun, Ziqi; Bartha, Robert

2007-09-01

A theoretical description and experimental validation of the enhanced diffusion weighting generated by selective adiabatic full passage (AFP) pulse trains is provided. Six phantoms (Ph-1-Ph-6) were studied on a 4 T Varian/Siemens whole body MRI system. Phantoms consisted of 2.8 cm diameter plastic tubes containing a mixture of 10 μm ORGASOL polymer beads and 2 mM Gd-DTPA dissolved in 5% agar (Ph-1) or nickel(II) ammonium sulphate hexahydrate doped (56.3-0.8 mM) water solutions (Ph-2-Ph-6). A customized localization by adiabatic selective refocusing (LASER) sequence containing slice selective AFP pulse trains and pulsed diffusion gradients applied in the phase encoding direction was used to measure 1H 2O diffusion. The b-value associated with the LASER sequence was derived using the Bloch-Torrey equation. The apparent diffusion coefficients measured by LASER were comparable to those measured by a conventional pulsed gradient spin-echo (PGSE) sequence for all phantoms. Image signal intensity increased in Ph-1 and decreased in Ph-2-Ph-6 as AFP pulse train length increased while maintaining a constant echo-time. These experimental results suggest that such AFP pulse trains can enhance contrast between regions containing microscopic magnetic susceptibility variations and homogeneous regions in which dynamic dephasing relaxation mechanisms are dominant.

Serum bactericidal assay for the evaluation of typhoid vaccine using a semi-automated colony-counting method.

PubMed

Jang, Mi Seon; Sahastrabuddhe, Sushant; Yun, Cheol-Heui; Han, Seung Hyun; Yang, Jae Seung

2016-08-01

Typhoid fever, mainly caused by Salmonella enterica serovar Typhi (S. Typhi), is a life-threatening disease, mostly in developing countries. Enzyme-linked immunosorbent assay (ELISA) is widely used to quantify antibodies against S. Typhi in serum but does not provide information about functional antibody titers. Although the serum bactericidal assay (SBA) using an agar plate is often used to measure functional antibody titers against various bacterial pathogens in clinical specimens, it has rarely been used for typhoid vaccines because it is time-consuming and labor-intensive. In the present study, we established an improved SBA against S. Typhi using a semi-automated colony-counting system with a square agar plate harboring 24 samples. The semi-automated SBA efficiently measured bactericidal titers of sera from individuals immunized with S. Typhi Vi polysaccharide vaccines. The assay specifically responded to S. Typhi Ty2 but not to other irrelevant enteric bacteria including Vibrio cholerae and Shigella flexneri. Baby rabbit complement was more appropriate source for the SBA against S. Typhi than complements from adult rabbit, guinea pig, and human. We also examined the correlation between SBA and ELISA for measuring antibody responses against S. Typhi using pre- and post-vaccination sera from 18 human volunteers. The SBA titer showed a good correlation with anti-Vi IgG quantity in the serum as determined by Spearman correlation coefficient of 0.737 (P < 0.001). Taken together, the semi-automated SBA might be efficient, accurate, sensitive, and specific enough to measure functional antibody titers against S. Typhi in sera from human subjects immunized with typhoid vaccines. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Powdered Chitin Agar as a Selective Medium for Enumeration of Actinomycetes in Water and Soil1

PubMed Central

Hsu, S. C.; Lockwood, J. L.

1975-01-01

Agar media made with 0.4% colloidal chitin plus mineral salts and adjusted to pH 8.0 was superior to four other commonly used media for the isolation and enumeration of actinomycetes from water samples. More actinomycetes developed on chitin agar, and the development of bacteria and fungi was suppressed. Frozen and vacuum-dried chitin from aqueous colloidal suspensions was finely divided and gave results comparable to those obtained with media prepared from colloidal suspensions. Images PMID:234719

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

Evaluation of the effectiveness of peracetic acid in the sterilization of dental equipment.

PubMed

Ceretta, R; Paula, M M S; Angioletto, Ev; Méier, M M; Mitellstädt, F G; Pich, C T; Junior, S A; Angioletto, E

2008-01-01

To evaluate the effectiveness of peracetic acid in the microbiological sterilisation of dental materials. Peracetic acid solution was evaluated at concentrations of 800, 1500 and 2500 ppm. At these concentrations, it was determined whether peracetic acid caused corrosion to dental instruments and induced cellular mutagenicity and cytotoxicity. In addition, the minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC), agar diffusion and diffusion by well method, were also verified. The corrosion rate, calculated from potentiodynamic assays was 10(-6) cm/year, indicating that the product does not damage equipment. The sterilisation capacity of peracetic acid at 2500 ppm was the best. The comet assay indicated genotoxic activity at 2500 ppm. This study demonstrated the effectiveness of peracetic acid for sterilizing dental equipment, providing another alternative for the prevention of infections in clinics.

Development of a rapid and simple immunochromatographic assay to identify Vibrio parahaemolyticus.

PubMed

Sakata, Junko; Kawatsu, Kentaro; Iwasaki, Tadashi; Kumeda, Yuko

2015-09-01

To rapidly and simply determine whether or not bacterial colonies growing on agar were Vibrio parahaemolyticus, we developed an immunochromatographic assay (VP-ICA) using two different monoclonal antibodies (designated mAb-VP34 and mAb-VP109) against the delta subunit of V. parahaemolyticus-F0F1 ATP synthase. The epitopes recognized by mAb-VP34 and mAb-VP109 were mapped to sequences of eight ((47)LLTSSFSA(54)) and six amino acid residues ((16)FDFAVD(21)), respectively. An amino acid sequence similarity search of the NCBI database using BLASTP showed that both epitopic amino acid sequences were present together only in V. parahaemolyticus. When 124 V. parahaemolyticus strains and 94 strains of 27 other Vibrio species or 35 non-Vibrio species were tested using the VP-ICA, the VP-ICA identified V. parahaemolyticus with 100% accuracy. The VP-ICA rapidly and simply identified the pathogen directly from a single agar colony within 30 min, indicating that VP-ICA will greatly reduce labor and time required to identify V. parahaemolyticus compared with conventional biochemical tests. Copyright © 2015. Published by Elsevier B.V.

Multiple Echo Diffusion Tensor Acquisition Technique (MEDITATE) on a 3T clinical scanner

PubMed Central

Baete, Steven H.; Cho, Gene; Sigmund, Eric E.

2013-01-01

This paper describes the concepts and implementation of an MRI method, Multiple Echo Diffusion Tensor Acquisition Technique (MEDITATE), which is capable of acquiring apparent diffusion tensor maps in two scans on a 3T clinical scanner. In each MEDITATE scan, a set of RF-pulses generates multiple echoes whose amplitudes are diffusion-weighted in both magnitude and direction by a pattern of diffusion gradients. As a result, two scans acquired with different diffusion weighting strengths suffice for accurate estimation of diffusion tensor imaging (DTI)-parameters. The MEDITATE variation presented here expands previous MEDITATE approaches to adapt to the clinical scanner platform, such as exploiting longitudinal magnetization storage to reduce T2-weighting. Fully segmented multi-shot Cartesian encoding is used for image encoding. MEDITATE was tested on isotropic (agar gel), anisotropic diffusion phantoms (asparagus), and in vivo skeletal muscle in healthy volunteers with cardiac-gating. Comparisons of accuracy were performed with standard twice-refocused spin echo (TRSE) DTI in each case and good quantitative agreement was found between diffusion eigenvalues, mean diffusivity, and fractional anisotropy derived from TRSE-DTI and from the MEDITATE sequence. Orientation patterns were correctly reproduced in both isotropic and anisotropic phantoms, and approximately so for in vivo imaging. This illustrates that the MEDITATE method of compressed diffusion encoding is feasible on the clinical scanner platform. With future development and employment of appropriate view-sharing image encoding this technique may be used in clinical applications requiring time-sensitive acquisition of DTI parameters such as dynamical DTI in muscle. PMID:23828606

Nutrient chemotaxis suppression of a diffusive instability in bacterial colony dynamics

NASA Astrophysics Data System (ADS)

Arouh, Scott; Levine, Herbert

2000-07-01

Bacteria grown on a semisolid agar surface have been observed to form branching patterns as the colony envelope propagates outward. The fundamental cause of this instability relates to the need for limited nutrient to diffuse towards the colony. Here, we investigate the effect on this instability of allowing the bacteria to move chemotactically in response to the nutrient gradient. Our results show that this additional effect has a tendency to suppress the instability. Our calculations are done within the context of a simple ``cutoff'' model of colony dynamics, but presumably remain valid for more complex and hence more realistic approaches.

Can the diagnosis of recurrent vulvovaginal candidosis be improved by use of vaginal lavage samples and cultures on chromogenic agar?

PubMed Central

Novikova, N; Rodrigues, A; Mårdh, P A

2002-01-01

OBJECTIVE: To investigate if introital and vaginal flushing samples inoculated on chromogenic agar could increase the recovery rate and rapid identification of Candida and non-albicans species, as compared to culture of posterior vaginal fornix samples on Sabouraud agar and speciation of isolates by biochemical tests. METHODS: Samples from the introitus and the posterior vaginal fornix and vaginal lavage samples were collected from 91 women with a history suggestive of recurrent vulvovaginal candidosis (RVVC), and with a suspected new attack of the condition. The specimens were cultured on Sabouraud and CHROMagar. Speciation of yeast isolates was made on the chromogenic agar by API 32C kits and by an atomized system (Vitek). RESULTS: Forty-six (51%) women were positive for Candida from one or more of the samples. The introital cultures were positive in 43 (47%) women, both on Sabouraud and chromogenic agar. From the posterior vaginal fomix, 42 (46%) women were positive on the Sabouraud and 43 (47%) on chromogenic agar cultures, while the vaginal lavage cultures yielded Candida on those two media in 40 (44%) and 41 (45%) cases, respectively. Candida albicans was the most frequent species recovered, from 40 (87%) cases, followed by C. krusei in 4 (9%), C. glabrata in 2 (4%), and C. parapsilosis in one case. There was only one woman who had a mixed yeast infection, by C. albicans and C. krusei. There was only one discrepancy in the speciation as demonstrated by mean of chromogenic agar and API 32C kit. CONCLUSIONS: Neither cultures of introital nor of vaginal lavage samples increases the detection rate of Candida in RVVC cases as compared to cultures of posterior vaginal fornix samples. Use of chromogenic agar is a convenient and reliable means to detect colonization by Candida and differentiate between C. albicans and non-albicans species. PMID:12530485

Antibacterial properties of Ag-exchanged Philippine natural zeolite-chitosan composites

NASA Astrophysics Data System (ADS)

Taaca, Kathrina Lois M.; Olegario, Eleanor M.; Vasquez, Magdaleno R.

2017-12-01

Zeolites are microporous minerals composed of silicon, aluminum and oxygen. These aluminosilicates consist of tetrahedral units which produce open framework structures to generate a system of pores and cavities of molecular dimensions. Zeolites are naturally abundant and can be mined in most parts of the world. In this study, natural zeolites (NaZ) which are locally-sourced here in the Philippines were investigated to determine its properties. An ion-exchange process was utilized, using the zeolite to silver (Ag) solution ratio of 1:20 (w/v), to incorporate Ag into the zeolite framework. Characterizations such as XRD, AAS, and Agar diffusion assay were used to evaluate the properties of the synthesized Ag-exchanged zeolites (AgZ). X-ray diffraction revealed that both NaZ and AgZ have peaks mostly corresponding to the clinoptilolite structure, with some trace peaks of the mordenite and quartz. Absorption spectroscopy revealed that the ion exchange process added about 0.61188g of silver into the zeolite structure. This Ag content was seen to be enough to make the AgZ sample exhibit an antibacterial effect where clearing zones against E. coli and S. aureus were observed in the agar diffusion assay, respectively. The AgZ sample was also tested as ceramic filler to a polymer matrix-chitosan. The diffusion assay revealed presence of antibacterial activity to the polymer composite with AgZ fillers. These results indicate that the Philippine natural zeolite, incorporated with metals such as Ag, can be used as an antibacterial agent and can be developed as a ceramic filler to improve the antibacterial property of composite materials for biomedical application.

Evaluation of the Double Agar Gel Immunodiffusion Test and of the Enzyme-Linked Immunosorbent Assay in the Diagnosis and Follow-Up of Patients with Chronic Pulmonary Aspergillosis

PubMed Central

de Azevedo, Priscila Zacarias; Sylvestre, Tatiane Fernanda; Cavalcante, Ricardo de Souza; de Carvalho, Lídia Raquel; Moris, Daniela Vanessa; de Oliveira, Maria Luiza Cotrim Sartor; Mendes, Rinaldo Poncio

2015-01-01

The diagnosis of chronic pulmonary aspergillosis (CPA) depends on the radiologic image and the identification of specific antibodies. The present study aimed to evaluate accuracy parameters of enzyme-linked immunosorbent assay (ELISA) and of the determination of serum galactomannan level in the diagnosis of patients with CPA, comparing these results with the double agar gel immunodiffusion (DID) test. In addition, the prevalence of cross-reactivity and the serological progression after treatment were evaluated by comparing DID and ELISA. Six study groups were formed: G1: 22 patients with CPA, 17 of whom had Aspergillus fungus ball, one chronic cavitary pulmonary aspergillosis (CCPA) and four chronic fibrosing pulmonary aspergillosis (CFPA); G2: 28 patients with pulmonary tuberculosis (TB); G3: 23 patients with histoplasmosis (HST); G4: 50 patients with paracoccidioidomycosis (PCM); G5: 20 patients with cryptococcosis (CRC); and G6: 200 healthy controls. Serum antibodies were measured by DID and ELISA, with two antigen preparations—Aspergillus fumigatus (DID1, ELISA1) and a pool of A. fumigatus, A. flavus and A. niger antigens (DID2, ELISA2). The Platélia Aspergillus Enzyme Immunoassay (EIA) kit was used to measure galactomannan. The cut-off points of ELISA were determined for each antigen preparation and for the 95% and 99% confidence intervals. Despite the low sensitivity, DID was the technique of choice due to its specificity, positive and negative predictive values and positive likelihood ratio–especially with the antigen pool and due to the low frequency of cross-reactivity. ELISA1 and a 0.090 cut-off showed high sensitivity, specificity and negative predictive value, but a high frequency of cross-reactivity with CRC. The best degree of agreement was observed between ELISA1 and ELISA2. The detection of serum galactomannan showed high sensitivity, comparable to ELISA2. The immunodiffusion test showed an excellent relationship with the progression after

Standardized methods and quality control limits for agar and broth microdilution susceptibility testing of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum.

PubMed

Waites, Ken B; Duffy, Lynn B; Bébéar, Cécile M; Matlow, Anne; Talkington, Deborah F; Kenny, George E; Totten, Patricia A; Bade, Donald J; Zheng, Xiaotian; Davidson, Maureen K; Shortridge, Virginia D; Watts, Jeffrey L; Brown, Steven D

2012-11-01

An international multilaboratory collaborative study was conducted to develop standard media and consensus methods for the performance and quality control of antimicrobial susceptibility testing of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum using broth microdilution and agar dilution techniques. A reference strain from the American Type Culture Collection was designated for each species, which was to be used for quality control purposes. Repeat testing of replicate samples of each reference strain by participating laboratories utilizing both methods and different lots of media enabled a 3- to 4-dilution MIC range to be established for drugs in several different classes, including tetracyclines, macrolides, ketolides, lincosamides, and fluoroquinolones. This represents the first multilaboratory collaboration to standardize susceptibility testing methods and to designate quality control parameters to ensure accurate and reliable assay results for mycoplasmas and ureaplasmas that infect humans.

The effect of lactic acid bacteria isolates on the urinary tract pathogens to infants in vitro.

PubMed

Lim, In Seok; Lee, Ho Seok; Kim, Won Yong

2009-01-01

Urinary tract infections are common clinical problems in children, even though lots of treatment strategies have been tried. Many studies of the application of probiotics for urinary tract infection in female adults exist, but there is a lack of studies in children. The aims of this study were to screen probiotic strains for inhibiting the uropathogens in vitro, to find candidates for in vivo study. Nine strains of E. coli were isolated from children with urinary tract infection and six uropathogens were obtained from Korean Collection for Type Cultures and American Type Culture Collection. Also 135 lactic acid bacteria (LAB) strains were isolated from healthy children, and were identified through physiologic, biochemical methods, 16S rDNA PCR, and data analysis. And with agar disk diffusion assay technique the antimicrobial activities of these LAB strains against those uropathogens were examined. Three strains of separated LAB strains demonstrated major antimicrobial activity against all the uropathogens. In the agar disk diffusion assay technique, antimicrobial activities increased most in the 4th day culture broth with separated Lactobacillus. In summary, some LAB can be used as candidates to develop the probiotic microorganisms that inhibit uropathogens in children, and are expected to be applied to treatment and prevention of pediatric urinary tract infection.

Antimicrobial Activity of Rhoeo discolor Phenolic Rich Extracts Determined by Flow Cytometry.

PubMed

García-Varela, Rebeca; García-García, Rebeca M; Barba-Dávila, Bertha A; Fajardo-Ramírez, Oscar R; Serna-Saldívar, Sergio O; Cardineau, Guy A

2015-10-14

Traditional medicine has led to the discovery of important active substances used in several health-related areas. Phytochemicals in Rhoeo discolor extracts have proven to have important antimicrobial activity. In the present study, our group determined the antimicrobial effects of extracts of Rhoeo discolor, a plant commonly used in Mexico for both medicinal and ornamental purposes. We evaluated the in vitro activity of phenolic rich extracts against specifically chosen microorganisms of human health importance by measuring their susceptibility via agar-disc diffusion assay and flow cytometry: Gram-positive Listeria innocua and Streptococcus mutans, Gram-negative Escherichia coli and Pseudomonas aeruginosa, and lastly a fungal pathogen Candida albicans. Ten different extracts were tested in eight different doses on all the microorganisms. Analytical data revealed a high content of phenolic compounds. Both agar-disc diffusion assay and flow cytometry results demonstrated that Pseudomonas aeruginosa was the least affected by extract exposure. However, low doses of these extracts (predominantly polar), in a range from 1 to 4 μg/mL, did produce a statistically significant bacteriostatic and bactericidal effect on the rest of the microorganisms. These results suggest the addition of certain natural extracts from Rhoeo discolor could act as antibacterial and antimycotic drugs or additives for foods and cosmetics.

Combination Studies of Oreganum Vulgare Extract Fractions and Volatile Oil along with Ciprofloxacin and Fluconazole against Common Fish Pathogens

PubMed Central

Bharti, Veni; Vasudeva, Neeru; Dhuhan, Joginder Singh

2013-01-01

Purpose: The study is aimed at finding new antibiotic therapy for aquaculture due to potential of bacteria to develop resistance to the existing therapies. Use of large quantities of synthetic antibiotics in aquaculture thus has the potential to be detrimental to fish health, to the environment and wildlife and to human health. Methods: Antimicrobial potential of volatile oil and fractions of chloroform extract of Oreganum vulgare was evaluated alone and in the presence of standard antimicrobials against common fish pathogens by disc-diffusion, agar well assay and two fold microdilution method by nanodrop spectrophotometric method. Results: The best results were represented by volatile oil followed by phenolic fraction by disc-diffusion, agar well and microdilution assays (Minimum inhibitory concentration). By the interaction studies, it was observed that the volatile oil and phenolic fraction were able to inhibit the pathogens at very low concentration compared to standard drugs. The fractional inhibitory concentration index (FICI) was calculated and volatile oil and phenolic fractions were found to be synergistic against Pseudomonas fluorescens and Candida albicans. Conclusion: The experimental data suggests the use of volatile oil and phenolic fraction in combination with standard antimicrobials to maintain healthy aquaculture with lesser adverse effects as compared to synthetic antibiotic therapy. PMID:24312842

An improved agar medium for growth of Geobacillus thermoglucosidarius strains.

PubMed

Javed, M; Baghaei-Yazdi, N; Qin, W; Amartey, S

2017-01-01

Geobacillus species have potential applications in many biotechnological processes. They are fastidious in their vitamin and amino acid requirements. A new semi-defined agar medium (SDM) was developed which gave consistently high viable cell counts of various G. thermoglucosidasius strains (5×10 8 -6×10 8 cfu/ml) under aerobic conditions at 70°C. Copyright © 2016 Elsevier B.V. All rights reserved.

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

[Thin layer agar represents a cost-effective alternative for the rapid diagnosis of multi-drug resistant tuberculosis].

PubMed

Hernández-Sarmiento, José M; Martínez-Negrete, Milton A; Castrillón-Velilla, Diana M; Mejía-Espinosa, Sergio A; Mejía-Mesa, Gloria I; Zapata-Fernández, Elsa M; Rojas-Jiménez, Sara; Marín-Castro, Andrés E; Robledo-Restrepo, Jaime A

2014-01-01

Using cost-benefit analysis for comparing the thin-layer agar culture method to the standard multiple proportion method used in diagnosing multidrug-resistant tuberculosis (MDR TB). A cost-benefit evaluation of two diagnostic tests was made at the Corporación para Investigaciones Biológicas (CIB) in Medellín, Colombia. 100 patients were evaluated; 10.8% rifampicin resistance and 14.3% isoniazid resistance were found. A computer-based decision tree model was used for cost-effectiveness analysis (Treeage Pro); the thin-layer agar culture method was most cost-effective, having 100% sensitivity, specificity and predictive values for detecting rifampicin and isoniazid resistance. The multiple proportion method value was calculated as being US$ 71 having an average 49 day report time compared to US$ 18 and 14 days for the thin-layer agar culture method. New technologies have been developed for diagnosing tuberculosis which are apparently faster and more effective; their operating characteristics must be evaluated as must their effectiveness in terms of cost-benefit. The present study established that using thin-layer agar culture was cheaper, equally effective and could provide results more quickly than the traditional method. This implies that a patient could receive MDR TB treatment more quickly.

A comparison of assays measuring the viability of Legionella ...

EPA Pesticide Factsheets

Background: The relatively high prevalence of Legionella pneumophila in premise plumbing systems has been widely reported. Published reports indicate Legionella has a comparatively high resistance to chlorine and moreover has the ability to grow in phagocytic amoeba which could provide additional protection in chlorinated drinking water distribution systems. Copper-Silver (Cu-Ag) ionization treatment systems are commercially available for use in large building water systems to help control the risks from Legionella bacteria. The objectives of this study were to develop and optimize Legionella viability assays and use them to investigate the viability of Legionella bacteria after exposure to water treated with coppper and silver ions. Methods: Log phase L. pneumophila cells were used in all experiments and were generated by incubation at 35C for 48 hours in buffered yeast extract broth. Viability assays used included plating on buffered charcoal yeast extract agar to determine the number of culturable cells and treating cells with propidium monoazide (PMA) or ethidium monoazide (EMA) followed by quantitative PCR targeting mip gene of L. pneumophila. The qPCR viability assays were optimized using L. pneumophila inactivated by heat treatment at 65C for 60 min. The effectiveness of Cu-Ag ionization treatment was studied by inoculating L. pneumonia at 105 CFU/mL in water collected directly from a building water system that employed this technology and incubat

A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate

PubMed Central

Levoye, Angélique; Zwier, Jurriaan M.; Jaracz-Ros, Agnieszka; Klipfel, Laurence; Cottet, Martin; Maurel, Damien; Bdioui, Sara; Balabanian, Karl; Prézeau, Laurent; Trinquet, Eric; Durroux, Thierry; Bachelerie, Françoise

2015-01-01

Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET) between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z′-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS) of compounds that may modulate GPCRs internalization. PMID:26617570

Near-infrared fluorescence imaging platform for quantifying in vivo nanoparticle diffusion from drug loaded implants.

PubMed

Markovic, Stacey; Belz, Jodi; Kumar, Rajiv; Cormack, Robert A; Sridhar, Srinivas; Niedre, Mark

2016-01-01

Drug loaded implants are a new, versatile technology platform to deliver a localized payload of drugs for various disease models. One example is the implantable nanoplatform for chemo-radiation therapy where inert brachytherapy spacers are replaced by spacers doped with nanoparticles (NPs) loaded with chemotherapeutics and placed directly at the disease site for long-term localized drug delivery. However, it is difficult to directly validate and optimize the diffusion of these doped NPs in in vivo systems. To better study this drug release and diffusion, we developed a custom macroscopic fluorescence imaging system to visualize and quantify fluorescent NP diffusion from spacers in vivo. To validate the platform, we studied the release of free fluorophores, and 30 nm and 200 nm NPs conjugated with the same fluorophores as a model drug, in agar gel phantoms in vitro and in mice in vivo. Our data verified that the diffusion volume was NP size-dependent in all cases. Our near-infrared imaging system provides a method by which NP diffusion from implantable nanoplatform for chemo-radiation therapy spacers can be systematically optimized (eg, particle size or charge) thereby improving treatment efficacy of the platform.

Evolutionary consequences of putative intra- and interspecific hybridiation in agaric fungi

Treesearch

Karen W. Hughes; Ronald H. Petersen; D. Jean Lodge; Sarah E. Bergemann; Kendra Baumgartner; Rodham E. Tulloss; Edgar Lickey; Joaquin. Cifuentes

2013-01-01

Agaric fungi of the southern Appalachian Mountains including Great Smoky Mountains National Park are often heterozygous for the rDNA internal transcribed spacer region (ITS) with .42% of collections showing some heterozygosity for indels and/or base-pair substitutions. For these collections, intra-individual haplotype divergence is typically less than 2%, but for 3% of...

A Modified Surface Killing Assay (MSKA) as a Functional in vitro Assay for Identifying Protective Antibodies Against Pneumococcal Surface Protein A (PspA)

PubMed Central

Genschmer, Kristopher R.; Accavitti-Loper, Mary Ann; Briles, David E.

2013-01-01

Streptococcus pneumoniae causes otitis media, meningitis and pneumonia in patients worldwide; predominantly affecting young children, the elderly, and the immune compromised. Current vaccines against invasive pneumococcal disease are based on the polysaccharide capsules of the most clinically relevant serotypes. Due to serotype replacement, non-vaccine serotypes of S. pneumoniae have become more clinically relevant and as a result pneumococcal vaccines are becoming increasingly complex. These events emphasize the need to evaluate the potential for pneumococcal cross-reactive proteins to contribute to future vaccines. Antibody elicited by the immunization of humans with pneumococcal surface protein A (PspA) can passively protect mice from infection. However, robust in vitro functional assays for antibody to PspA are not available to predict the protective capacity of immune serum. For polysaccharide based vaccines, a standardized opsonophagocytosis killing assay (OPKA) is used. Antibody to PspA, however, does not work well in the standard OPKA. The present studies take advantage of past observations that phagocytosis is more efficient on tissue surfaces than in solution. In a modified surface killing assay (MSKA), monoclonal antibody to PspA, in the presence of complement, opsonized pneumococci for killing by phagocytes on an agar surface. Five monoclonal antibodies to PspA were tested; three demonstrated increased amounts of killing compared to the diluent control and protected mice by passive protection against type 3 pneumococci. The two antibodies that were not functional in the MSKA also failed to protect mice. Thus, an MSKA might be useful as a functional assay for immunity to PspA. PMID:24211169

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.

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

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.

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.

Cytotoxicity of Light-Cured Dental Materials according to Different Sample Preparation Methods

PubMed Central

Lee, Myung-Jin; Kim, Mi-Joo; Kwon, Jae-Sung; Lee, Sang-Bae; Kim, Kwang-Mahn

2017-01-01

Dental light-cured resins can undergo different degrees of polymerization when applied in vivo. When polymerization is incomplete, toxic monomers may be released into the oral cavity. The present study assessed the cytotoxicity of different materials, using sample preparation methods that mirror clinical conditions. Composite and bonding resins were used and divided into four groups according to sample preparation method: uncured; directly cured samples, which were cured after being placed on solidified agar; post-cured samples were polymerized before being placed on agar; and “removed unreacted layer” samples had their oxygen-inhibition layer removed after polymerization. Cytotoxicity was evaluated using an agar diffusion test, MTT assay, and confocal microscopy. Uncured samples were the most cytotoxic, while removed unreacted layer samples were the least cytotoxic (p < 0.05). In the MTT assay, cell viability increased significantly in every group as the concentration of the extracts decreased (p < 0.05). Extracts from post-cured and removed unreacted layer samples of bonding resin were less toxic than post-cured and removed unreacted layer samples of composite resin. Removal of the oxygen-inhibition layer resulted in the lowest cytotoxicity. Clinicians should remove unreacted monomers on the resin surface immediately after restoring teeth with light-curing resin to improve the restoration biocompatibility. PMID:28772647

Antioxidant and antibacterial activities on foodborne pathogens of Artocarpus heterophyllus Lam. (Moraceae) leaves extracts.

PubMed

Loizzo, M R; Tundis, R; Chandrika, U G; Abeysekera, A M; Menichini, F; Frega, N G

2010-06-01

Total water extract, ethyl acetate, and aqueous fractions from the leaves of Artocarpus heterophyllus were evaluated for phenolic content, antioxidant, and antibacterial activities against some foodborne pathogens such as E. coli, Listeria monocytogenes, Salmonella typhimurium, Salmonella enterica, Bacillus cereus, Enterococcus faecalis, and Staphylococcus aureus. The minimum inhibitory concentration (MICs) of extract and fractions determined by the agar dilution method were ranged from 221.9 microg/mL for ethyl acetate fraction to 488.1 microg/mL for total extract. In the agar diffusion method the diameters of inhibition were 12.2 for the total extract, 10.7 and 11.5 for ethyl acetate and aqueous fractions, respectively. A. heterophyllus showed significant antioxidant activity tested in different in vitro systems (DPPH, ABTS, FRAP, and Fe(2+) chelating activity assay). In particular, in DPPH assay A. heterophyllus total extract exhibited a strong antiradical activity with an IC(50) value of 73.5 microg/mL while aqueous fraction exerted the highest activity in FRAP assay (IC(50) value of 72.0 microg/mL). The total phenols content by Folin-Ciocalteau method was determined with the purpose of testing its relationship with the antioxidant and antibacterial activities.

Eliminating Size-Associated Diffusion Constraints for Rapid On-Surface Bioassays with Nanoparticle Probes.

PubMed

Li, Junwei; Zrazhevskiy, Pavel; Gao, Xiaohu

2016-02-24

Nanoparticle probes enable implementation of advanced on-surface assay formats, but impose often underappreciated size-associated constraints, in particular on assay kinetics and sensitivity. The present study highlights substantially slower diffusion-limited assay kinetics due to the rapid development of a nanoprobe depletion layer next to the surface, which static incubation and mixing of bulk solution employed in conventional assay setups often fail to disrupt. In contrast, cyclic solution draining and replenishing yields reaction-limited assay kinetics irrespective of the probe size. Using common surface bioassays, enzyme-linked immunosorbent assays and immunofluorescence, this study shows that this conceptually distinct approach effectively "erases" size-dependent diffusion constraints, providing a straightforward route to rapid on-surface bioassays employing bulky probes and procedures involving multiple labeling cycles, such as multicycle single-cell molecular profiling. For proof-of-concept, the study demonstrates that the assay time can be shortened from hours to minutes with the same probe concentration and, at a typical incubation time, comparable target labeling can be achieved with up to eight times lower nanoprobe concentration. The findings are expected to enable realization of novel assay formats and stimulate development of rapid on-surface bioassays with nanoparticle probes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Pump-probe imaging of nanosecond laser-induced bubbles in agar gel.

PubMed

Evans, R; Camacho-López, S; Pérez-Gutiérrez, F G; Aguilar, G

2008-05-12

In this paper we show results of Nd:YAG laser-induced bubbles formed in a one millimeter thick agar gel slab. The nine nanosecond duration pulse with a wave length of 532 nm was tightly focused inside the bulk of the gel sample. We present for the first time a pump-probe laser-flash shadowgraphy system that uses two electronically delayed Nd:YAG lasers to image the the bubble formation and shock wave fronts with nanosecond temporal resolution and up to nine seconds of temporal range. The shock waves generated by the laser are shown to begin at an earlier times within the laser pulse as the pulse energy increases. The shock wave velocity is used to infer a shocked to unshocked material pressure difference of up to 500 MPa. The bubble created settles to a quasi-stable size that has a linear relation to the maximum bubble size. The energy stored in the bubble is shown to increase nonlinearly with applied laser energy, and corresponds in form to the energy transmission in the agar gel. We show that the interaction is highly nonlinear, and most likely is plasma-mediated.

Biodegradation and bioconversion of coals by fungi: Technical report No. 6 for the period December 1, 1986-February 28, 1987. [Candida sp. , Cunninghamella sp. , Acremonium sp. , and Trametes versicolor

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

Ward, B.

We continued our tests for biosolubilization activity on lignite, subbituminous, and bituminous coals, using both surface solubilization and agar diffusion assays for selected lignite-solubilizing strains challenged with untreated and treated coals shown in earlier tests to be resistant to biosolubilization. These latter were subjected to treatments with heat or pressurized oxygen atmosphere, or to combinations of the same. We have observed biosolubilization of some weathered high-rank coals by some of the fungal isolates tested. 1 tab.

Simplified Quantitative Assay System for Measuring Activities of Drugs against Intracellular Legionella pneumophila

PubMed Central

Higa, Futoshi; Kusano, Nobuchika; Tateyama, Masao; Shinzato, Takashi; Arakaki, Noriko; Kawakami, Kazuyoshi; Saito, Atsushi

1998-01-01

We developed a new simple assay for the quantitation of the activities of drugs against intracellular Legionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death. The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h. The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-α agar plates. Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L. pneumophila were determined by the colorimetric assay system. The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-α broth. The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs. According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study. Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples. PMID:9574712

[Short-term screening of anticarcinogenic ingredients of tea by cell biology assays].

PubMed

Liu, L; Han, C; Chen, J

1998-01-01

By using a panel of short term cell biology assays, several ingredients of tea (tea pigments, caffeine, tea polysaccharide, tea polyphenols tablet and mixed tea) were screened in order to investigate their anticarcinogenic effects. The cytokinesis block micronuclei test in V79 cells induced by mitomycin, the test of metabolic cooperation between V79 and M cells and the test of growth ability of Hela cells in soft agar were used in the screening. The results showed that the six kinds of tea ingredients tested were effective in the test involved in different stages of carcinogenesis, i.e. initiation, promotion and progression. The effects of mixed tea and tea pigments were the strongest among the ingredients tested.

Vibrios from Fish Pen Slime Which Mimic Escherichia coli on Violet Red Bile Agar

PubMed Central

Rosen, A.; Levin, R. E.

1970-01-01

Organisms from fish pen slime which mimicked coliforms and Escherichia coli on Violet Red Bile Agar were identified as members of the genus Vibrio on the basis of metabolic and morphological characteristics. Images PMID:4195607

Colwellia agarivorans sp. nov., an agar-digesting marine bacterium isolated from coastal seawater

USDA-ARS?s Scientific Manuscript database

A novel Gram-stain-negative, facultatively anaerobic, yellowish and agar-digesting marine bacterium, designated strain QM50**T, was isolated from coastal seawater in an aquaculture site near Qingdao, China. Phylogenetic analysis based on 16S rDNA sequences revealed that the novel isolate represented...

Development of hyperspectral imaging technique for salmonella enteritidis and typhimurium on agar plates

USDA-ARS?s Scientific Manuscript database

Salmonella is a common cause of foodborne disease resulting from the consumption of contaminated food products. Although a direct plating method is widely used for presumptive positive screening of pathogenic Salmonella colonies on agar plates, it is labor-intensive, time-consuming and also prone to...

Thermal inactivation and sublethal injury kinetics of Salmonella enterica and Listeria monocytogenes in broth versus agar surface.

PubMed

Wang, Xiang; Devlieghere, Frank; Geeraerd, Annemie; Uyttendaele, Mieke

2017-02-21

The objective of the present study was to compare the thermal inactivation and sublethal injury kinetics of Salmonella enterica and Listeria monocytogenes in broth (suspended cells) and on solid surface (agar-seeded cells). A 3-strain cocktail of S. enterica or L. monocytogenes inoculated in broth or on agar was subjected to heating in a water bath at various set temperatures (55.0, 57.5 and 60.0°C for S. enterica and 60.0, 62.5 and 65°C for L. monocytogenes). The occurrence of sublethally injured cells was determined by comparing enumerations on nonselective (TSAYE) and selective (XLD or ALOA) media. Results showed that the inactivation curves obtained from selective media were log-linear, and significant shoulders (p<0.05) were observed on some of the inactivation curves from TSAYE media. The D-values derived from the total population were higher than those from the uninjured cells. Generally, cells on agar surface exhibited higher heat resistance than those in broth. For S. enterica, cell injury increased with the exposure time, no difference was observed when treated at temperatures from 55.0 to 60.0°C, while for L. monocytogenes, cell injury increased significantly with heating time and treatment temperature (from 60.0 to 65°C). Moreover, the degree of sublethal injury affected by thermal treatment in broth or on agar surface depended upon the target microorganism. Higher proportions of injured S. enterica cells were observed for treatment in broth than on agar surface, while the opposite was found for L. monocytogenes. The provided information may be used to assess the efficacy of thermal treatment processes on surfaces for inactivation of S. enterica and L. monocytogenes, and it provides insight into the sublethally injured survival state of S. enterica and L. monocytogenes treated in liquid or on solid food. Copyright © 2016 Elsevier B.V. All rights reserved.

Equivalency testing of TTC Tergitol 7 agar (ISO 9308-1:2000) with five culture media for the detection of E. coli in water samples in Greece.

PubMed

Mavridou, A; Smeti, E; Mandilara, G; Mandilara, G; Boufa, P; Vagiona-Arvanitidou, M; Vantarakis, A; Vassilandonopoulou, G; Pappa, O; Roussia, V; Tzouanopoulos, A; Livadara, M; Aisopou, I; Maraka, V; Nikolaou, E; Mandilara, G

2010-01-01

In this study ten laboratories in Greece compared the performance of reference method TTC Tergitol 7 Agar (with the additional test of beta-glucuronidase production) with five alternative methods, to detect E. coli in water, in line with European Water Directive recommendations. The samples were prepared by spiking drinking water with sewage effluent following a standard protocol. Chlorinated and non-chlorinated samples were used. The statistical analysis was based on the mean relative difference of confirmed counts and was performed in line with ISO 17994. The results showed that in total, three of the alternative methods (Chromocult Coliform agar, Membrane Lauryl Sulfate agar and Trypton Bilex-glucuronidase medium) were not different from TTC Tergitol 7 agar (TTC Tergitol 7 agar vs Chromocult Coliform agar, 294 samples, mean RD% 5.55; vs MLSA, 302 samples, mean RD% 1; vs TBX, 297 samples, mean RD% -2.78). The other two alternative methods (Membrane Faecal coliform medium and Colilert 18/ Quantitray) gave significantly higher counts than TTC Tergitol 7 agar (TTC Tergitol 7 agar vs MFc, 303 samples, mean RD% 8.81; vs Colilert-18/Quantitray, 76 samples, mean RD% 18.91). In other words, the alternative methods generated performance that was as reliable as, or even better than, the reference method. This study will help laboratories in Greece overcome culture and counting problems deriving from the EU reference method for E. coli counts in water samples.

Metal-amplified Density Assays, (MADAs), including a Density-Linked Immunosorbent Assay (DeLISA).

PubMed

Subramaniam, Anand Bala; Gonidec, Mathieu; Shapiro, Nathan D; Kresse, Kayleigh M; Whitesides, George M

2015-02-21

This paper reports the development of Metal-amplified Density Assays, or MADAs - a method of conducting quantitative or multiplexed assays, including immunoassays, by using Magnetic Levitation (MagLev) to measure metal-amplified changes in the density of beads labeled with biomolecules. The binding of target analytes (i.e. proteins, antibodies, antigens) to complementary ligands immobilized on the surface of the beads, followed by a chemical amplification of the binding in a form that results in a change in the density of the beads (achieved by using gold nanoparticle-labeled biomolecules, and electroless deposition of gold or silver), translates analyte binding events into changes in density measureable using MagLev. A minimal model based on diffusion-limited growth of hemispherical nuclei on a surface reproduces the dynamics of the assay. A MADA - when performed with antigens and antibodies - is called a Density-Linked Immunosorbent Assay, or DeLISA. Two immunoassays provided a proof of principle: a competitive quantification of the concentration of neomycin in whole milk, and a multiplexed detection of antibodies against Hepatitis C virus NS3 protein and syphilis T. pallidum p47 protein in serum. MADAs, including DeLISAs, require, besides the requisite biomolecules and amplification reagents, minimal specialized equipment (two permanent magnets, a ruler or a capillary with calibrated length markings) and no electrical power to obtain a quantitative readout of analyte concentration. With further development, the method may be useful in resource-limited or point-of-care settings.

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.

Irradiation of silver and agar/silver nanoparticles with argon, oxygen glow discharge plasma, and mercury lamp.

PubMed

Ahmad, Mahmoud M; Abdel-Wahab, Essam A; El-Maaref, A A; Rawway, Mohammed; Shaaban, Essam R

2014-01-01

The irradiation effect of argon, oxygen glow discharge plasma, and mercury lamp on silver and agar/silver nanoparticle samples is studied. The irradiation time dependence of the synthesized silver and agar/silver nanoparticle absorption spectra and their antibacterial effect are studied and compared. In the agar/silver nanoparticle sample, as the irradiation time of argon glow discharge plasma or mercury lamp increases, the peak intensity and the full width at half maximum, FWHM, of the surface plasmon resonance absorption band is increased, however a decrease of the peak intensity with oxygen glow plasma has been observed. In the silver nanoparticle sample, as the irradiation time of argon, oxygen glow discharge plasma or mercury lamp increases, the peak intensity of the surface plasmon resonance absorption band is increased, however, there is no significant change in the FWHM of the surface plasmon resonance absorption band. The SEM results for both samples showed nanoparticle formation with mean size about 50 nm and 40 nm respectively. Throughout the irradiation time with the argon, oxygen glow discharge plasma or mercury lamp, the antibacterial activity of several kinds of Gram-positive and Gram-negative bacteria has been examined.

Direct growth inhibition assay of total airborne fungi with application of biocide-treated malt extract agar.

PubMed

Er, Chin Ming; Sunar, N M; Leman, A M; Othman, N

2015-01-01

Indoor air pollution by airborne fungi has risen to become a common issue all over the world and it is hazardous to indoor occupants' health as it is associated with a series of respiratory-related and skin-related diseases. Selected bioactive compounds from the food industry have been suggested to be effective against individual fungus isolated from indoor environment. However, the techniques used to evaluate these compounds were lengthy and unsuitable against total airborne fungi. Therefore, this paper describes an assay to assess the effectiveness of a bioactive compound to inhibit growth of total airborne fungi.•A combination and modification of previous methods and the NIOSH Manual Analytical Standard Method (NMAM 0800) is proposed.•This method concurrently samples the total airborne fungi and evaluates the ability of bioactive compounds (potassium sorbate in this paper), as a biocide, to treat these indoor airborne fungi.•The current method shortens the time of evaluation from 30 days to only 5 days and employs the counting of colony forming units (CFUs) to ease the measurement of the growth of fungi.

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.

Generation and precise control of dynamic biochemical gradients for cellular assays

NASA Astrophysics Data System (ADS)

Saka, Yasushi; MacPherson, Murray; Giuraniuc, Claudiu V.

2017-03-01

Spatial gradients of diffusible signalling molecules play crucial roles in controlling diverse cellular behaviour such as cell differentiation, tissue patterning and chemotaxis. In this paper, we report the design and testing of a microfluidic device for diffusion-based gradient generation for cellular assays. A unique channel design of the device eliminates cross-flow between the source and sink channels, thereby stabilizing gradients by passive diffusion. The platform also enables quick and flexible control of chemical concentration that makes highly dynamic gradients in diffusion chambers. A model with the first approximation of diffusion and surface adsorption of molecules recapitulates the experimentally observed gradients. Budding yeast cells cultured in a gradient of a chemical inducer expressed a reporter fluorescence protein in a concentration-dependent manner. This microfluidic platform serves as a versatile prototype applicable to a broad range of biomedical investigations.

Prunus mume extract exhibits antimicrobial activity against pathogenic oral bacteria.

PubMed

Seneviratne, Chamida J; Wong, Ricky W K; Hägg, Urban; Chen, Yong; Herath, Thanuja D K; Samaranayake, P Lakshman; Kao, Richard

2011-07-01

Prunus mume is a common fruit in Asia, which has been used in traditional Chinese medicine. In this study, we focused on the antimicrobial properties of Prunus mume extract against oral pathogens related to dental caries and periodontal diseases. A total of 15 oral pathogens including Streptococcus mutans, S. sobrinus, S. mitis, S. sanguinis, Lactobacillus acidophilus, P. gingivalis, Aggregatibacter actinomycetemcomitans, and Candida species were included in the study. Initially, agar diffusion assay was performed to screen the antimicrobial activities of Prunus mume extract. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were then determined for sensitive species. Effect of Prunus mume extract on human oral keratinocytes (HOK) viability was also tested. In the agar diffusion assay, drug suspension of 2 g/mL was able to inhibit all the bacterial species tested, but not the fungal species. MIC and MBC range of Prunus mume extract against the oral bacteria was 0.15625-0.0003 g/mL and P. gingivalis being the most susceptible species. Prune extract did not cause any detrimental effect on HOK. Prunus mume extract may be a potential candidate for developing an oral antimicrobial agent to control or prevent dental diseases associated with oral pathogenic bacteria. © 2011 The Authors. International Journal of Paediatric Dentistry © 2011 BSPD, IAPD and Blackwell Publishing Ltd.

Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

PubMed Central

Moran, Josephine C.; Crank, Emma L.; Ghabban, Hanaa A.; Horsburgh, Malcolm J.

2016-01-01

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential nutrient or produces antimicrobial compounds that its competitor is not resistant to. Here we describe a deferred growth inhibition assay, a method for assessing the ability of one bacterium to inhibit the growth of another through the production of antimicrobial compounds or through competition for nutrients. This technique has been used to investigate the correlation of nasal isolates with the exclusion of particular species from a community. This technique can also be used to screen for lantibiotic producers or potentially novel antimicrobials. The assay is performed by first culturing the test inhibitor-producing strain overnight on an agar plate, then spraying over the test competitor strain and incubating again. After incubation, the extent of inhibition can be measured quantitatively, through the size of the zone of clearing around the inhibitor-producing strain, and qualitatively, by assessing the clarity of the inhibition zone. Here we present the protocol for the deferred inhibition assay, describe ways to minimize variation between experiments, and define a clarity scale that can be used to qualitatively assess the degree of inhibition. PMID:27684443

INTERLABORATORY EVALUATION OF MI AGAR AND THE US ENVIRONMENTAL PROTECTION AGENCY-APPROVED MEMBRANE FILTER METHOD FOR THE RECOVERY OF TOTAL COLIFORMS AND ESCHERICHIA COLI FROM DRINKING WATER

EPA Science Inventory

A new membrane filter (MF) medium, MI agar, recently validated for use in recovering chlorine-damaged total coloiforms (TC) and Escherichia coli from drinking water, was compared to the US Environmental Protection Agency (EPA)-approved MF method(mEndo agar and nutrient agar suppl...

Inactivation of pathogenic bacteria inoculated onto a Bacto™ agar model surface using TiO2-UVC photocatalysis, UVC and chlorine treatments.

PubMed

Yoo, S; Ghafoor, K; Kim, S; Sun, Y W; Kim, J U; Yang, K; Lee, D-U; Shahbaz, H M; Park, J

2015-09-01

The aim of this study was to study inactivation of different pathogenic bacteria on agar model surface using TiO2-UV photocatalysis (TUVP). A unified food surface model was simulated using Bacto(™) agar, a routinely used microbial medium. The foodborne pathogenic bacteria Escherichia coli K12 (as a surrogate for E. coli O157:H7), Salmonella Typhimurium, Staphylococcus aureus and Listeria monocytogenes were inoculated onto the agar surface, followed by investigation of TUVP-assisted inactivation and morphological changes in bacterial cells. The TUVP process showed higher bacterial inactivation, particularly for Gram-negative bacteria, than UVC alone and a control (dark reaction). A TUVP treatment of 17·2 mW cm(-2) (30% lower than the UVC light intensity) reduced the microbial load on the agar surface by 4·5-6·0 log CFU cm(-2). UVC treatment of 23·7 mW cm(-2) caused 3·0-5·3 log CFU cm(-2) reduction. The use of agar model surface is effective for investigation of bacterial disinfection and TUVP is a promising nonthermal technique. The results showing effects of photocatalysis and other treatments for inactivation of bacterial pathogens on model surface can be useful for applying such processes for disinfection of fruit, vegetables and other similar surfaces. © 2015 The Society for Applied Microbiology.

Rapid detection of microbial DNA by a novel isothermal genome exponential amplification reaction (GEAR) assay.

PubMed

Prithiviraj, Jothikumar; Hill, Vincent; Jothikumar, Narayanan

2012-04-20

In this study we report the development of a simple target-specific isothermal nucleic acid amplification technique, termed genome exponential amplification reaction (GEAR). Escherichia coli was selected as the microbial target to demonstrate the GEAR technique as a proof of concept. The GEAR technique uses a set of four primers; in the present study these primers targeted 5 regions on the 16S rRNA gene of E. coli. The outer forward and reverse Tab primer sequences are complementary to each other at their 5' end, whereas their 3' end sequences are complementary to their respective target nucleic acid sequences. The GEAR assay was performed at a constant temperature 60 °C and monitored continuously in a real-time PCR instrument in the presence of an intercalating dye (SYTO 9). The GEAR assay enabled amplification of as few as one colony forming units of E. coli per reaction within 30 min. We also evaluated the GEAR assay for rapid identification of bacterial colonies cultured on agar media directly in the reaction without DNA extraction. Cells from E. coli colonies were picked and added directly to GEAR assay mastermix without prior DNA extraction. DNA in the cells could be amplified, yielding positive results within 15 min. Published by Elsevier Inc.

In vitro/in vivo evaluation of agar nanospheres for pulmonary delivery of bupropion HCl.

PubMed

Varshosaz, Jaleh; Minaiyan, Mohsen; Zaki, Mohammad Reza; Fathi, Milad; Jaleh, Hossein

2016-07-01

Bupropion HCl is an atypical antidepressant drug with rapid and high first-pass metabolism. Sustained release dosage form of this drug is suggested for reducing its side effects which are mainly seizures. The aim of the present study was to design pulmonary agar nanospheres of bupropion HCl with effective systemic absorption and extended release properties. Bupropion HCl was encapsulated in agar nanospheres by ionic gelation, and characterized for physical and release properties. Pharmacokinetic studies on nanospheres were performed on rats by intratracheal spraying of 5 mg/kg of drug in form of nanospheres compared to intravenous and pulmonary delivery of the same dose as simple solution of the drug. The optimized nanoparticles showed particle size of 320 ± 90 nm with polydispersity index of 0.85, the zeta potential of -29.6 mV, drug loading efficiency of 43.1 ± 0.28% and release efficiency of 66.7 ± 2%. The area under the serum concentration-time profile for the pulmonary nanospheres versus simple solution was 10 237.84 versus 28.8 µg/ml min, Tmax of 360 versus 60 min and the Cmax of 1927.93 versus9.93 ng/ml, respectively. The absolute bioavailability of the drug was 86.69% for nanospheres and 0.25% for pulmonary simple solution. Our results indicate that pulmonary delivery of bupropion loaded agar nanospheres achieves systemic exposure and extends serum levels of the drug.

Development of blood-yolk-polymyxin B-trimethoprim agar for the enumeration of Bacillus cereus in various foods.

PubMed

Kim, Dong-Hyeon; Kim, Hyunsook; Chon, Jung-Whan; Moon, Jin-San; Song, Kwang-Young; Seo, Kun-Ho

2013-07-15

Blood-yolk-polymyxin B-trimethoprim agar (BYPTA) was developed by the addition of egg yolk, laked horse blood, sodium pyruvate, polymyxin B, and trimethoprim, and compared with mannitol-yolk-polymyxin B agar (MYPA) for the isolation and enumeration of Bacillus cereus (B. cereus) in pure culture and various food samples. In pure culture, there was no statistical difference (p>0.05) between the recoverability and sensitivity of MYPA and BYPTA, whereas BYPTA exhibited higher specificity (p<0.05). To evaluate BYPTA agar with food samples, B. cereus was experimentally spiked into six types of foods, triangle kimbab, sandwich, misugaru, Saengsik, red pepper powder, and soybean paste. No statistical difference was observed in recoverability (p>0.05) between MYPA and BYPTA in all tested foods, whereas BYPTA exhibited higher selectivity than MYPA, especially in foods with high background microflora, such as Saengsik, red pepper powder, and soybean paste. The newly developed selective medium BYPTA could be a useful enumeration tool to assess the level of B. cereus in foods, particularly with high background microflora. Copyright © 2013 Elsevier B.V. All rights reserved.

[Variations in hyperbilirrubinemia in low birth weight newborns under phototherapy and continous or discontinous agar oral administration (author's transl)].

PubMed

Colomer, J; Moya, M; Marco, V; De Paredes, C; Escrivá, F; Vila, R

1975-06-01

Therapeutic attitude in hyperbilirrubinemia is always worth because other infrequent complications but not for this, less important. Phototherapy innocuousness, largely demonstrated, fosters its profilactic use at beginning and not only for those babies with serum bilirrubin over 10 mg % in the first day of life. Previously we have reported positive results with agar oral administration without collateral effects. On this grounds we have planned the following experience in a homogenous group of L.B.W.: one group was fed with agar previously to each formula administration; other group received the same amount of agar but divided in only three administrations in 24 hours; the last group received continuous phototherapy for 96 hours with a white cold fluorescent light from a source of 8-Vita-lite lamp of 40 watts with a intensity of 500 foot candle and 30 lumens. All of these babies weighed less than 2.500 g. and were between 10 and 90 percentil of Lubschenko diagram. They were fed with the same formula and same time table with no infusions, rejecting all that presented any type of pathology. Obstetric conditions were basically identical. This population was randomly divided in four groups. 1) Control group with no profilaxis, but with identical bilirrubin andhematocrit determinations. 2) Group with continuous agar oral administration, 125 mg. before each of the seven formula feeding. 3) Group with discontinuous agar administration, 250 mg. before three of the seven formula feeding. 4) Group with continuous phototherapy for 96 hours. These is initial identification of the groups with statistic signification, and after that a quantitative and sequential evolution of bilirrubin is analized in each group.

Fish meal extract bile esculin agar (FMBE) a selective medium for Bacteroides fragilis group.

PubMed

Beena, V K; Rao, S; Kotian, M; Shivananda, P G

1997-07-01

Fish meal extract bile esculin agar (FMBE) is prepared using Fish meal extract concentrate as the basal substance, for the selective isolation and presumptive identification of B.fragilis group. The efficiency of the medium was evaluated by growing stock cultures of B.fragilis groups as well as inoculating clinical specimens and comparing the results with Bacteroides bile esculin agar (BBE). All the 87 stock cultures of B.fragilis grew on FMBE and BBE. No other anaerobes tested grew on the medium. However 7 out of 65 neomycin resistant aerobes grew on the FMBE. From the 100 clinical samples, 62 strains of B. Fragilis group were recovered on FMBE and BBE, and 53 strains on supplemented BHIBA. The cost effectiveness, selectivity and the ability to detect esculin hydrolysis will enable FMBE as a suitable medium as comparable to that of BBE, if not superior.

Naphtho[1,2-b]furan-4,5-dione is a potent anti-MRSA agent against planktonic, biofilm and intracellular bacteria.

PubMed

Yang, Shih-Chun; Yen, Feng-Lin; Wang, Pei-Wen; Aljuffali, Ibrahim A; Weng, Yi-Han; Tseng, Chih-Hua; Fang, Jia-You

2017-09-01

Naphtho[1,2-b]furan-4,5-dione (N12D) and naphtho[2,3-b]furan-4,9-dione (N23D) are furanonaphthoquinone derivatives from natural resources. We examined the antimicrobial activity of N12D and N23D against drug-resistant Staphylococcus aureus. Minimum inhibitory concentration, minimum bactericidal concentration, bacterial viability and agar diffusion assay were conducted against methicillin-resistant S. aureus (MRSA) and clinical isolates of vancomycin-resistant S. aureus. The minimum inhibitory concentration of N12D and N23D against MRSA was 4.9-9.8 and 39 μM, respectively. With regard to the agar diffusion test, the inhibition zone of the quinone compounds was threefold larger than that of oxacillin. N12D was found to inhibit MRSA biofilm thickness from 24 to 16 μm as observed by confocal microscopy. N12D showed a significant reduction of the intracellular MRSA burden without decreasing the macrophage viability. The antibacterial mechanisms of N12D may be bacterial wall/membrane damage and disturbance of gluconeogenesis and the tricarboxylic acid cycle.

The growth of Steroidobacter agariperforans sp. nov., a novel agar-degrading bacterium isolated from soil, is enhanced by the diffusible metabolites produced by bacteria belonging to Rhizobiales.

PubMed

Sakai, Masao; Hosoda, Akifumi; Ogura, Kenjiro; Ikenaga, Makoto

2014-01-01

An agar-degrading bacterium was isolated from soil collected in a vegetable cropping field. The growth of this isolate was enhanced by supplying culture supernatants of bacteria belonging to the order Rhizobiales. Phylogenetic analysis based on 16S rRNA gene sequences indicated the novel bacterium, strain KA5-B(T), belonged to the genus Steroidobacter in Gammaproteobacteria, but differed from its closest relative, Steroidobacter denitrificans FS(T), at the species level with 96.5% similarity. Strain KA5-B(T) was strictly aerobic, Gram-negative, non-motile, non-spore forming, and had a straight to slightly curved rod shape. Cytochrome oxidase and catalase activities were positive. The strain grew on media containing culture supernatants in a temperature range of 15-37°C and between pH 4.5 and 9.0, with optimal growth occurring at 30°C and pH 6.0-8.0. No growth occurred at 10 or 42°C or at NaCl concentrations more than 3% (w/v). The main cellular fatty acids were iso-C15:0, C16:1ω7c, and iso-C17:1ω9c. The main quinone was ubiquinone-8 and DNA G+C content was 62.9 mol%. In contrast, strain FS(T) was motile, did not grow on the agar plate, and its dominant cellular fatty acids were C15:0 and C17:1ω8c. Based on its phylogenetic and phenotypic properties, strain KA5-B(T) (JCM 18477(T) = KCTC 32107(T)) represents a novel species in genus Steroidobacter, for which the name Steroidobacter agariperforans sp. nov. is proposed.

Inhibition of Granulopoiesis in Diffusion Chambers by a Granulocyte Chalone

DTIC Science & Technology

1974-07-01

culture has o 5_g a been well documented. ’ Breivik et al. , using chamber to chamber transfers in nonpretreated hosts, have illustrated the...1972. 7. Breivik , H. and Benestad, H. B. Regulation of granulocyte and macrophage formation in diffusion chamber cultures of mouse...haematopoietic cells. Exptl. Cell Res. 70:340-348, 1972. 8. Breivik , H., Benestad, H. B. and B^yum, A. Diffusion chamber and spleen colony assay of murine

Bacteriophage-based assays for the rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a meta-analysis.

PubMed

Pai, Madhukar; Kalantri, Shriprakash; Pascopella, Lisa; Riley, Lee W; Reingold, Arthur L

2005-10-01

To summarize, using meta-analysis, the accuracy of bacteriophage-based assays for the detection of rifampicin resistance in Mycobacterium tuberculosis. By searching multiple databases and sources we identified a total of 21 studies eligible for meta-analysis. Of these, 14 studies used phage amplification assays (including eight studies on the commercial FASTPlaque-TB kits), and seven used luciferase reporter phage (LRP) assays. Sensitivity, specificity, and agreement between phage assay and reference standard (e.g. agar proportion method or BACTEC 460) results were the main outcomes of interest. When performed on culture isolates (N=19 studies), phage assays appear to have relatively high sensitivity and specificity. Eleven of 19 (58%) studies reported sensitivity and specificity estimates > or =95%, and 13 of 19 (68%) studies reported > or =95% agreement with reference standard results. Specificity estimates were slightly lower and more variable than sensitivity; 5 of 19 (26%) studies reported specificity <90%. Only two studies performed phage assays directly on sputum specimens; although one study reported sensitivity and specificity of 100 and 99%, respectively, another reported sensitivity of 86% and specificity of 73%. Current evidence is largely restricted to the use of phage assays for the detection of rifampicin resistance in culture isolates. When used on culture isolates, these assays appear to have high sensitivity, but variable and slightly lower specificity. In contrast, evidence is lacking on the accuracy of these assays when they are directly applied to sputum specimens. If phage-based assays can be directly used on clinical specimens and if they are shown to have high accuracy, they have the potential to improve the diagnosis of MDR-TB. However, before phage assays can be successfully used in routine practice, several concerns have to be addressed, including unexplained false positives in some studies, potential for contamination and indeterminate

THE MICROGARDENING COOKBOOK, DIRECTIONS FOR PREPARING DISHES AND TUBES OF STERILE NUTRIENT AGAR.

ERIC Educational Resources Information Center

CHANDLER, MARION N.

THIS BOOKLET WAS PREPARED FOR TEACHER USE IN ASSOCIATION WITH THE ELEMENTARY SCIENCE STUDY UNIT "MICROGARDENING." IT CONTAINS DIRECTIONS FOR PREPARING CULTURE DISHES AND TUBES OF NUTRIENT STERILE AGAR FOR FUNGAL AND/OR BACTERIAL GROWTH. IT INCLUDES (1) LISTS OF NEEDED SUPPLIES AND EQUIPMENT, (2) DIRECTIONS FOR THE PREPARATION AND…

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.

Variation in the excitability of developed D. discoideum cells as a function of agar concentration in the substrate

NASA Astrophysics Data System (ADS)

Oikawa, Noriko; Bae, Albert; Amselem, Gabriel; Bodenschatz, Eberhard

2010-03-01

In the absence of nutrients, Dictyostelium discoideum cells enter a developmental cycle--they signal each other, aggregate, and ultimately form fruiting bodies. During the signaling stage, the cells relay waves of cyclic adenosine 3',5' monophosphate (cAMP). We observed a transition from spiral to circular patterns in the signaling wave, depending on the agar concentration of the substrate. In this talk we will present the changes in the times for the onset of signaling and synchronization versus agar concentration, as measured by spectral entropy. We also will discuss the origin of these effects.

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

Evaluation of the BD Max Cdiff assay for the detection of toxigenic Clostridium difficile in human stool specimens.

PubMed

Putsathit, Papanin; Morgan, Justin; Bradford, Damien; Engelhardt, Nelly; Riley, Thomas V

2015-02-01

The Becton Dickinson (BD) PCR-based GeneOhm Cdiff assay has demonstrated a high sensitivity and specificity for detecting Clostridium difficile. Recently, the BD Max platform, using the same principles as BD GeneOhm, has become available in Australia. This study aimed to investigate the sensitivity and specificity of BD Max Cdiff assay for the detection of toxigenic C. difficile in an Australian setting. Between December 2013 and January 2014, 406 stool specimens from 349 patients were analysed with the BD Max Cdiff assay. Direct and enrichment toxigenic culture were performed on bioMérieux ChromID C. difficile agar as a reference method. isolates from specimens with discrepant results were further analysed with an in-house PCR to detect the presence of toxin genes. The overall prevalence of toxigenic C. difficile was 7.2%. Concordance between the BD Max assay and enrichment culture was 98.5%. The sensitivity, specificity, positive predictive value and negative predictive value for the BD Max Cdiff assay were 95.5%, 99.0%, 87.5% and 99.7%, respectively, when compared to direct culture, and 91.7%, 99.0%, 88.0% and 99.4%, respectively, when compared to enrichment culture. The new BD Max Cdiff assay appeared to be an excellent platform for rapid and accurate detection of toxigenic C. difficile.

Draft genome of agar-degrading marine bacterium Gilvimarinus agarilyticus JEA5.

PubMed

Lee, Youngdeuk; Lee, Su-Jin; Park, Gun-Hoo; Heo, Soo-Jin; Umasuthan, Navaneethaiyer; Kang, Do-Hyung; Oh, Chulhong

2015-06-01

Gilvimarinus agarilyticus JEA5, which effectively degrades agar, was isolated from the seawater of Jeju Island, Republic of Korea. Here, we report the draft genome sequence of G. agarilyticus JEA5 with a total genome size of 4,179,438bp from 2 scaffolds (21 contigs) with 53.15% G+C content. Various polysaccharidases including 11 predicted agarases were observed from the draft genome of G. agarilyticus JEA5. Copyright © 2015 Elsevier B.V. All rights reserved.

Kinetics and equilibrium modelling of lead uptake by algae Gelidium and algal waste from agar extraction industry.

PubMed

Vilar, Vítor J P; Botelho, Cidália M S; Boaventura, Rui A R

2007-05-08

Pb(II) biosorption onto algae Gelidium, algal waste from agar extraction industry and a composite material was studied. Discrete and continuous site distribution models were used to describe the biosorption equilibrium at different pH (5.3, 4 and 3), considering competition among Pb(II) ions and protons. The affinity distribution function of Pb(II) on the active sites was calculated by the Sips distribution. The Langmuir equilibrium constant was compared with the apparent affinity calculated by the discrete model, showing higher affinity for lead ions at higher pH values. Kinetic experiments were conducted at initial Pb(II) concentrations of 29-104 mgl(-1) and data fitted to pseudo-first Lagergren and second-order models. The adsorptive behaviour of biosorbent particles was modelled using a batch mass transfer kinetic model, which successfully predicts Pb(II) concentration profiles at different initial lead concentration and pH, and provides significant insights on the biosorbents performance. Average values of homogeneous diffusivity, D(h), are 3.6 x 10(-8); 6.1 x 10(-8) and 2.4 x 10(-8)cm(2)s(-1), respectively, for Gelidium, algal waste and composite material. The concentration of lead inside biosorbent particles follows a parabolic profile that becomes linear near equilibrium.

CD3-T cell receptor modulation is selectively induced in CD8 but not CD4 lymphocytes cultured in agar.

PubMed Central

Oudrhiri, N; Farcet, J P; Gourdin, M F; M'Bemba, E; Gaulard, P; Katz, A; Divine, M; Galazka, A; Reyes, F

1990-01-01

The CD3-T cell receptor (TcR) complex is central to the immune response. Upon binding by specific ligands, internalized CD3-TcR molecules increase, and either T cell response or unresponsiveness may ensue depending on the triggering conditions. Using semi-solid agar culture, we have shown previously that quiescent CD4 but not CD8 lymphocytes generate clonal colonies under phytohaemagglutinin stimulation. Here we have demonstrated that the agar induces selective CD3-TcR modulation in the CD8 and not in the CD4 subset. CD8 lymphocytes preactivated in liquid culture and recultured in agar with exogenous recombinant interleukin-2 generate colonies with a modulated CD3-TcR surface expression. The peptides composing the CD3-TcR complex are synthesized in CD8 colonies as well as in CD4; however, the CD3 gamma chain is phosphorylated at a higher level in CD8 colonies. A component of the agar polymer, absent in agarose, appears to be the ligand that induces differential CD3-TcR modulation in the CD8 subset. In contrast to agar culture, CD8 colonies can be derived from quiescent CD8 lymphocytes in agarose. These CD8 colonies express unmodulated CD-TcR. CD3-TcR modulation with anti-CD3 monoclonal antibody prior to culturing in agarose inhibits the colony formation. We conclude that given triggering conditions can result in both CD3-TcR modulation and inhibition of the proliferative response selectively in the CD8 lymphocyte subset and not in the CD4. Images Fig. 3 Fig. 4 Fig. 5 PMID:2146997

Use of culture- and ELISA-based toxin assay for detecting Clostridium Difficile, a neglected pathogen: A single-center study from a tertiary care setting.

PubMed

Lall, Sujata; Nataraj, Gita; Mehta, Preeti

2017-01-01

Clostridium difficile is a Gram-positive spore-bearing anaerobic bacillus increasingly associated with both community- and hospital-acquired colitis and diarrhea. It is the most common identifiable bacterial cause of healthcare-associated diarrhea associated with antibiotic use and one of the most common anaerobic infections. The diagnosis of C. difficile infection includes detection of toxin A/B in stool specimens by direct enzyme immunoassay, culture of pathogen from the stool specimens using a selective agar Cycloserine-Cefoxitin fructose agar (CCFA), tissue culture assay, and detection of glutamate dehydrogenase an enzyme produced by C. difficile. With few reports from India on this disease, the present study was planned to throw more light on the prevalence and utility of laboratory diagnostic methods for C. difficile-associated diarrhea (CDAD). After taking approval from the Ethics Committee, 150 patients with antibiotic-associated diarrhea were taken as a study group and fifty patients with exposure to antibiotics but who did not develop diarrhea were taken as controls. Stool specimen was processed for both culture on CCFA and toxin detection by IVD Tox A + B ELISA. Only four specimens were culture positive, whereas 13 were ELISA positive. All culture-positive isolates were toxigenic. C. difficile was neither isolated nor its toxin detected in the control group. Culture- and toxin-based assays may not detect all cases of CDAD. Based on the results of the present study, culture does not provide any additional yield over toxin assay. Better diagnostic modalities would be required to prove CDAD.

Spin echo versus stimulated echo diffusion tensor imaging of the in vivo human heart

PubMed Central

von Deuster, Constantin; Stoeck, Christian T.; Genet, Martin; Atkinson, David

2015-01-01

Purpose To compare signal‐to‐noise ratio (SNR) efficiency and diffusion tensor metrics of cardiac diffusion tensor mapping using acceleration‐compensated spin‐echo (SE) and stimulated echo acquisition mode (STEAM) imaging. Methods Diffusion weighted SE and STEAM sequences were implemented on a clinical 1.5 Tesla MR system. The SNR efficiency of SE and STEAM was measured (b = 50–450 s/mm2) in isotropic agar, anisotropic diffusion phantoms and the in vivo human heart. Diffusion tensor analysis was performed on mean diffusivity, fractional anisotropy, helix and transverse angles. Results In the isotropic phantom, the ratio of SNR efficiency for SE versus STEAM, SNRt(SE/STEAM), was 2.84 ± 0.08 for all tested b‐values. In the anisotropic diffusion phantom the ratio decreased from 2.75 ± 0.05 to 2.20 ± 0.13 with increasing b‐value, similar to the in vivo decrease from 2.91 ± 0.43 to 2.30 ± 0.30. Diffusion tensor analysis revealed reduced deviation of helix angles from a linear transmural model and reduced transverse angle standard deviation for SE compared with STEAM. Mean diffusivity and fractional anisotropy were measured to be statistically different (P < 0.001) between SE and STEAM. Conclusion Cardiac DTI using motion‐compensated SE yields a 2.3–2.9× increase in SNR efficiency relative to STEAM and improved accuracy of tensor metrics. The SE method hence presents an attractive alternative to STEAM based approaches. Magn Reson Med 76:862–872, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. PMID:26445426

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

The Mediterranean red alga Asparagopsis taxiformis has antifungal activity against Aspergillus species.

PubMed

Genovese, Giuseppa; Leitner, Sandra; Minicante, Simona A; Lass-Flörl, Cornelia

2013-09-01

The red algae Asparagopsis taxiformis collected from the Straits of Messina (Italy) were screened for antifungal activity against Aspergillus species. EUCAST methodology was applied and extracts showed antifungal activity against A. fumigatus, A. terreus and A. flavus. The lowest minimum inhibitory concentrations observed were <0.15 mg ml(-1) and the highest were >5 mg ml(-1) for Aspergillus spp. tested. Agar diffusion assays confirmed antifungal activity of A. taxiformis extracts in Aspergillus species. © 2013 Blackwell Verlag GmbH.

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

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

The thin-layer agar method for direct phenotypic detection of multi- and extensively drug-resistant tuberculosis.

PubMed

Ardizzoni, E; Mulders, W; Kotrikadze, T; Aspindzelashvili, R; Goginashvili, L; Pangtey, H; Varaine, F; Bastard, M; Rigouts, L; de Jong, B C

2015-12-01

Molecular techniques rapidly detect resistance to rifampicin (RMP) and isoniazid (INH), but do not eliminate the need for culture-based drug susceptibility testing (DST) against other drugs. The thin-layer agar (TLA) test, a non-commercial direct DST method, has demonstrated good performance for INH and RMP; however, evidence is still limited, and its applicability for DST of ofloxacin (OFX) and kanamycin (KM) is unknown. We compared 279 TLA DST results with those of MGIT for INH and RMP, and 280 results for OFX and KM with those of the 7H11 agar proportion method, obtained from 320 smear-positive samples from 165 Georgian TB patients. Discrepancies were solved by comparison with a composite reference standard. The prevalence of multidrug-resistant tuberculosis (TB) was 30 of 164 patients (18.3%), 2 (6.7%) of whom had extensively drug-resistant TB. TLA showed 94.7%, 98.2%, 100% and 78.9% sensitivity, respectively, for INH, RMP, OFX and KM, with 100% specificity. Average time to results was 7 days in TLA, 23 in MGIT and 49 for 7H11 agar. In low-resource settings, TLA can be applied for the rapid detection of resistance to INH, RMP and fluoroquinolones. Further studies are necessary to improve sensitivity to KM and further assess its performance for OFX and other drugs and its applicability in field conditions.

[Evaluation of four methods for detecting methicillin-resistant Staphylococcus aureus isolates from clinical specimens at a regional hospital in Mexico].

PubMed

Acosta-Pérez, Gabriel; Rodríguez-Ábrego, Gabriela; Longoria-Revilla, Ernesto; Castro-Mussot, María Eugenia

2012-01-01

To estimate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in clinical isolates and to compare different methods for detection of MRSA in a lab with limited available personnel and resources. 140 Staphylococcus aureus strains isolated from patients in several departments were assayed for β-lactamase production, MIC-Vitek 2 oxacillin, ChromID MRSA, disk diffusion in agar for cefoxitin 30 μg and PBP2a detection. The results of conventional tests were compared with the "gold standard" PCR test for mecA gene. Cohen´s kappa index was also calculated in order to evaluate the intra assay agreement between the used methods. The found prevalence was 90.7%. Sensitivity and specificity were: disk diffusion for cefoxitin 97 and 92% respectively, MIC Vitek 2-XL 97 and 69%, ChromoID MRSA 97 and 85%, and PBP2a detection 98 and 100%. All methods are very good for detecting MRSA, choosing a method to use will depend on each laboratory infrastructure.

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae)

PubMed Central

Valencia, Laura Carolina; García, Adriana; Ramírez-Pinilla, Martha Patricia; Fuentes, Jorge Luis

2011-01-01

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies. PMID:22215974

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae).

PubMed

Valencia, Laura Carolina; García, Adriana; Ramírez-Pinilla, Martha Patricia; Fuentes, Jorge Luis

2011-10-01

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies.

Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes.

PubMed

Bienert, Gerd P; Møller, Anders L B; Kristiansen, Kim A; Schulz, Alexander; Møller, Ian M; Schjoerring, Jan K; Jahn, Thomas P

2007-01-12

The metabolism of aerobic organisms continuously produces reactive oxygen species. Although potentially toxic, these compounds also function in signaling. One important feature of signaling compounds is their ability to move between different compartments, e.g. to cross membranes. Here we present evidence that aquaporins can channel hydrogen peroxide (H2O2). Twenty-four aquaporins from plants and mammals were screened in five yeast strains differing in sensitivity toward oxidative stress. Expression of human AQP8 and plant Arabidopsis TIP1;1 and TIP1;2 in yeast decreased growth and survival in the presence of H2O2. Further evidence for aquaporin-mediated H2O2 diffusion was obtained by a fluorescence assay with intact yeast cells using an intracellular reactive oxygen species-sensitive fluorescent dye. Application of silver ions (Ag+), which block aquaporin-mediated water diffusion in a fast kinetics swelling assay, also reversed both the aquaporin-dependent growth repression and the H2O2-induced fluorescence. Our results present the first molecular genetic evidence for the diffusion of H2O2 through specific members of the aquaporin family.

Development of edible films from tapioca starch and agar, enriched with red cabbage (Brassica oleracea) as a sausage deterioration bio-indicator

NASA Astrophysics Data System (ADS)

Aditya Wardana, Ata; Dewanti Widyaningsih, Tri

2017-12-01

Sausage spoilage has been identified as a cause of some food poisoning cases. Development of a bioindicator film is one of the alternative methods to detect sausage deterioration. The objectives of this paper were to develop a bioindicator edible films (BEF) from tapioca starch (TS), agar, and red cabbage juice (RC), and to evaluate its performance on sausage deterioration detection. The experiment had a 3x3 randomized factorial experimental design (agar: 3, 5, 7% by weight of TS; RC: 10, 15, 20% v/v based on 100% of suspension). Glycerol was used as the plasticizer. The results showed that the addition of agar into the film solution increased the thickness, elongation, and tensile strength, and decreased water vapour transmission rate (WVTR). While the addition of RC increased the thickness, but decreased elongation, tensile strength, and WVTR. BEF consisting of 2% tapioca starch, 7% (w/w) agar and 10 % (v/v) RC was chosen to apply on sausage. It could detect an increase in the microbial population and in the pH variations as result of sausage deterioration at 24, 48, and 72 h shown through color changes of BEF from bright purple at 0 h to light purple, dark purple-blue, and purple-green color respectively.

High density diffusion-free nanowell arrays.

PubMed

Takulapalli, Bharath R; Qiu, Ji; Magee, D Mitchell; Kahn, Peter; Brunner, Al; Barker, Kristi; Means, Steven; Miersch, Shane; Bian, Xiaofang; Mendoza, Alex; Festa, Fernanda; Syal, Karan; Park, Jin G; LaBaer, Joshua; Wiktor, Peter

2012-08-03

Proteomics aspires to elucidate the functions of all proteins. Protein microarrays provide an important step by enabling high-throughput studies of displayed proteins. However, many functional assays of proteins include untethered intermediates or products, which could frustrate the use of planar arrays at very high densities because of diffusion to neighboring features. The nucleic acid programmable protein array (NAPPA) is a robust in situ synthesis method for producing functional proteins just-in-time, which includes steps with diffusible intermediates. We determined that diffusion of expressed proteins led to cross-binding at neighboring spots at very high densities with reduced interspot spacing. To address this limitation, we have developed an innovative platform using photolithographically etched discrete silicon nanowells and used NAPPA as a test case. This arrested protein diffusion and cross-binding. We present confined high density protein expression and display, as well as functional protein-protein interactions, in 8000 nanowell arrays. This is the highest density of individual proteins in nanovessels demonstrated on a single slide. We further present proof of principle results on ultrahigh density protein arrays capable of up to 24000 nanowells on a single slide.

High Density Diffusion-Free Nanowell Arrays

PubMed Central

Takulapalli, Bharath R; Qiu, Ji; Magee, D. Mitchell; Kahn, Peter; Brunner, Al; Barker, Kristi; Means, Steven; Miersch, Shane; Bian, Xiaofang; Mendoza, Alex; Festa, Fernanda; Syal, Karan; Park, Jin; LaBaer, Joshua; Wiktor, Peter

2012-01-01

Proteomics aspires to elucidate the functions of all proteins. Protein microarrays provide an important step by enabling high-throughput studies of displayed proteins. However, many functional assays of proteins include untethered intermediates or products, which could frustrate the use of planar arrays at very high densities because of diffusion to neighboring features. The nucleic acid programmable protein array (NAPPA), is a robust, in situ synthesis method for producing functional proteins just-in-time, which includes steps with diffusible intermediates. We determined that diffusion of expressed proteins led to cross-binding at neighboring spots at very high densities with reduced inter-spot spacing. To address this limitation, we have developed an innovative platform using photolithographically-etched discrete silicon nanowells and used NAPPA as a test case. This arrested protein diffusion and cross-binding. We present confined high density protein expression and display, as well as functional protein-protein interactions, in 8,000 nanowell arrays. This is the highest density of individual proteins in nano-vessels demonstrated on a single slide. We further present proof of principle results on ultra-high density protein arrays capable of up to 24,000 nanowells on a single slide. PMID:22742968

Significant antibacterial activity and synergistic effects of camel lactoferrin with antibiotics against methicillin-resistant Staphylococcus aureus (MRSA).

PubMed

Redwan, Elrashdy M; El-Baky, Nawal Abd; Al-Hejin, Ahmed M; Baeshen, Mohammed N; Almehdar, Hussein A; Elsaway, Abdulrahman; Gomaa, Abu-Bakr M; Al-Masaudi, Saad Berki; Al-Fassi, Fahad A; AbuZeid, Isam Eldin; Uversky, Vladimir N

2016-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) causes major healthcare problems in many countries, as it is present as several hospital- and community-associated strains. Hospital-associated MRSA is one of the most prevalent nosocomial pathogens throughout the world and infections caused by community-acquired MRSA are rising. This emphasizes the need for new and efficient anti-MRSA agents. We evaluated the antibacterial effects of camel lactoferrin (cLf) and human lactoferrin (hLf) alone and in combination with several antibiotics against MRSA. Antimicrobials were tested against MRSA and an S. aureus control strain by the agar disc diffusion method. The minimum inhibitory concentration (MIC) was determined for antimicrobials by the broth microdilution method. Synergy between cLf or hLf and antibiotics was examined by checkerboard and time-kill assays. The agar disc diffusion assay showed that MRSA growth was inhibited by cLf at 0.25-3 mg/ml and hLf at 1-3 mg/ml. cLf demonstrated 3 times higher inhibitory activity against MRSA than hLf in terms of MIC values (250 vs. 750 μg/ml, respectively). Biotinylated cLf was recognized by two membrane proteins of MRSA, 66-67 KDa. Combinations of cLf or hLf and oxacillin or vancomycin at sub-MIC levels enhanced in vitro antibacterial activity against MRSA compared with each agent alone. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Characterization of internal structure of hydrated agar and gelatin matrices by cryo-SEM.

PubMed

Rahbani, Janane; Behzad, Ali R; Khashab, Niveen M; Al-Ghoul, Mazen

2013-02-01

There has been a considerable interest in recent years in developing polymer gel matrices for many important applications such as 2DE for quantization and separation of a variety of proteins and drug delivery system to control the release of active agents. However, a well-defined knowledge of the ultrastructures of the gels has been elusive. In this study, we report the characterization of two different polymers used in 2DE: Gelatin, a naturally occurring polymer derived from collagen (protein) and agar, a polymer of polysaccharide (sugar) origin. Low-temperature SEM is used to examine the internal structure of these gels in their frozen natural hydrated states. Results of this study show that both polymers have an array of hollow cells that resembles honeycomb structures. While agar pores are almost circular, the corresponding Gaussian curve is very broad exhibiting a range of radii from nearly 370 to 700 nm. Gelatin pores are smaller and more homogeneous reflecting a narrower distribution from nearly 320 to 650 nm. Overall, these ultrastructural findings could be used to correlate with functions of the polymers. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

QIN DAWG Validation of Gradient Nonlinearity Bias Correction Workflow for Quantitative Diffusion-Weighted Imaging in Multicenter Trials.

PubMed

Malyarenko, Dariya I; Wilmes, Lisa J; Arlinghaus, Lori R; Jacobs, Michael A; Huang, Wei; Helmer, Karl G; Taouli, Bachir; Yankeelov, Thomas E; Newitt, David; Chenevert, Thomas L

2016-12-01

Previous research has shown that system-dependent gradient nonlinearity (GNL) introduces a significant spatial bias (nonuniformity) in apparent diffusion coefficient (ADC) maps. Here, the feasibility of centralized retrospective system-specific correction of GNL bias for quantitative diffusion-weighted imaging (DWI) in multisite clinical trials is demonstrated across diverse scanners independent of the scanned object. Using corrector maps generated from system characterization by ice-water phantom measurement completed in the previous project phase, GNL bias correction was performed for test ADC measurements from an independent DWI phantom (room temperature agar) at two offset locations in the bore. The precomputed three-dimensional GNL correctors were retrospectively applied to test DWI scans by the central analysis site. The correction was blinded to reference DWI of the agar phantom at magnet isocenter where the GNL bias is negligible. The performance was evaluated from changes in ADC region of interest histogram statistics before and after correction with respect to the unbiased reference ADC values provided by sites. Both absolute error and nonuniformity of the ADC map induced by GNL (median, 12%; range, -35% to +10%) were substantially reduced by correction (7-fold in median and 3-fold in range). The residual ADC nonuniformity errors were attributed to measurement noise and other non-GNL sources. Correction of systematic GNL bias resulted in a 2-fold decrease in technical variability across scanners (down to site temperature range). The described validation of GNL bias correction marks progress toward implementation of this technology in multicenter trials that utilize quantitative DWI.

QIN DAWG Validation of Gradient Nonlinearity Bias Correction Workflow for Quantitative Diffusion-Weighted Imaging in Multicenter Trials

PubMed Central

Malyarenko, Dariya I.; Wilmes, Lisa J.; Arlinghaus, Lori R.; Jacobs, Michael A.; Huang, Wei; Helmer, Karl G.; Taouli, Bachir; Yankeelov, Thomas E.; Newitt, David; Chenevert, Thomas L.

2017-01-01

Previous research has shown that system-dependent gradient nonlinearity (GNL) introduces a significant spatial bias (nonuniformity) in apparent diffusion coefficient (ADC) maps. Here, the feasibility of centralized retrospective system-specific correction of GNL bias for quantitative diffusion-weighted imaging (DWI) in multisite clinical trials is demonstrated across diverse scanners independent of the scanned object. Using corrector maps generated from system characterization by ice-water phantom measurement completed in the previous project phase, GNL bias correction was performed for test ADC measurements from an independent DWI phantom (room temperature agar) at two offset locations in the bore. The precomputed three-dimensional GNL correctors were retrospectively applied to test DWI scans by the central analysis site. The correction was blinded to reference DWI of the agar phantom at magnet isocenter where the GNL bias is negligible. The performance was evaluated from changes in ADC region of interest histogram statistics before and after correction with respect to the unbiased reference ADC values provided by sites. Both absolute error and nonuniformity of the ADC map induced by GNL (median, 12%; range, −35% to +10%) were substantially reduced by correction (7-fold in median and 3-fold in range). The residual ADC nonuniformity errors were attributed to measurement noise and other non-GNL sources. Correction of systematic GNL bias resulted in a 2-fold decrease in technical variability across scanners (down to site temperature range). The described validation of GNL bias correction marks progress toward implementation of this technology in multicenter trials that utilize quantitative DWI. PMID:28105469

Synthesis and characterization of high-surface-area millimeter-sized silica beads with hierarchical multi-modal pore structure by the addition of agar

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

Han, Yosep; Choi, Junhyun; Tong, Meiping, E-mail: tongmeiping@iee.pku.edu.cn

2014-04-01

Millimeter-sized spherical silica foams (SSFs) with hierarchical multi-modal pore structure featuring high specific surface area and ordered mesoporous frameworks were successfully prepared using aqueous agar addition, foaming and drop-in-oil processes. The pore-related properties of the prepared spherical silica (SSs) and SSFs were systematically characterized by field emission-scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), small-angle X-ray diffraction (SAXRD), Hg intrusion porosimetry, and N{sub 2} adsorption–desorption isotherm measurements. Improvements in the BET surface area and total pore volume were observed at 504 m{sup 2} g{sup −1} and 5.45 cm{sup 3} g{sup −1}, respectively, after an agar addition and foaming process. Despitemore » the increase in the BET surface area, the mesopore wall thickness and the pore size of the mesopores generated from the block copolymer with agar addition were unchanged based on the SAXRD, TEM, and BJH methods. The SSFs prepared in the present study were confirmed to have improved BET surface area and micropore volume through the agar loading, and to exhibit interconnected 3-dimensional network macropore structure leading to the enhancement of total porosity and BET surface area via the foaming process. - Highlights: • Millimeter-sized spherical silica foams (SSFs) are successfully prepared. • SSFs exhibit high BET surface area and ordered hierarchical pore structure. • Agar addition improves BET surface area and micropore volume of SSFs. • Foaming process generates interconnected 3-D network macropore structure of SSFs.« less

Dexamethasone diffusion across contact lenses is inhibited by Staphylococcus epidermidis biofilms in vitro.

PubMed

Brothers, Kimberly M; Nau, Amy C; Romanowski, Eric G; Shanks, Robert M Q

2014-10-01

This study was designed to measure the impact of bacterial biofilms on diffusion of an ocular therapeutic through silicone hydrogel bandage lenses in vitro. An assay was designed to study the passage of a commonly used steroid, dexamethasone, through silicone hydrogel soft contact lenses. Diffused dexamethasone was measured using a spectrophotometer over a period of 18 hours and quantified using a standard curve. This assay was performed with control and Staphylococcus epidermidis biofilm-coated contact lenses comprised of lotrafilcon A and methafilcon. Biofilms were formed in brain heart infusion broth supplemented with D-glucose. The presented data validate a simple in vitro model that can be used to measure the penetration of a topical therapeutic through silicone hydrogel soft contact lenses. Using this model, we measured a reduction in dexamethasone diffusion up to 88% through S. epidermidis biofilm-coated silicone hydrogel lenses compared with control lenses. The results of this in vitro study demonstrate that bacterial biofilms impede dexamethasone diffusion through silicone hydrogel contact lenses and warrant future studies regarding the clinical benefit of using ocular therapeutics in the setting of bandage contact lens use for corneal epithelial defects.

Dexamethasone diffusion across contact lenses is inhibited by Staphylococcus epidermidis biofilms in vitro

PubMed Central

Brothers, Kimberly M.; Nau, Amy C.; Romanowski, Eric G.; Shanks, Robert M. Q.

2014-01-01

Purpose This study was designed to measure the impact of bacterial biofilms on diffusion of an ocular therapeutic through silicone hydrogel bandage lenses in vitro. Methods An assay was designed to study the passage of a commonly used steroid dexamethasone through the silicone hydrogel soft contact lenses. Diffused dexamethasone was measured using a spectrophotometer over a period of 18 hours and quantified using a standard curve. This assay was performed with control and Staphylococcus epidermidis biofilm-coated contact lenses composed of lotrafilcon A and methafilcon. Biofilms were formed in brain heart infusion broth supplemented with D-glucose. Results The presented data validate a simple in vitro model that can be used to measure penetration of a topical therapeutic through silicone hydrogel soft contact lenses. Using this model we measured a reduction of dexamethasone diffusion by up to 88% through S. epidermidis biofilm-coated silicon hydrogel lenses compared to control lenses. Conclusions The results of this in vitro study demonstrate that bacterial biofilms impede dexamethasone diffusion through silicon hydrogel contact lenses, and warrant future studies regarding the clinical benefit of using ocular therapeutics in the setting of bandage contact lens use for corneal epithelial defects. PMID:25090165

Oral chronic ethanol administration to rodents by agar gel diet.

PubMed

Bykov, I; Palmén, M; Piirainen, L; Lindros, K O

2004-01-01

Chronic ethanol administration to rodents requires specially designed equipment and is labor intensive. Here we report a new procedure. A commercial liquid diet preparation was made into a gel by addition of 0.5% agar. The gel, containing 5.3% ethanol, was offered in Falcon tubes equipped with a feeding opening. The gel consumption by C57/Bl mice resulted in high blood ethanol levels (average 43 mM). After 6 weeks, marked liver steatosis and significantly increased serum alanine aminotransferase levels had developed. Administration of ethanol in a nutritionally adequate gel provides a simple method for studies on chronic ethanol effects in rodents.

Antimicrobial Activity of Lippia Species from the Brazilian Semiarid Region Traditionally Used as Antiseptic and Anti-Infective Agents

PubMed Central

Pinto, Cristiana da Purificação; Rodrigues, Velize Dias; Pinto, Fernanda da Purificação; Pinto, Renata da Purificação; Uetanabaro, Ana Paula Trovatti; Pinheiro, Carla Santos Ribeiro; Gadea, Suzana Ferreira Magalhães; Silva, Tânia Regina dos Santos; Lucchese, Angélica Maria

2013-01-01

Lippia origanoides Kunth, Lippia alnifolia Schauer, and Lippia thymoides Martius and Schauer are shrubs used in the traditional Brazilian medicine as antiseptics, as well as in the treatment of infectious diseases. This study was designed to investigate the antibacterial and antifungal activities of the methanolic extracts of these species, as new potential sources of antimicrobial drugs. The antimicrobial activity of methanolic extracts was investigated against resistant yeasts and bacteria by agar disk diffusion. Then, the MIC determination of the most active species and its fractions in hexane, dichloromethane, ethyl acetate, and water was performed. By the agar diffusion assay, all species were active against at least two microorganisms, giving evidence to support their use in the popular medicine. L. origanoides leaves exhibited the widest antimicrobial action, inhibiting the growth of two Gram-positive bacteria and two yeasts; this activity was also confirmed by the MIC evaluation. The fractionation of L. origanoides crude extracts improved the activity in spectrum and intensity. The results obtained in this study indicate that L. origanoides may be a promising alternative in the treatment of bacterial and fungal infections and in the seeking of new antimicrobial drugs. PMID:24109492

Effect of permeable flow on cyclic layering in solidifying magma bodies: Insights from an analog experiment of diffusion-precipitation systems

NASA Astrophysics Data System (ADS)

Toramaru, A.; Yamauchi, S.

2012-04-01

Characteristic structures such as rhythmic layering, cress cumulate, cross bedding, perpendicular feldspar rock etc, are commonly observed in layered intrusion or shallow magmatic intrusions. These structures result from complex processes including thermal and compositional diffusions, crystallization, crystal settling, convection and interaction among three phases (crystals, bubble, melt). In order to understand how the differentiation proceeds in solidifying magma bodies from each characteristic structure together with chemical signatures, it is necessary to evaluate the relative importance among these elemental processes on structures. As an attempt to evaluate the effect of advection on a diffusion-related structure, we carried out an analog experiment of Liesegang system using lead-iodide (PbI2) crystallization in agar media which have been normally used to prohibit convection. In the ordinary Liesegang band formation experiments including only diffusion and crystallization kinetics without any advection and convection, the precipitation bands develop with regular spacing following a geometric progression due to two-component diffusion and reaction with supersaturation. This type of banding structure has been advocated as the same type of cyclic layering or vesicle layering (a sort of rhythmic layering) in dykes or sills. In order to see the effect of one-directional advection on Liesegang band, we apply the electric field (5 V to 25 V for a distance 15 cm) along the concentration gradient in agar media, thereby counteracting flows of lead anion Pb2+ and iodide ion I- are driven at constant velocities. The flows of anions and ions are equivalent to the permeable flows in porous media of crystal mush. The resultant precipitation structures exhibit very curious banding structure in which band spacings do not change with distance, are nearly constant and quite narrow, depending on the voltage, unlike those in ordinary Liesegang bands in which band spacings

Characterization of a novel alkaline arylsulfatase from Marinomonas sp. FW-1 and its application in the desulfation of red seaweed agar.

PubMed

Wang, Xueyan; Duan, Delin; Xu, Jiachao; Gao, Xin; Fu, Xiaoting

2015-10-01

A bacterial strain capable of hydrolyzing sulfate ester bonds of p-nitrophenyl sulfate (pNPS) and agar was isolated from the coast area of Qingdao, China. It was identified as Marinomonas based on its 16S rRNA gene sequence and named as Marinomonas sp. FW-1. An arylsulfatase with a recovery of 13 % and a fold of 12 was purified to a homogeneity using ion exchange and gel filtration chromatographies. The enzyme was composed of a single polypeptide chain with the molecular mass of 33 kDa estimated using SDS-PAGE. The optimal pH and temperature of arylsulfatase were pH 9.0 and 45, respectively. Arylsulfatase was stable over pH 8-11 and at temperature below 55 °C. The K m and V max of this enzyme for the hydrolysis of pNPS were determined to be 13.73 and 270.27 μM/min, respectively. The desulfation ratio against agar from red seaweed Gelidium amansii and Gracilaria lemaneiformis were 86.11 and 89.61 %, respectively. There was no difference between the DNA electrophoresis spectrum on the gel of the arylsulfatase-treated G. amansii agar and that of the commercial agarose. Therefore, this novel alkaline arylsulfatase might have a great potential for application in enzymatic conversion of agar to agarose.

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.

Quantitative Assessment of Antimicrobial Activity of PLGA Films Loaded with 4-Hexylresorcinol

PubMed Central

Kemme, Michael; Heinzel-Wieland, Regina

2018-01-01

Profound screening and evaluation methods for biocide-releasing polymer films are crucial for predicting applicability and therapeutic outcome of these drug delivery systems. For this purpose, we developed an agar overlay assay embedding biopolymer composite films in a seeded microbial lawn. By combining this approach with model-dependent analysis for agar diffusion, antimicrobial potency of the entrapped drug can be calculated in terms of minimum inhibitory concentrations (MICs). Thus, the topical antiseptic 4-hexylresorcinol (4-HR) was incorporated into poly(lactic-co-glycolic acid) (PLGA) films at different loadings up to 3.7 mg/cm2 surface area through a solvent casting technique. The antimicrobial activity of 4-HR released from these composite films was assessed against a panel of Gram-negative and Gram–positive bacteria, yeasts and filamentous fungi by the proposed assay. All the microbial strains tested were susceptible to PLGA-4-HR films with MIC values down to 0.4% (w/w). The presented approach serves as a reliable method in screening and quantifying the antimicrobial activity of polymer composite films. Moreover, 4-HR-loaded PLGA films are a promising biomaterial that may find future application in the biomedical and packaging sector. PMID:29324696

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.

Investigating plasmodesmata genetics with virus-induced gene silencing and an agrobacterium-mediated GFP movement assay.

PubMed

Brunkard, Jacob O; Burch-Smith, Tessa M; Runkel, Anne M; Zambryski, Patricia

2015-01-01

Plasmodesmata (PD) are channels that connect the cytoplasm of adjacent plant cells, permitting intercellular transport and communication. PD function and formation are essential to plant growth and development, but we still know very little about the genetic pathways regulating PD transport. Here, we present a method for assaying changes in the rate of PD transport following genetic manipulation. Gene expression in leaves is modified by virus-induced gene silencing. Seven to ten days after infection with Tobacco rattle virus carrying a silencing trigger, the gene(s) of interest is silenced in newly arising leaves. In these new leaves, individual cells are then transformed with Agrobacterium to express GFP, and the rate of GFP diffusion via PD is measured. By measuring GFP diffusion both within the epidermis and between the epidermis and mesophyll, the assay can be used to study the effects of silencing a gene(s) on PD transport in general, or transport through secondary PD specifically. Plant biologists working in several fields will find this assay useful, since PD transport impacts plant physiology, development, and defense.

Antibacterial activity of selected plant essential oils against Escherichia coli O157:H7.

PubMed

Burt, S A; Reinders, R D

2003-01-01

To quantify the antibacterial properties of five essential oils (EO) on a non-toxigenic strain of Escherichia coli O157:H7 in the presence and absence of a stabilizer and an emulsifier and at three different temperatures. Five EOs known to exhibit antibacterial properties were screened by disc diffusion assay and the most active were selected for further study in microdilution colorimetric assays. Oregano (Origanum vulgare) and thyme (Thymus vulgaris; light and red varieties) EO had the strongest bacteriostatic and bactericidal properties, followed by bay (Pimenta racemosa) and clove bud (Eugenia caryophyllata synonym: Syzygium aromaticum) EO. Oregano oil was colicidal at 625 microl l(-1) at 10, 20 and 37 degrees C. The addition of 0.05% (w/v) agar as stabilizer reinforced the antibacterial properties, particularly at 10 degrees C, whereas 0.25% (w/v) lecithin reduced antibacterial activity. Scanning electron micrographs showed extensive morphological changes to treated cells. Oregano and thyme EO possess significant in vitro colicidal and colistatic properties, which are exhibited in a broad temperature range and substantially improved by the addition of agar as stabilizer. Bay and clove bud EO are less active. Lecithin diminished antibacterial properties. The bactericidal concentration of oregano EO irreversibly damaged E. coli O157:H7 cells within 1 min. Oregano and light thyme EO, particularly when enhanced by agar stabilizer, may be effective in reducing the number or preventing the growth of E. coli O157:H7 in foods.

Evaluation of VIDAS UP Listeria assay (LPT) for the detection of Listeria in a variety of foods and environmental surfaces: First Action 2013.10.

PubMed

Crowley, Erin; Bird, Patrick; Flannery, Jonathan; Benzinger, M Joseph; Fisher, Kiel; Boyle, Megan; Huffman, Travis; Bastin, Ben; Bedinghaus, Paige; Judd, William; Hoang, Thao; Agin, James; Goins, David; Johnson, Ronald L

2014-01-01

The VIDAS UP Listeria (LPT) is an automated rapid screening enzyme phage-ligand based assay for the detection of Listeria species in human food products and environmental samples. The VIDAS LPT method was compared in a multi-laboratory collaborative study to AOAC Official Method 993.12 Listeria monocytogenes in Milk and Dairy Products reference method following current AOAC guidelines. A total of 14 laboratories participated, representing government and industry, throughout the United States. One matrix, queso fresco (soft Mexican cheese), was analyzed using two different test portion sizes, 25 and 125 g. Samples representing each test portion size were artificially contaminated with Listeria species at three levels, an uninoculated control level [0 colony-forming units (CFU)/test portion], a low-inoculum level (0.2-2 CFU/test portion), and a high-inoculum level (2-5 CFU/test portion). For this evaluation, 1800 unpaired replicate test portions were analyzed by either the VIDAS LPT or AOAC 993.12. Each inoculation level was analyzed using the Probability of Detection (POD) statistical model. For the low-level inoculated test portions, difference in collaborator POD (dLPOD) values of 0.01, (-0.10, 0.13), with 95% confidence intervals, were obtained for both 25 and 125 g test portions. The range of the confidence intervals for dLPOD values for both the 25 and 125 g test portions contains the point 0.0 indicating no statistically significant difference in the number of positive samples detected between the VIDAS LPT and the AOAC methods. In addition to Oxford agar, VIDAS LPT test portions were confirmed using Agar Listeria Ottavani and Agosti (ALOA), a proprietary chromogenic agar for the identification and differentiation of L. monocytogenes and Listeria species. No differences were observed between the two selective agars. The VIDAS LPT method, with the optional ALOA agar confirmation method, was adopted as Official First Action status for the detection of Listeria

A modified MacConkey agar for selective enumeration of necrotoxigenic E. coli O55 and probiotic E. coli Nissle 1917.

PubMed

Splichalova, Alla; Splichal, Igor; Sonnenborn, Ulrich; Rada, Vojtech

2014-09-01

An agar selective enumeration of necrotoxigenic Escherichia coli O55 (NTEC2) and probiotic E. coli Nissle 1917, using modified MacConkey agar, was developed to study bacterial interference between these E. coli strains in a gnotobiotic piglet model. Replacement of lactose with saccharose in the agar enables the direct visual enumeration of red colonies of E. coli O55 and yellow colonies of E. coli Nissle 1917 that are co-cultured in the same Petri dish. A total of 336 colonies (168 for each color) were subjected to strain-specific PCR identification with LNA probes. Sensitivity, specificity, and positive and negative predictive values were 96.43%, 95.83%, 95.86% and 96.41% respectively in E. coli O55, and 98.21%, 97.02%, 97.06% and 98.19% respectively in E. coli Nissle 1917. Color-based enumeration of both E. coli strains in colonic contents and mesenteric lymph nodes homogenates of gnotobiotic piglets demonstrated the applicability of this method for the gnotobiotic piglet model of enteric diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of a modified Cefsulodin-Irgasan-Novobiocin agar for isolation of Yersinia spp.

PubMed

Tan, Lai Kuan; Ooi, Peck Toung; Carniel, Elisabeth; Thong, Kwai Lin

2014-01-01

Y. enterocolitica and Y. pseudotuberculosis are important food borne pathogens. However, the presence of competitive microbiota makes the isolation of Y. enterocolitica and Y. pseudotuberculosis from naturally contaminated foods difficult. We attempted to evaluate the performance of a modified Cefsulodin-Irgasan-Novobiocin (CIN) agar in the differentiation of Y. enterocolitica from non-Yersinia species, particularly the natural intestinal microbiota. The modified CIN enabled the growth of Y. enterocolitica colonies with the same efficiency as CIN and Luria-Bertani agar. The detection limits of the modified CIN for Y. enterocolitica in culture medium (10 cfu/ml) and in artificially contaminated pork (10(4) cfu/ml) were also comparable to those of CIN. However, the modified CIN provided a better discrimination of Yersinia colonies from other bacteria exhibiting Yersinia-like colonies on CIN (H2S-producing Citrobacter freundii, C. braakii, Enterobacter cloacae, Aeromonas hydrophila, Providencia rettgeri, and Morganella morganii). The modified CIN exhibited a higher recovery rate of Y. enterocolitica from artificially prepared bacterial cultures and naturally contaminated samples compared with CIN. Our results thus demonstrated that the use of modified CIN may be a valuable means to increase the recovery rate of food borne Yersinia from natural samples, which are usually contaminated by multiple types of bacteria.

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.

Applying Agar's Concept of "Languaculture" to Explain Asian Students' Experiences in the Australian Tertiary Context

ERIC Educational Resources Information Center

Norris, Lindy; Tsedendamba, Nara

2015-01-01

This paper reports part of a broader qualitative case study of Asian students "translation" (Agar, 2006) to study in an Australian university. The paper is concerned with the experiences of eight participants and their involvement in a training programme in the use of language learning strategies (LLS) to support their engagement with…

Initial cytotoxicity assays of media for sulfate-reducing bacteria: An endodontic biopharmaceutical product under development.

PubMed

Heggendorn, Fabiano Luiz; Silva, Gabriela Cristina de Carvalho; Cardoso, Elisama Azevedo; Castro, Helena Carla; Gonçalves, Lúcio Souza; Dias, Eliane Pedra; Lione, Viviane de Oliveira Freitas; Lutterbach, Márcia Teresa Soares

2016-01-01

This study assessed the cell viability of the inoculation vehicle of BACCOR (a combination of sulfate-reducing bacteria plus a culture media for bacteria), a biopharmaceutical product under development for dental use as aid in fractured endodontic file removal from the root canal. Different culture media for bacteria were evaluated: modified Postgate E (MCP-E mod), Modified Postgate E without Agar-agar (MCP-E w/Ag), Postgate C with Agar-agar (MCP-C Ag) and Postgate C without Agar-agar (MCP-C w/Ag). Cytotoxicity was quantified by the MTT test, exposing L929 and Vero cell lines to the vehicles over 24 h. The exposure of L929 cell line to MCP-E w/Ag resulted in biocompatibility (52% cell viability), while the exposure of the Vero kidney line revealed only MCP-E mod as cytotoxic. When diluted, all the vehicles showed biocompatibility with both cell lines. MCP-E w/Ag was the vehicle chosen for BACCOR, because of its biocompatibility with the cells used.

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.

Can Ceftazidime-Avibactam and Aztreonam Overcome β-Lactam Resistance Conferred by Metallo-β-Lactamases in Enterobacteriaceae?

PubMed

Marshall, Steven; Hujer, Andrea M; Rojas, Laura J; Papp-Wallace, Krisztina M; Humphries, Romney M; Spellberg, Brad; Hujer, Kristine M; Marshall, Emma K; Rudin, Susan D; Perez, Federico; Wilson, Brigid M; Wasserman, Ronald B; Chikowski, Linda; Paterson, David L; Vila, Alejandro J; van Duin, David; Kreiswirth, Barry N; Chambers, Henry F; Fowler, Vance G; Jacobs, Michael R; Pulse, Mark E; Weiss, William J; Bonomo, Robert A

2017-04-01

Based upon knowledge of the hydrolytic profile of major β-lactamases found in Gram-negative bacteria, we tested the efficacy of the combination of ceftazidime-avibactam (CAZ-AVI) with aztreonam (ATM) against carbapenem-resistant enteric bacteria possessing metallo-β-lactamases (MBLs). Disk diffusion and agar-based antimicrobial susceptibility testing were initially performed to determine the in vitro efficacy of a unique combination of CAZ-AVI and ATM against 21 representative Enterobacteriaceae isolates with a complex molecular background that included bla IMP , bla NDM , bla OXA-48 , bla CTX-M , bla AmpC , and combinations thereof. Time-kill assays were conducted, and the in vivo efficacy of this combination was assessed in a murine neutropenic thigh infection model. By disk diffusion assay, all 21 isolates were resistant to CAZ-AVI alone, and 19/21 were resistant to ATM. The in vitro activity of CAZ-AVI in combination with ATM against diverse Enterobacteriaceae possessing MBLs was demonstrated in 17/21 isolates, where the zone of inhibition was ≥21 mm. All isolates demonstrated a reduction in CAZ-AVI agar dilution MICs with the addition of ATM. At 2 h, time-kill assays demonstrated a ≥4-log 10 -CFU decrease for all groups that had CAZ-AVI with ATM (8 μg/ml) added, compared to the group treated with CAZ-AVI alone. In the murine neutropenic thigh infection model, an almost 4-log 10 -CFU reduction was noted at 24 h for CAZ-AVI (32 mg/kg every 8 h [q8h]) plus ATM (32 mg/kg q8h) versus CAZ-AVI (32 mg/kg q8h) alone. The data presented herein require us to carefully consider this new therapeutic combination to treat infections caused by MBL-producing Enterobacteriaceae . Copyright © 2017 American Society for Microbiology.

Performance of Chromogenic Candida agar and CHROMagar Candida in recovery and presumptive identification of monofungal and polyfungal vaginal isolates.

PubMed

Ozcan, Kadri; Ilkit, Macit; Ates, Aylin; Turac-Bicer, Aygul; Demirhindi, Hakan

2010-02-01

Chromogenic Candida agar (OCCA) is a novel medium facilitating isolation and identification of Candida albicans, C. tropicalis, and C. krusei, as well as indicating polyfungal population in clinical samples. We compare the performance of OCCA, to CHROMagar Candida (CAC) and Sabouraud chloramphenicol agar (SCA). Vaginal swab samples from 392 women were simultaneously inoculated onto three study media. A total of 161 (41.1%) were found to be positive for fungi of which 140 (87%) were monofungal, and 21 (13%) polyfungal. One-hundred and fifty-seven samples (97.5%) were positive on CAC, 156 (96.9%) on OCCA, 148 (91.9%) on SCA and 144 (89.4%) samples were positive on all three media. The yeasts were identified by conventional methods including germ tube test, microscopic morphology on cornmeal-Tween 80 agar, and the commercial API 20C AUX. The 182 isolates were C. albicans (n = 104), C. glabrata (n = 51), C. krusei (n = 7), C. tropicalis (n = 5), C. famata (n = 3), C. kefyr (n = 3), C. zeylanoides (n = 3), C. colliculosa (n = 2), and other species of Candida (n = 4). Among the 21 polyfungal populations, 20 (95.2%) were detected in OCCA, 14 (66.7%) in CAC, and 13 (61.9%) in CAC and OCCA (P <0.05). Most polyfungal populations (47.6%) yielded C. albicans + C. glabrata. The efficiency of both chromogenic media for C. albicans was >or=92.9% at 72 h. OCCA is more efficient and reliable for rapidly identifying C. albicans and polyfungal populations than CAC. However, CAC is more efficient for identifying C. krusei and C. tropicalis. A chromogenic agar with a higher isolation rate of yeasts and better detection of polyfungal populations than SCA, is suggested as a medium of first choice when available.

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 Microplate Growth Inhibition Assay for Screening Bacteriocins against Listeria monocytogenes to Differentiate Their Mode-of-Action.

PubMed

Vijayakumar, Paul Priyesh; Muriana, Peter M

2015-06-11

Lactic acid bacteria (LAB) have historically been used in food fermentations to preserve foods and are generally-recognized-as-safe (GRAS) by the FDA for use as food ingredients. In addition to lactic acid; some strains also produce bacteriocins that have been proposed for use as food preservatives. In this study we examined the inhibition of Listeria monocytogenes 39-2 by neutralized and non-neutralized bacteriocin preparations (Bac+ preps) produced by Lactobacillus curvatus FS47; Lb. curvatus Beef3; Pediococcus acidilactici Bac3; Lactococcus lactis FLS1; Enterococcus faecium FS56-1; and Enterococcus thailandicus FS92. Activity differences between non-neutralized and neutralized Bac+ preps in agar spot assays could not readily be attributed to acid because a bacteriocin-negative control strain was not inhibitory to Listeria in these assays. When neutralized and non-neutralized Bac+ preps were used in microplate growth inhibition assays against L. monocytogenes 39-2 we observed some differences attributed to acid inhibition. A microplate growth inhibition assay was used to compare inhibitory reactions of wild-type and bacteriocin-resistant variants of L. monocytogenes to differentiate bacteriocins with different modes-of-action (MOA) whereby curvaticins FS47 and Beef3, and pediocin Bac3 were categorized to be in MOA1; enterocins FS92 and FS56-1 in MOA2; and lacticin FLS1 in MOA3. The microplate bacteriocin MOA assay establishes a platform to evaluate the best combination of bacteriocin preparations for use in food applications as biopreservatives against L. monocytogenes.

A Microplate Growth Inhibition Assay for Screening Bacteriocins against Listeria monocytogenes to Differentiate Their Mode-of-Action

PubMed Central

Vijayakumar, Paul Priyesh; Muriana, Peter M.

2015-01-01

Lactic acid bacteria (LAB) have historically been used in food fermentations to preserve foods and are generally-recognized-as-safe (GRAS) by the FDA for use as food ingredients. In addition to lactic acid; some strains also produce bacteriocins that have been proposed for use as food preservatives. In this study we examined the inhibition of Listeria monocytogenes 39-2 by neutralized and non-neutralized bacteriocin preparations (Bac+ preps) produced by Lactobacillus curvatus FS47; Lb. curvatus Beef3; Pediococcus acidilactici Bac3; Lactococcus lactis FLS1; Enterococcus faecium FS56-1; and Enterococcus thailandicus FS92. Activity differences between non-neutralized and neutralized Bac+ preps in agar spot assays could not readily be attributed to acid because a bacteriocin-negative control strain was not inhibitory to Listeria in these assays. When neutralized and non-neutralized Bac+ preps were used in microplate growth inhibition assays against L. monocytogenes 39-2 we observed some differences attributed to acid inhibition. A microplate growth inhibition assay was used to compare inhibitory reactions of wild-type and bacteriocin-resistant variants of L. monocytogenes to differentiate bacteriocins with different modes-of-action (MOA) whereby curvaticins FS47 and Beef3, and pediocin Bac3 were categorized to be in MOA1; enterocins FS92 and FS56-1 in MOA2; and lacticin FLS1 in MOA3. The microplate bacteriocin MOA assay establishes a platform to evaluate the best combination of bacteriocin preparations for use in food applications as biopreservatives against L. monocytogenes. PMID:26111195

Arthrornyces and Blastosporella, two new genera of conidia-producing lyophylloid agarics (Agaricales, Basidiornycota) from the neotropics

Treesearch

Timothy J. Baroni; Ana Esperanza Franco-molano; D. Jean Lodge; Daniel L. Lindner; Egon Horak; Valerie Hofstetter

2007-01-01

Two new genera encompassing three new species of lyophylloid agarics that produce conidia on the basidiomata are described. Arthromyces is a genus comprised of two very different arthrospore-producing mushroom species found in the Greater Antilles and Central America. Blastosporella is a monotypic genus with spherical balls of...

Comparison of effectiveness of wood decay fungi maintained by annual subculture on agar and stored in sterile water for 18 years.

PubMed

Richter, Dana L; Kangas, Laura C; Smith, Jill K; Laks, Peter E

2010-03-01

Fourteen isolates of basidiomycete decay fungi (12 species) were maintained for 18 years on agar slants transferred annually and also stored as mycelium-agar cores under cold sterile water without subculture. Isolates stored by each method were evaluated for decay effectiveness using a standard laboratory accelerated soil-block decay test. Effectiveness was measured by mean percent mass loss of wood blocks. There was no significant difference (p < or = 0.05) in decay effectiveness between storage methods for 12 of the fungus isolates tested. For the 2 fungi that showed a significant difference in the amount of decay with respect to storage method, 1 fungus (Fomitopsis lilacinogilva) produced more decay by the strain maintained as an agar slant, while the other fungus (Trametes versicolor) produced more decay by the strain stored in sterile water. Results suggested that storage under sterile water is an easy and effective method to store isolates of decay fungi for long periods, but as with any microbial storage method, careful monitoring of isolates upon revival is necessary.

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.

Calibration of the BASS acoustic current meter with carrageenan agar

USGS Publications Warehouse

Morrison, A.T.; Williams, A.J.; Martini, M.

1993-01-01

The BASS current meter can measure currents down to the millimeter per second range. Due to the dependence of zero offset on pressure, determining a sensor referenced velocity requires accurate in situ zeroing of the meter. Previously, flow was restricted during calibration by placing plastic bags around the acoustic volume. In this paper, bacterial grade and carrageenan agars are used in the laboratory to create a zero flow condition during calibration and are shown to be acoustically transparent. Additionally, the results of open ocean and dockside carrageenan and plastic bag comparisons are presented. Carrageenan is shown to reliably provide a low noise, zero mean flow environment that is largely independent of ambient conditions. The improved zeros make millimeter per second accuracy possible under field conditions.

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.

Effect of EDTA on Pb(II) Uptake and Translocation by Tumbleweed (Salsola Kali): Agar and Hydroponics Studies

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

de la Rosa, Guadalupe; Gardea-Torresdey, Jorge L.; Peralta-Videa, Jose R.

Environmental accumulation of Pb represents a worldwide health hazard. While conventional cleanup techniques are generally expensive and soil disturbing, phytoremediation represents an inexpensive friendly option for the removal of contaminants from soil and water. In this research, tumbleweed (Salsola kali) plants exposed for 15 days to Pb(NO3)2 at 80 and 125 ppm in hydroponics and agar media, demonstrated a high capacity to uptake lead. The results showed that the plants cultivated in agar accumulated 25563, 5534 and 2185 mg Pb kg-1 DW in roots, stems and leaves, respectively. Moreover, Pb concentrations found in hydroponically grown tumbleweed plants tissues were 30744,more » 1511 and 1421 mg kg-1 DW in roots, stems and leaves, respectively. It was observed that EDTA enhanced Pb translocation. No Pb phytotoxic effects were observed during the experimental time period. Cellular structural features were also observed using TEM.« less

Novel Single-Tube Agar-Based Test System for Motility Enhancement and Immunocapture of Escherichia coli O157:H7 by H7 Flagellar Antigen-Specific Antibodies

PubMed Central

Murinda, Shelton E.; Nguyen, Lien T.; Ivey, Susan J.; Almeida, Raul A.; Oliver, Stephen P.

2002-01-01

This paper describes a novel single-tube agar-based technique for motility enhancement and immunoimmobilization of Escherichia coli O157:H7. Motility indole ornithine medium and agar (0.4%, wt/vol) media containing either nutrient broth, tryptone broth, or tryptic soy broth (TSBA) were evaluated for their abilities to enhance bacterial motility. Twenty-six E. coli strains, including 19 O157:H7 strains, 1 O157:H− strain, and 6 generic E. coli strains, were evaluated. Test bacteria were stab inoculated in the center of the agar column, and tubes were incubated at 37°C for 18 to 96 h. Nineteen to 24 of the 26 test strains (73.1 to 92.3%) were motile in the different media. TSBA medium performed best and was employed in subsequent studies of motility enhancement and H7 flagellar immunocapture. H7 flagellar antiserum (30 and 60 μl) mixed with TSBA was placed as a band (1 ml) in the middle of an agar column separating the top (3-ml) and bottom (3-ml) agar layers. The top agar layer was inoculated with the test bacterial strains. The tubes were incubated at 37°C for 12 to 18 h and for 18 to 96 h. The specificity and sensitivity of the H7 flagellar immunocapture tests were 75 and 100%, respectively. The procedure described is simple and sensitive and could be adapted easily for routine use in laboratories that do not have sophisticated equipment and resources for confirming the presence of H7 flagellar antigens. Accurate and rapid identification of H7 flagellar antigen is critical for the complete characterization of E. coli O157:H7, owing to the immense clinical, public health, and economic significance of this food-borne pathogen. PMID:12454173

Biologically active traditional medicinal herbs from Balochistan, Pakistan.

PubMed

Zaidi, Mudassir A; Crow, Sidney A

2005-01-04

The biological activities of the following four important medicinal plants of Balochistan, Pakistan were checked; Grewia erythraea Schwein f. (Tiliaceae), Hymenocrater sessilifolius Fisch. and C.A. Mey (Lamiaceae), Vincetoxicum stocksii Ali and Khatoon (Asclepiadaceae) and Zygophyllum fabago L. (Zygophyllaceae). The methanolic extracts were fractionated into hexane, ethyl acetate, chloroform, butanol and water. The antifungal and antibacterial activities of these plants were determined against 12 fungal and 12 bacterial strains by agar well diffusion and disk diffusion assays. The extract of Zygophyllum fabago was found to be highly effective against Candida albicans and Escherichia coli. The extract of Vincetoxicum stocksii was also found to be significantly active against Candida albicans, Bacillus subtilis and Bacillus cereus. Extracts of Hymenocrater sessilifolius and Grewia erythraea showed good activity only against Pseudomonas aeruginosa.

Performance of a contact textile-based light diffuser for photodynamic therapy.

PubMed

Khan, Tania; Unternährer, Merthan; Buchholz, Julia; Kaser-Hotz, Barbara; Selm, Bärbel; Rothmaier, Markus; Walt, Heinrich

2006-03-01

Medical textiles offer a unique contact opportunity that could provide value-added comfort, reliability, and safety for light or laser-based applications. We investigated a luminous textile diffuser for use in photodynamic therapy. Textile diffusers are produced by an embroidery process. Plastic optical fibers are bent and sewn into textile to release light by macrobending. A reflective backing is incorporated to improve surface homogeneity, intensity, and safety. Clonogenic assay (MCF-7 cells) and trypan blue exclusion (NuTu19 cells) tests were performed in vitro using 0.1μg/ml m-THPC with three textile diffusers and a standard front lens diffuser. Heating effects were studied in solution and on human skin. PDT application in vivo was performed with the textile diffuser on equine sarcoids (three animals, 50mW/cm(2), 10-20J) and eight research animals. Lastly, computer simulations were performed to see how the textile diffuser might work on a curved object. At low fluency rate, there is a trend for the textile diffuser to have lower survival rates than the front lens diffuser for both cell lines. The textile diffuser was observed to retain more heat over a long period (>1min). All animals tolerated the treatments well and showed similar initial reactions. The simulations showed a likely focusing effect in a curved geometry. The initial feasibility and application using a textile-based optical diffuser has been demonstrated. Possibilities that provide additional practical advantages of the textile diffuser are discussed.

Orientation of Anopheles gambiae (Diptera: Culicidae) to Plant-Host Volatiles in a Novel Diffusion-Cage Olfactometer

PubMed Central

Otienoburu, Philip E.; Nikbakhtzadeh, Mahmood R.; Foster, Woodbridge A.

2016-01-01

A novel diffusion-cage olfactometer tested the responses of Anopheles gambiae Giles to plant volatiles. Green-leaf volatiles are often released from cut or injured plant tissue and may alter the headspace of plants used in olfactometer assays. The diffusion-cage olfactometer is designed for use with whole, intact plants, hence giving a more realistic behavioral assay. Its simple plastic construction, ease of assembly, and accommodation to whole plants makes it a useful tool for measuring mosquito orientation to plant volatiles within large enclosures. We compared its performance to that of the more commonly used T-tube wind-tunnel olfactometer, by testing the orientation of mosquitoes to volatiles of a few prevalent plants of eastern Africa reportedly utilized by An. gambiae for sugar: Parthenium hysterophorus (Asteraceae), Ricinus communis (Euphorbiaceae), Lantana camara (Verbenaceae), and Senna occidentalis (Fabaceae). Results indicate that the diffusion-cage olfactometer is an effective alternative to conventional wind-tunnel olfactometers, to test mosquito orientation to plant volatiles under seminatural conditions. PMID:26502752

Arthromyces and Blastosporella, two new genera of conidia-producing lyophylloid agarics (Agaricales, Basidiomycota) from the neotropics

Treesearch

Timothy J. Baroni; Ana Esperanza Franco-Molano; D. Jean Lodge; Daniel L. Lindner; Egon Horak; Valerie Hofstetter

2007-01-01

Two new genera encompassing three new species of lyophylloid agarics that produce conidia on the basidiomata are described. Arthromyces is a genus comprised of two very different arthrospore-producing mushroom species found in the Greater Antilles and Central America. Blastosporella is a monotypic genus with spherical balls of blastospores covering the pileus surface...

Diffuse reflectance imaging for non-melanoma skin cancer detection using laser feedback interferometry

NASA Astrophysics Data System (ADS)

Mowla, Alireza; Taimre, Thomas; Lim, Yah L.; Bertling, Karl; Wilson, Stephen J.; Prow, Tarl W.; Soyer, H. P.; Rakić, Aleksandar D.

2016-04-01

We propose a compact, self-aligned, low-cost, and versatile infrared diffuse-reflectance laser imaging system using a laser feedback interferometry technique with possible applications in in vivo biological tissue imaging and skin cancer detection. We examine the proposed technique experimentally using a three-layer agar skin phantom. A cylindrical region with a scattering rate lower than that of the surrounding normal tissue was used as a model for a non-melanoma skin tumour. The same structure was implemented in a Monte Carlo computational model. The experimental results agree well with the Monte Carlo simulations validating the theoretical basis of the technique. Results prove the applicability of the proposed technique for biological tissue imaging, with the capability of depth sectioning and a penetration depth of well over 1.2 mm into the skin phantom.

Fabrication, Characterization, and Evaluation of Bionanocomposites Based on Natural Polymers and Antibiotics for Wound Healing Applications.

PubMed

Rădulescu, Marius; Holban, Alina Maria; Mogoantă, Laurențiu; Bălşeanu, Tudor-Adrian; Mogoșanu, George Dan; Savu, Diana; Popescu, Roxana Cristina; Fufă, Oana; Grumezescu, Alexandru Mihai; Bezirtzoglou, Eugenia; Lazar, Veronica; Chifiriuc, Mariana Carmen

2016-06-10

The aim of our research activity was to obtain a biocompatible nanostructured composite based on naturally derived biopolymers (chitin and sodium alginate) loaded with commercial antibiotics (either Cefuroxime or Cefepime) with dual functions, namely promoting wound healing and assuring the local delivery of the loaded antibiotic. Compositional, structural, and morphological evaluations were performed by using the thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and fourier transform infrared spectroscopy (FTIR) analytical techniques. In order to quantitatively and qualitatively evaluate the biocompatibility of the obtained composites, we performed the tetrazolium-salt (MTT) and agar diffusion in vitro assays on the L929 cell line. The evaluation of antimicrobial potential was evaluated by the viable cell count assay on strains belonging to two clinically relevant bacterial species (i.e., Escherichia coli and Staphylococcus aureus).

A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication.

PubMed

Thayanithy, Venugopal; O'Hare, Patrick; Wong, Phillip; Zhao, Xianda; Steer, Clifford J; Subramanian, Subbaya; Lou, Emil

2017-11-13

Tunneling nanotubes (TNTs) are naturally-occurring filamentous actin-based membranous extensions that form across a wide spectrum of mammalian cell types to facilitate long-range intercellular communication. Valid assays are needed to accurately assess the downstream effects of TNT-mediated transfer of cellular signals in vitro. We recently reported a modified transwell assay system designed to test the effects of intercellular transfer of a therapeutic oncolytic virus, and viral-activated drugs, between cells via TNTs. The objective of the current study was to demonstrate validation of this in vitro approach as a new method for effectively excluding diffusible forms of long- and close-range intercellular transfer of intracytoplasmic cargo, including exosomes/microvesicles and gap junctions in order to isolate TNT-selective cell communication. We designed several steps to effectively reduce or eliminate diffusion and long-range transfer via these extracellular vesicles, and used Nanoparticle Tracking Analysis to quantify exosomes following implementation of these steps. The experimental approach outlined here effectively reduced exosome trafficking by >95%; further use of heparin to block exosome uptake by putative recipient cells further impeded transfer of these extracellular vesicles. This validated assay incorporates several steps that can be taken to quantifiably control for extracellular vesicles in order to perform studies focused on TNT-selective communication.

An electrochemical approach to monitor pH change in agar media during plant tissue culture.

PubMed

Wang, Min; Ha, Yang

2007-05-15

In this work, metal oxide microelectrodes were developed to monitor pH change in agar media during plant tissue culture. An antimony wire was produced by a new approach "capillary melt method". The surface of the obtained antimony wire was oxidized in a potassium nitrate melt to fabricate an antimony oxide film for pH sensing. Characterization results show that the oxide layer grown on the wire surface consists of Sb(2)O(3) crystal phase. The sensing response, open-circuit potential, of the electrode has a good linear relationship (R(2)=1.00) with pH value of the test solution. Adding organic compounds into the test media would not affect the linear relationship, although the slope of the lines varied with different ingredients added. The antimony oxide electrodes were employed to continuously monitor pH change of agar culture media during a 2-week plant tissue culture of Dendrobium candidum. The antimony oxide electrode fabricated this way has the advantages of low cost, easy fabrication, fast response, and almost no contamination introduced into the system. It would be suitable for in situ and continuous pH measurement in many bio applications.

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

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

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.

Cytotoxicity of dental alloys, metals, and ceramics assessed by millipore filter, agar overlay, and MTT tests.

PubMed

Sjögren, G; Sletten, G; Dahl, J E

2000-08-01

Biocompatibility of dental materials is dependent on the release of elements from the materials. In addition, the composition, pretreatment, and handling of the materials influence the element release. This study evaluated the cytotoxicity of dental alloys, metals, and ceramics, with specific emphasis on the effects of altering the composition and the pretreatment. By using cells from a mouse fibroblast cell line and the agar overlay test, Millipore filter test, and MTT test, cytotoxicity of various metals, metal alloys, and ceramics for dental restoration were studied. Effects of altering the composition of a high noble gold alloy and of pretreatment of a ceramic-bonding alloy were also studied. In addition, the release of elements into the cell culture medium by the materials studied was measured using an inductively coupled plasma optical emission spectrophotometer. The results of the MTT test were analyzed statistically using ANOVA and Scheffé test at a significance level of P <.05. Specimens manufactured from materials intended for dental restorations and handled in accordance with the manufacturers' instructions were ranked from "noncytotoxic" to "mildly cytotoxic" according to the agar overlay and Millipore filter tests. For the MTT test, no significant differences were observed between these materials and controls, with the exception of JS C-gold and unalloyed titanium. The modified materials were ranked from "mildly cytotoxic" to "moderately cytotoxic" in the agar overlay and Millipore filter tests and from "noncytotoxic" to "moderately cytotoxic" in the MTT test. Thus, cytotoxicity was related to the alloy composition and treatment. The release of Cu and Zn seemed to be important for the cytotoxic effect. Alterations in the composition and the pretreatment can greatly influence the cytotoxicity, and the results stress the importance of carefully following the manufacturers' instructions when handling dental materials.

Antibacterial activities of extracts from Ugandan medicinal plants used for oral care.

PubMed

Ocheng, Francis; Bwanga, Freddie; Joloba, Moses; Borg-Karlson, Ann-Karin; Gustafsson, Anders; Obua, Celestino

2014-08-08

Medicinal plants are widely used for treatment of oral/dental diseases in Uganda. To investigate antibacterial activities of 16 commonly used medicinal plants on microorganisms associated with periodontal diseases (PD) and dental caries (DC). Pulp juice and solvent extracts (hexane, methanol and water) from the plants were tested against Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia associated with PD and Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus associated with DC. Tests were done using agar well-diffusion (pulp juice) and agar-dilution (Solvent extracts) assays. Pulp juice from Zanthoxylum chalybeum and Euclea latidens showed activity against all the bacteria, Zanthoxylum chalybeum being most active. Hexane extract from aerial part of Helichrysum odoratissimum was most active (MIC: 0.125-0.5 mg/ml). Methanol extract from leaves of Lantana trifolia showed activity against all bacteria (MIC: 0.25-1 mg/ml). Several of the tested plants showed antibacterial activities against bacteria associated with PD and DC, meriting further investigations. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Development of a selective myclobutanil agar (MBA) medium for the isolation of Fusarium species from asparagus fields.

PubMed

Vujanovic, Vladimir; Hamel, Chantal; Jabaji-Hare, Suha; St-Arnaud, Marc

2002-09-01

A new selective myclobutanil agar medium for the detection of Fusarium, species is proposed. Ten media formulations based on various selective agents (pentachloronitrobenzene (PCNB), Rose Bengal, malachite green, sodium hypochlorite, captan, benomyl, chlorotalonil, myclobutanil, thiram, and cupric sulfate) were compared. First, mycelium growth and colony appearance of Alternaria alternata, Aspergillus flavus, Cladosporium cladosporioides, Epicoccum nigrum, Fusarium sp., Fuisarium solani, Fusarium moniliforme, Fusarium oxysporum f.sp. dianthi, Penicillium sp., and Trichoderma viride isolates were compared. Second, the ability of the different media to isolate and enumerate fusaria from asparagus fields was evaluated. The myclobutanil-based medium showed the highest selectivity to Fusarium spp. growth but required a slightly longer incubation time (>5 d) than peptone-pentachloronitrobenzene-based agar (PPA) (< 5 d). PPA allowed a faster fusaria growth but also permited the growth of other moulds. The other media were less selective and did not allow to isolate fusaria or to differenciate them from other growing fungi.

Malt-yeast extract-sucrose agar, a suitable medium for enumeration and isolation of fungi from silage.

PubMed Central

Skaar, I; Stenwig, H

1996-01-01

A general medium named malt-yeast extract-sucrose agar (MYSA) containing oxgall was designed. The medium was intended for the enumeration and isolation of molds and yeasts in routine examinations of animal feed stuffs. In this study MYSA was tested as a general medium for mycological examination of silage. The medium was compared with dichloran-rose bengal medium (DRBC) in an examination of more than 500 specimens of big bale grass silage. Selected characteristics of known fungal species commonly isolated from feeds were examined after growth on MYSA and DRBC and on malt extract agar, used as a noninhibitory control medium. MYSA suppressed bacterial growth, without affecting the growth of fungi common in feeds. The fungi growing on MYSA were easily recognized, and the medium seemed to slow radial growth of fungal colonies, which permitted, easy counting. The number of species found was higher on MYSA than on DRBC. When we compared MYSA with DRBC for mycological examination of grass silage samples, MYSA was found to be the medium of choice. PMID:8837416

Melatonin Protects Human Cells from Clustered DNA Damages, Killing and Acquisition of Soft Agar Growth Induced by X-rays or 970 MeV/n Fe ions

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

Das, B.; Sutherland, B.; Bennett, P. V.

We tested the ability of melatonin (N-acetyl-5 methoxytryptamine), a highly effective radical scavenger and human hormone, to protect DNA in solution and in human cells against induction of complex DNA clusters and biological damage induced by low or high linear energy transfer radiation (100 kVp X-rays, 970 MeV/nucleon Fe ions). Plasmid DNA in solution was treated with increasing concentrations of melatonin (0.0-3.5 mM) and were irradiated with X-rays. Human cells (28SC monocytes) were also irradiated with X-rays and Fe ions with and without 2 mM melatonin. Agarose plugs containing genomic DNA were subjected to Contour Clamped Homogeneous Electrophoretic Field (CHEF)more » followed by imaging and clustered DNA damages were measured by using Number Average length analysis. Transformation experiments on human primary fibroblast cells using soft agar colony assay were carried out which were irradiated with Fe ions with or without 2 mM melatonin. In plasmid DNA in solution, melatonin reduced the induction of single- and double-strand breaks. Pretreatment of human 28SC cells for 24 h before irradiation with 2 mM melatonin reduced the level of X-ray induced double-strand breaks by {approx}50%, of abasic clustered damages about 40%, and of Fe ion-induced double-strand breaks (41% reduction) and abasic clusters (34% reduction). It decreased transformation to soft agar growth of human primary cells by a factor of 10, but reduced killing by Fe ions only by 20-40%. Melatonin's effective reduction of radiation-induced critical DNA damages, cell killing, and striking decrease of transformation suggest that it is an excellent candidate as a countermeasure against radiation exposure, including radiation exposure to astronaut crews in space travel.« less

Comparison of Neisseria gonorrhoeae MICs obtained by Etest and agar dilution for ceftriaxone, cefpodoxime, cefixime and azithromycin.

PubMed

Gose, Severin; Kong, Carol J; Lee, Yer; Samuel, Michael C; Bauer, Heidi M; Dixon, Paula; Soge, Olusegun O; Lei, John; Pandori, Mark

2013-12-01

We evaluated Neisseria gonorrhoeae Etest minimum inhibitory concentrations (MICs) relative to agar dilution MICs for 664 urethral isolates for ceftriaxone (CRO) and azithromycin (AZM), 351 isolates for cefpodoxime (CPD) and 315 isolates for cefixime (CFM). Etest accurately determined CPD, CFM and AZM MICs, but resulted in higher CRO MICs.

Effect of Antioxidant Mixtures on Growth and Ochratoxin A Production of Aspergillus Section Nigri Species under Different Water Activity Conditions on Peanut Meal Extract Agar

PubMed Central

Barberis, Carla; Astoreca, Andrea; Fernandez-Juri, María Guillermina; Dalcero, Ana María; Magnoli, Carina

2010-01-01

The effect of mixtures of antioxidants butylated hydroxyanisol (BHA) and propyl paraben (PP) on lag phase, growth rate and ochratoxin A (OTA) production by four Aspergillus section Nigri strains was evaluated on peanut meal extract agar (PMEA) under different water activities (aw). The antioxidant mixtures used were: BHA + PP (mM), M1 (0.5 + 0.5), M2 (1.0 + 0.5), M3 (2.5 + 0.5), M4 (0.5 + 1.0), M5 (1.0 + 1.0), M6 (2.5 + 1.0), M7 (5.0 + 2.5) and M8 (10 + 2.5). The mixture M8 completely suppressed mycelial growth for all strains. A significant stimulation in OTA production was observed with mixtures M1 to M5 mainly at the highest aw; whereas M6, M7 and M8 completely inhibited OTA production in all strains assayed; except M6 in A. carbonarius strain (RCP G). These results could enable a future intervention strategy to minimize OTA contamination. PMID:22069644

Enhanced catalytic efficiency of endo-β-agarase I by fusion of carbohydrate-binding modules for agar prehydrolysis.

PubMed

Alkotaini, Bassam; Han, Nam Soo; Kim, Beom Soo

2016-11-01

Recently, Microbulbifer thermotolerans JAMB-A94 endo-β-agarase I was expressed as catalytic domain (GH16) without a carbohydrate-binding module (CBM). In this study, we successfully constructed different fusions of GH16 with its original CBM6 and CBM13 derived from Catenovulum agarivorans. The optimum temperature and pH for fusions GH16-CBM6, GH16-CBM13, GH16-CBM6-CBM13 and GH16-CBM13-CBM6 were similar to GH16, at 55°C and pH 7. All the constructed fusions significantly enhanced the GH16 affinity (Km) and the catalytic efficiency (Kcat/Km) toward agar. Among them, GH16-CBM6-CBM13 exhibited the highest agarolytic activity, for which Km decreased from 3.67 to 2.11mg/mL and Kcat/Km increased from 98.6 (mg/mL) -1 sec -1 to 400.6 (mg/mL) -1 sec -1 . Moreover, all fusions selectively increased GH16 binding ability to agar, in which the highest binding ability of 95% was obtained with fusion GH16-CBM6-CBM13. Melted agar was prehydrolyzed with GH16-CBM6-CBM13, resulting in a degree of liquefaction of 45.3% and reducing sugar yield of 14.2%. Further addition of Saccharophagus degradans agarolytic enzymes resulted in mono-sugar yields of 35.4% for galactose and 31.5% for 3,6-anhydro-l-galactose. There was no pH neutralization step required and no 5-hydroxymethylfurfural detected, suggesting the potential of a new enzymatic prehydrolysis process for efficient production of bio-products such as biofuels. Copyright © 2016 Elsevier Inc. All rights reserved.

Optimization of modified Middlebrook 7H11 agar for isolation of Mycobacterium bovis from raw milk cheese.

PubMed

Forgrave, R; Donaghy, J A; Fisher, A; Rowe, M T

2014-10-01

Reports have highlighted the absence of contemporary peer reviewed publications pertaining to Mycobacterium bovis culture from raw milk and cheese. By replicating traditional methods, cheese-making methodology and equipment were devised to produce Cheddar (n = 6) and Caerphilly (n = 3) artificially contaminated with M. bovis (three genotypes) under stringent laboratory-containment guidelines for handling hazardous microbiological material. Middlebrook 7H11, modified for M. bovis isolation, was assessed for capacity to enumerate M. bovis despite changing cheese microflora and prolonged M. bovis exposure to the cheese matrix using maturing cheese test portions (n = 63; up to 16 weeks). Malachite green (MG) containing media isolated M. bovis at significantly (P < 0·05) lower levels than unmodified Middlebrook 7H11 agar despite MG being a common adjunct of Middlebrook 7H11 agar modified for M. bovis growth. Subsequently, a selective MG-free Middlebrook 7H11 agar modified using haemolysed red cells and calf serum was demonstrated as the best performing (P < 0·05) medium for recovery of M. bovis from typical UK cheese types, Cheddar and Caerphilly. Significance and impact of the study: Following increased M. bovis infection of UK cattle, the risk posed to consumers from consumption of unpasteurized milk and dairy products has changed. Furthermore, published methods for the culture and molecular detection of M. bovis in raw milk products are limited. Cheese-making protocols and M. bovis culture media reported here provide tools for further investigation of M. bovis survival during all stages of cheese manufacture and could inform future assessment of the risk to consumers from M. bovis contamination of unpasteurized dairy products. © 2014 The Society for Applied Microbiology.

CPTAC Assay Portal: a repository of targeted proteomic assays

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

Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.

2014-06-27

To address these issues, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as a public repository of well-characterized quantitative, MS-based, targeted proteomic assays. The purpose of the CPTAC Assay Portal is to facilitate widespread adoption of targeted MS assays by disseminating SOPs, reagents, and assay characterization data for highly characterized assays. A primary aim of the NCI-supported portal is to bring together clinicians or biologists and analytical chemists to answer hypothesis-driven questions using targeted, MS-based assays. Assay content is easily accessed through queries and filters, enabling investigatorsmore » to find assays to proteins relevant to their areas of interest. Detailed characterization data are available for each assay, enabling researchers to evaluate assay performance prior to launching the assay in their own laboratory.« less

Two Variants of a High-Throughput Fluorescent Microplate Assay of Polysaccharide Endotransglycosylases.

PubMed

Kováčová, Kristína; Farkaš, Vladimír

2016-04-01

Polysaccharide endotransglycosylases (PETs) are the cell wall-modifying enzymes of fungi and plants. They catalyze random endo-splitting of the polysaccharide donor molecule and transfer of the newly formed reducing sugar residue to the nonreducing end of an acceptor molecule which can be a polysaccharide or an oligosaccharide. Owing to their important role in the cell wall formation, the inhibition of PETs represents an attractive strategy in the fight against fungal infections. We have elaborated two variants of a versatile high-throughput microplate fluorimetric assay that could be used for effective identification of PETs and screening of their inhibitors. Both assays use the respective polysaccharides as the donors and sulforhodamine-labeled oligosaccharides as the acceptors but differ from each other by mode of how the labeled polysaccharide products of transglycosylation are separated from the unreacted oligosaccharide acceptors. In the first variant, the reactions take place in a layer of agar gel laid on the bottoms of the wells of a microtitration plate. After the reaction, the high-Mr transglycosylation products are precipitated with 66 % ethanol and retained within the gel while the low-Mr products and the unreacted acceptors are washed out. In the second variant, the donor polysaccharides are adsorbed to the surface of a microplate well and remain adsorbed there also after becoming labeled in the course of the transglycosylation reaction whereas the unused low-Mr acceptors are washed out. As a proof of versatility, assays of heterologously expressed transglycosylases ScGas1, ScCrh1, and ScCrh2 from the yeast Saccharomyces cerevisiae, CaPhr1 and CaPhr2 from Candida albicans, and of a plant xyloglucan endotransglycosylase (XET) are demonstrated.

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

Fractional Diffusion Equations and Anomalous Diffusion

NASA Astrophysics Data System (ADS)

Evangelista, Luiz Roberto; Kaminski Lenzi, Ervin

2018-01-01

Preface; 1. Mathematical preliminaries; 2. A survey of the fractional calculus; 3. From normal to anomalous diffusion; 4. Fractional diffusion equations: elementary applications; 5. Fractional diffusion equations: surface effects; 6. Fractional nonlinear diffusion equation; 7. Anomalous diffusion: anisotropic case; 8. Fractional Schrödinger equations; 9. Anomalous diffusion and impedance spectroscopy; 10. The Poisson–Nernst–Planck anomalous (PNPA) models; References; Index.

Evaluating the Effect of Peptoid Lipophilicity on Antimicrobial Potency, Cytotoxicity, and Combinatorial Library Design.

PubMed

Turkett, Jeremy A; Bicker, Kevin L

2017-04-10

Growing prevalence of antibiotic resistant bacterial infections necessitates novel antimicrobials, which could be rapidly identified from combinatorial libraries. We report the use of the peptoid library agar diffusion (PLAD) assay to screen peptoid libraries against the ESKAPE pathogens, including the optimization of assay conditions for each pathogen. Work presented here focuses on the tailoring of combinatorial peptoid library design through a detailed study of how peptoid lipophilicity relates to antibacterial potency and mammalian cell toxicity. The information gleaned from this optimization was then applied using the aforementioned screening method to examine the relative potency of peptoid libraries against Staphylococcus aureus, Acinetobacter baumannii, and Enterococcus faecalis prior to and following functionalization with long alkyl tails. The data indicate that overall peptoid hydrophobicity and not simply alkyl tail length is strongly correlated with mammalian cell toxicity. Furthermore, this work demonstrates the utility of the PLAD assay in rapidly evaluating the effect of molecular property changes in similar libraries.

Inhibition of norsolorinic acid accumulation to Aspergillus parasiticus by marine actinomycetes

NASA Astrophysics Data System (ADS)

Yan, Peisheng; Shi, Cuijuan; Shen, Jihong; Wang, Kai; Gao, Xiujun; Li, Ping

2014-11-01

Thirty-six strains of marine actinomycetes were isolated from a sample of marine sediment collected from the Yellow Sea and evaluated in terms of their inhibitory activity on the growth of Aspergillus parasiticus and the production of norsolorinic acid using dual culture plate assay and agar diffusion methods. Among them, three strains showed strong antifungal activity and were subsequently identified as Streptomyces sp. by 16S rRNA gene sequencing analysis. The supernatant from the fermentation of the MA01 strain was extracted sequentially with chloroform and ethyl acetate, and the activities of the extracts were determined by tip culture assay. The assay results show that both extracts inhibited mycelium growth and toxin production, and the inhibitory activities of the extracts increased as their concentrations increased. The results of this study suggest that marine actinomycetes are biologically important for the control of mycotoxins, and that these bacteria could be used as novel biopesticides against mycotoxins.

Development and Validation of a Microtiter Plate-Based Assay for Determination of Bacteriophage Host Range and Virulence.

PubMed

Xie, Yicheng; Wahab, Laith; Gill, Jason J

2018-04-12

Bacteriophages, which are the natural predators of bacteria, have re-emerged as an attractive alternative to combat antibiotic resistant bacteria. Phages are highly specific at the species and strain level and measurement of the phage host range plays an important role in utilizing the phage as antimicrobials. The most common method for phage host range determination has been to spot phage lysates on soft agar overlays and observe plaque formation. In this study, a liquid culture-based assay was developed in a 96-well microtiter plate format to measure the phage host range and virulence for a collection of 15 Salmonella phages against a panel of 20 Salmonella strains representing 11 serovars. This method was compared to a traditional spot method. The majority of the host range results from two methods were in agreement including in cases where a bacterial strain was insensitive to the phage. Each method produced a false-negative result in 19/300 (6%) of the measured phage-host combinations when compared to the other method. The spot method tended to indicate greater phage sensitivity than the microtiter assay even though direct comparisons of the response magnitude between the two methods is difficult since they operate on different mechanisms. The microtiter plate assay was able to provide data on both the phage host range and virulence in greater resolution in a high-throughput format.

Development and Validation of a Microtiter Plate-Based Assay for Determination of Bacteriophage Host Range and Virulence

PubMed Central

Xie, Yicheng; Wahab, Laith

2018-01-01

Bacteriophages, which are the natural predators of bacteria, have re-emerged as an attractive alternative to combat antibiotic resistant bacteria. Phages are highly specific at the species and strain level and measurement of the phage host range plays an important role in utilizing the phage as antimicrobials. The most common method for phage host range determination has been to spot phage lysates on soft agar overlays and observe plaque formation. In this study, a liquid culture-based assay was developed in a 96-well microtiter plate format to measure the phage host range and virulence for a collection of 15 Salmonella phages against a panel of 20 Salmonella strains representing 11 serovars. This method was compared to a traditional spot method. The majority of the host range results from two methods were in agreement including in cases where a bacterial strain was insensitive to the phage. Each method produced a false-negative result in 19/300 (6%) of the measured phage-host combinations when compared to the other method. The spot method tended to indicate greater phage sensitivity than the microtiter assay even though direct comparisons of the response magnitude between the two methods is difficult since they operate on different mechanisms. The microtiter plate assay was able to provide data on both the phage host range and virulence in greater resolution in a high-throughput format. PMID:29649135

[Preparing oral dosage form of ketamine in the hospital for simplicity and patient compliance--preparations using agar].

PubMed

Kaneuchi, Miki; Kohri, Naonori; Senbongi, Kaname; Sakai, Hideo; Iseki, Ken

2005-02-01

Ketamine has been widely used in the operation as intravenous and intramuscular injections, since ketamine has dissociative anesthetic properties. When it is given in sub-anesthetic dose, ketamine is known to have an analgesic effect. The analgesic effect is observed for patients with neuropathic pain when administrated not only by injection but also orally. In Japan, since ketamine is not commercially available except injection forms, patients have to take it as solution of injections for the oral medication. Since the solution of injections has extremely bitter taste, patients intensely desire the development of preparations without the bitterness. In the present study, we prepared oral gel dosage forms of ketamine using agar. It is simple to prepare this dosage form, and most pharmacists can prepare it easily in many hospitals. This gel dosage form met content uniformity requirements and the shape of that was maintained intact during the dissolution test (for 10 hours). The release rate was reduced by additions of additives such as sugar and a flavor in the gel. The reason for the reduction in release could be the suppression of ketamine diffusion depended on the micro-viscosity of solution in the gel. The ketamine contents and the release profile of the gel preparations were unchanged at the room temperature for 12-week storage. The gel preparations in this study would be useful for the oral medication of ketamine, since it is easy for patients to carry them when they go out and the intensely bitter taste could be improved by the addition of a flavor.

Tobacco Agar, a New Medium for Differentiating Candida dubliniensis from Candida albicans

PubMed Central

Khan, Zia U.; Ahmad, Suhail; Mokaddas, Eiman; Chandy, Rachel

2004-01-01

Isolates of Candida dubliniensis may be misidentified as Candida albicans in microbiological laboratories if only the germ tube and/or the chlamydospore test is used for identification to the species level. In this study, we have evaluated the efficacy of tobacco agar for the differentiation of C. dubliniensis from C. albicans. On this medium at 28°C, all 30 C. dubliniensis isolates produced yellowish-brown colonies with hyphal fringes and abundant chlamydospores, whereas 54 C. albicans isolates formed smooth, white-to-cream-colored colonies with no chlamydospore production. This medium provides a simple tool for presumptive differentiation of C. dubliniensis from C. albicans. PMID:15472343

Historical sources about diseases, death and embalming regarding the family of Jean Antoine Michel Agar, Minister of Finance of Gioacchino Murat.

PubMed

Marinozzi, S; Gazzaniga, V; Giuffra, V; Fornaciari, G

2011-06-01

Among the mummies preserved in the Basilica of San Domenico Maggiore in Naples, there are the bodies of the wife and three children of Jean Antoine Michel Agar, Minister of Finance of Naple's Kingdom during the Monarchy of Joachim Murat (1808-1815). Between 1983 and 1987 paleopathological analyses were performed; in particular, X-ray examination allowed investigation of the health status of the Agar family members and reconstruction of the embalming processes used to preserve the bodies. In addition, an analysis of the historical and archival documents was carried out, to formulate hypotheses about the causes of death, demonstrating how these sources could become important instruments to obtain diagnoses and pathological histories.

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.

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.

Identification of hemolysin BL-producing Bacillus cereus isolates by a discontinuous hemolytic pattern in blood agar.

PubMed Central

Beecher, D J; Wong, A C

1994-01-01

Bacillus cereus causes distinct exotoxin-mediated diarrheal and emetic food poisoning syndromes and a variety of nongastrointestinal infections. Evidence is accumulating that hemolysin BL is a major B. cereus virulence factor. We describe two methods for detection of hemolysin BL in crude samples and on primary culture media. In the first method, the highly unusual discontinuous hemolysis pattern that is characteristic of pure hemolysin BL was produced in sheep and calf blood agar around wells filled with crude culture supernatant from hemolysin BL-producing strains. In the second method, the pattern was formed surrounding colonies of hemolysin BL-producing strains grown on media consisting of nutrient agar, 0.15 M NaCl, 2% calf serum, and sheep or calf blood. Hemolysin BL production was detected with these methods in 41 of 62 (66%) previously identified B. cereus isolates and in 46 of 136 (34%) presumptive B. cereus isolates from soil. All nine isolates tested that were associated with diarrhea or nongastrointestinal illness were positive for hemolysin BL. The methods presented here are specific, simple, inexpensive, and applicable to the screening of large numbers of samples or isolates. Images PMID:8017944

Multiparametric comparison of chromogenic-based culture methods used to assess the microbiological quality of drinking water and the mFC method combined with a molecular confirmation procedure.

PubMed

Maheux, Andrée F; Dion-Dupont, Vanessa; Bisson, Marc-Antoine; Bouchard, Sébastien; Jubinville, Éric; Nkuranga, Martine; Rodrigue, Lynda; Bergeron, Michel G; Rodriguez, Manuel J

2015-03-01

MI agar and Colilert(®), as well as mFC agar combined with an Escherichia coli-specific molecular assay (mFC + E. coli rtPCR), were compared in terms of their sensitivity, ease of use, time to result and affordability. The three methods yielded a positive E. coli signal for 11.5, 10.8, and 11.5% of the 968 well water samples tested, respectively. One hundred and thirty-six (136) samples gave blue colonies on mFC agar and required confirmation. E. coli-specific rtPCR showed false-positive results in 23.5% (32/136) of cases. In terms of ease of use, Colilert was the simplest method to use while the MI method provided ease of use comparable to all membrane filtration methods. However, the mFC + E. coli rtPCR assay required highly trained employees for confirmation purposes. In terms of affordability, and considering contamination rate of well water samples tested, the Colilert method and the mFC + E. coli rtPCR assay were at least five times more costly than the MI agar method. Overall, compared with the other two methods tested, the MI agar method offers the most advantages to assess drinking water quality.

Chromium and zinc uptake by algae Gelidium and agar extraction algal waste: kinetics and equilibrium.

PubMed

Vilar, Vítor J P; Botelho, Cidália M S; Boaventura, Rui A R

2007-11-19

Biosorption of chromium and zinc ions by an industrial algal waste, from agar extraction industry has been studied in a batch system. This biosorbent was compared with the algae Gelidium itself, which is the raw material for agar extraction, and the industrial waste immobilized with polyacrylonitrile (composite material). Langmuir and Langmuir-Freundlich equilibrium models describe well the equilibrium data. The parameters of Langmuir equilibrium model at pH 5.3 and 20 degrees C were for the algae, q(L)=18 mg Cr(III)g(-1) and 13 mgZn(II)g(-1), K(L) = 0.021l mg(-1)Cr(III) and 0.026l mg(-1) Zn(II); for the algal waste, q(L)=12 mgCr(III)g(-1) and 7mgZn(II)g(-1), K(L)=0.033lmg(-1) Cr(III) and 0.042l mg(-1) Zn(II); for the composite material, q(L) = 9 mgCr(III)g(-1) and 6 mgZn(II)g(-1), K(L)=0.032l mg(-1)Cr(III) and 0.034l mg(-1)Zn(II). The biosorbents exhibited a higher preference for Cr(III) ions and algae Gelidium is the best one. The pseudo-first-order Lagergren and pseudo-second-order models fitted well the kinetic data for the two metal ions. Kinetic constants and equilibrium uptake concentrations given by the pseudo-second-order model for an initial Cr(III) and Zn(II) concentration of approximately 100 mgl(-1), at pH 5.3 and 20 degrees C were k(2,ads)=0.04 g mg(-1)Cr(III)min(-1) and 0.07 g mg(-1)Zn(II)min(-1), q(eq)=11.9 mgCr(III)g(-1) and 9.5 mgZn(II)g(-1) for algae; k(2,ads)=0.17 g mg(-1)Cr(III)min(-1) and 0.19 g mg(-1)Zn(II)min(-1), q(eq)=8.3 mgCr(III)g(-1) and 5.6 mgZn(II)g(-1) for algal waste; k(2,ads)=0.01 g mg(-1)Cr(III)min(-1) and 0.18 g mg(-1)Zn(II)min(-1), q(eq)=8.0 mgCr(III)g(-1) and 4.4 mgZn(II)g(-1) for composite material. Biosorption was modelled using a batch adsorber mass transfer kinetic model, which successfully predicts Cr(III) and Zn(II) concentration profiles. The calculated average homogeneous diffusivities, D(h), were 4.2 x 10(-8), 8.3 x 10(-8) and 1.4 x 10(-8)cm(2)s(-1) for Cr(III) and 4.8 x 10(-8), 9.7 x 10(-8) and 6.2 x 10(-8)cm(2)s(-1

Development of an in vitro chemo-radiation response assay for cervical carcinoma.

PubMed

Monk, Bradley J; Burger, Robert A; Parker, Ricardo; Radany, Eric H; Redpath, Leslie; Fruehauf, John P

2002-11-01

To determine if synergistic effects of radiation (RT) and chemotherapy (chemo) on human cervical carcinoma cell lines and fresh tumor explants could be determined using an in vitro assay. In vitro radiation response was determined for 4 cell lines and 26 fresh tumor explants in an agar-based assay. Cells were exposed to increasing doses of RT with or without cisplatin (CDDP), carmustine (BCNU), buthionine sulfoximine (BSO), or paclitaxel (Tax). Cell suspensions were cultured for 5 days, with [(3)H]thymidine added on day 3 and proliferation was measured. Results were reported as the fraction of proliferation compared to control (FC). For each combination of irradiation and drug, synergy was tested using the Chou analysis, where a combination index (CI) <1 indicated synergistic interaction. In simple correlation analysis, an R value of >0.7 indicated cross-resistance. RT dose-dependent proliferation inhibition was observed for 2 of the 4 cell lines, and for all but 1 of the fresh specimens. Significant heterogeneity of tumor response to RT was seen. Four specimens that were 1 standard deviation below the median FC response after exposure to 300 cGy were classified as extremely radiation resistant. Twenty-one tumors were evaluated for synergistic response using the combination of chemo and RT with a median FC of 0.27 (+/-0.27) for 6.0 Gy of RT alone, 0.22 (+/-0.21) for CDDP alone, and 0.05 (+/-0.08) for the combination. A CI of 0.35 and an R value of 0.09 demonstrated synergy between chemo and RT without cross-resistance. Similar synergy without cross-resistance was found for RT in combination with BCNU, BSO, and TAX. Heterogeneous RT dose-response relationships in the in vitro assay were demonstrated. Explants were more sensitive to RT than cell lines. Unlike cell lines, fresh tumor cells consistently displayed synergy with RT and chemo. The synergy between RT and BSO suggests that glutathione depletion may enhance the effect of RT. The assay was feasible for

Production and Characterization of Antifungal Compounds Produced by Lactobacillus plantarum IMAU10014

PubMed Central

Wang, HaiKuan; Yan, YanHua; Wang, JiaMing; Zhang, HePing; Qi, Wei

2012-01-01

Lactobacillus plantarum IMAU10014 was isolated from koumiss that produces a broad spectrum of antifungal compounds, all of which were active against plant pathogenic fungi in an agar plate assay. Two major antifungal compounds were extracted from the cell-free supernatant broth of L. plantarum IMAU10014. 3-phenyllactic acid and Benzeneacetic acid, 2-propenyl ester were carried out by HPLC, LC-MS, GC-MS, NMR analysis. It is the first report that lactic acid bacteria produce antifungal Benzeneacetic acid, 2-propenyl ester. Of these, the antifungal products also have a broad spectrum of antifungal activity, namely against Botrytis cinerea, Glomerella cingulate, Phytophthora drechsleri Tucker, Penicillium citrinum, Penicillium digitatum and Fusarium oxysporum, which was identified by the overlay and well-diffusion assay. F. oxysporum, P. citrinum and P. drechsleri Tucker were the most sensitive among molds. PMID:22276116

Comparison of Neisseria gonorrhoeae MICs Obtained by Etest and Agar Dilution for Ceftriaxone, Cefpodoxime, Cefixime and Azithromycin.

PubMed

Gose, Severin; Kong, Carol J; Lee, Yer; Samuel, Michael C; Bauer, Heidi M; Dixon, Paula; Soge, Olusegun O; Lei, John; Pandori, Mark

2013-10-24

We evaluated Neisseria gonorrhoeae Etest minimum inhibitory concentrations (MICs) relative to agar dilution MICs for 664 urethral isolates for ceftriaxone (CRO) and azithromycin (AZM), 351 isolates for cefpodoxime (CPD) and 315 isolates for cefixime (CFM). Etest accurately determined CPD, CFM and AZM MICs, but resulted in higher CRO MICs. © 2013. Published by Elsevier B.V. All rights reserved.

Offline and online capillary electrophoresis enzyme assays of β-N-acetylhexosaminidase.

PubMed

Křížek, Tomáš; Doubnerová, Veronika; Ryšlavá, Helena; Coufal, Pavel; Bosáková, Zuzana

2013-03-01

Enzyme assays of β-N-acetylhexosaminidase from Aspergillus oryzae using capillary electrophoresis in the offline and online setup have been developed. The pH value and concentration of the borate-based background electrolyte were optimized in order to achieve baseline separation of N,N',N″-triacetylchitotriose, N,N'-diacetylchitobiose, and N-acetyl-D-glucosamine. The optimized method using 25 mM tetraborate buffer, pH 10.0, was evaluated in terms of repeatability, limits of detection, quantification, and linearity. The method was successfully applied to the offline enzyme assay of β-N-acetylhexosaminidase, which was demonstrated by monitoring the hydrolysis of N,N',N″-triacetylchitotriose. The presented method was also utilized to study the pH dependence of enzyme activity. An online assay with N,N'-diacetylchitobiose as a substrate was developed using the Transverse Diffusion of Laminar Flow Profiles model to optimize the injection sequence and in-capillary mixing of substrate and enzyme plugs. The experimental results were in good agreement with predictions of the model. The online assay was successfully used to observe the inhibition effect of N,N'-dimethylformamide on the activity of β-N-acetylhexosaminidase with nanoliter volumes of reagents used per run and a high degree of automation. After adjustment of background electrolyte pH, an online assay with N,N',N″-triacetylchitotriose as a substrate was also performed.

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.

GAL4 transactivation-based assay for the detection of selective intercellular protein movement.

PubMed

Kumar, Dhinesh; Chen, Huan; Rim, Yeonggil; Kim, Jae-Yean

2015-01-01

Several plant proteins function as intercellular messenger to specify cell fate and coordinate plant development. Such intercellular communication can be achieved by direct, selective, or nonselective (diffusion-based) trafficking through plasmodesmata (PD), the symplasmic membrane-lined nanochannels adjoining two cells. A trichome rescue trafficking assay was reported to allow the detection of protein movement in Arabidopsis leaf tissue using transgenic gene expression. Here, we provide a protocol to dissect the mode of intercellular protein movement in Arabidopsis root. This assay system involves a root ground tissue-specific GAL4/UAS transactivation expression system in combination with fluorescent reporter proteins. In this system, mCherry, a red fluorescent protein, can move cell to cell via diffusion, while mCherry-H2B is tightly cell autonomous. Thus, a protein fused to mCherry-H2B that can move out from the site of synthesis likely contains a selective trafficking signal to impart a cell-to-cell gain-of-trafficking function to the cell-autonomous mCherry-H2B. This approach can be adapted to investigate the cell-to-cell trafficking properties of any protein of interest.

Nutrient limitation leads to penetrative growth into agar and affects aroma formation in Pichia fabianii, P. kudriavzevii and Saccharomyces cerevisiae.

PubMed

van Rijswijck, Irma M H; Dijksterhuis, Jan; Wolkers-Rooijackers, Judith C M; Abee, Tjakko; Smid, Eddy J

2015-01-01

Among fermentative yeast species, Saccharomyces cerevisiae is most frequently used as a model organism, although other yeast species may have special features that make them interesting candidates to apply in food-fermentation processes. In this study, we used three yeast species isolated from fermented masau (Ziziphus mauritiana) fruit, S. cerevisiae 131, Pichia fabianii 65 and Pichia kudriavzevii 129, and determined the impact of nitrogen and/or glucose limitation on surface growth mode and the production of volatile organic compounds (VOCs). All three species displayed significant changes in growth mode in all nutrient-limited conditions, signified by the formation of metafilaments or pseudohyphae. The timing of the transition was found to be species-specific. Transition in growth mode is suggested to be linked to the production of certain fusel alcohols, such as phenylethyl alcohol, which serve as quorum-sensing molecules. Interestingly, we did not observe concomitant increased production of phenylethyl alcohol and filamentous growth. Notably, a broader range of esters was found only for the Pichia spp. grown on nitrogen-limited agar for 21 days compared to nutrient-rich agar, and when grown on glucose- and glucose- plus nitrogen-limited agar. Our data suggest that for the Pichia spp., the formation of esters may play an important role in the switch in growth mode upon nitrogen limitation. Further biological or ecological implications of ester formation are discussed. Copyright © 2014 John Wiley & Sons, Ltd.

Early responses of mature Arabidopsis thaliana plants to reduced water potential in the agar-based polyethylene glycol infusion drought model.

PubMed

Frolov, Andrej; Bilova, Tatiana; Paudel, Gagan; Berger, Robert; Balcke, Gerd U; Birkemeyer, Claudia; Wessjohann, Ludger A

2017-01-01

Drought is one of the most important environmental stressors resulting in increasing losses of crop plant productivity all over the world. Therefore, development of new approaches to increase the stress tolerance of crop plants is strongly desired. This requires precise and adequate modeling of drought stress. As this type of stress manifests itself as a steady decrease in the substrate water potential (ψ w ), agar plates infused with polyethylene glycol (PEG) are the perfect experimental tool: they are easy in preparation and provide a constantly reduced ψ w , which is not possible in soil models. However, currently, this model is applicable only to seedlings and cannot be used for evaluation of stress responses in mature plants, which are obviously the most appropriate objects for drought tolerance research. To overcome this limitation, here we introduce a PEG-based agar infusion model suitable for 6-8-week-old A. thaliana plants, and characterize, to the best of our knowledge for the first time, the early drought stress responses of adult plants grown on PEG-infused agar. We describe essential alterations in the primary metabolome (sugars and related compounds, amino acids and polyamines) accompanied by qualitative and quantitative changes in protein patterns: up to 87 unique stress-related proteins were annotated under drought stress conditions, whereas further 84 proteins showed a change in abundance. The obtained proteome patterns differed slightly from those reported for seedlings and soil-based models. Copyright © 2016 Elsevier GmbH. All rights reserved.

High frequency lateral flow affinity assay using superparamagnetic nanoparticles

NASA Astrophysics Data System (ADS)

Lago-Cachón, D.; Rivas, M.; Martínez-García, J. C.; Oliveira-Rodríguez, M.; Blanco-López, M. C.; García, J. A.

2017-02-01

Lateral flow assay is one of the simplest and most extended techniques in medical diagnosis for point-of-care testing. Although it has been traditionally a positive/negative test, some work has been lately done to add quantitative abilities to lateral flow assay. One of the most successful strategies involves magnetic beads and magnetic sensors. Recently, a new technique of superparamagnetic nanoparticle detection has been reported, based on the increase of the impedance induced by the nanoparticles on a RF-current carrying copper conductor. This method requires no external magnetic field, which reduces the system complexity. In this work, nitrocellulose membranes have been installed on the sensor, and impedance measurements have been carried out during the sample diffusion by capillarity along the membrane. The impedance of the sensor changes because of the presence of magnetic nanoparticles. The results prove the potentiality of the method for point-of-care testing of biochemical substances and nanoparticle capillarity flow studies.

Anti-Candida activity and brine shrimp toxicity assay of Ganoderma boninense.

PubMed

Daruliza, K M A; Fernandez, L; Jegathambigai, R; Sasidharan, S

2012-01-01

Ganoderma (G.) boninense is a white rot fungus, which can be found in the palm oil tree. Several studies have shown that G. boninense has antimicrobial and antagonistic properties. However, there is limited information reported on antifungal properties especially on Candida (C) albicans. Hence, this study was conducted to determine the anti-Candida activity of G. boninense against C albicans. Crude methanolic extracts of G. boninense was obtained by maceration method with 70% methanol. Anti-Candida test was carried out using disc diffusion assay, broth dilution method, time killing profile and brine shrimp toxicity assay. Anti-Candida activity indicated that the mean zone of inhibition was 12.5 +/- 0.6 mm. The MIC value for C. albicans found to be 3.125 mg/ml. The result from time-killing profile showed that the growth of C albicans was inhibited hence decreases its exponential phase. For brine shrimp toxicity assay, the LC50 value was 3.59 mg/ml which proved that the extract of G. boninense is not toxic.

Choline chloride based ionic liquid analogues as tool for the fabrication of agar films with improved mechanical properties

USDA-ARS?s Scientific Manuscript database

In the present paper, we test the suitability of Choline-Cl/urea (DES-U) and Choline-Cl/glycerol (DES-G) eutectic mixtures at 1:2 molar ratios for the production of agar biodegradable films. A three-step process is proposed: pre-solubilization of polymer in DES followed by compression-molding and s...

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.

Preparation of nanocellulose from micro-crystalline cellulose: The effect on the performance and properties of agar-based composite films.

PubMed

Shankar, Shiv; Rhim, Jong-Whan

2016-01-01

A facile approach has been performed to prepare nanocellulose (NC) from micro-crystalline cellulose (MCC) and test their effect on the performance properties of agar-based composite films. The NC was characterized by STEM, XRD, FTIR, and TGA. The NC was well dispersed in distilled water after sonication and their size was in the range of 100-500nm. The XRD results revealed the crystallinity of NC. The crystallinity index of NC (0.71) was decreased compared to the MCC (0.81). The effect of NC or MCC content (1, 3, 5 and 10wt% based on agar) on the mechanical, water vapor permeability (WVP), and thermal properties of the composites were studied. The NC obtained from MCC can be used as a reinforcing agent for the preparation of biodegradable composites films for their potential use in the development of biodegradable food packaging materials. Copyright © 2015 Elsevier Ltd. All rights reserved.

Diffusion archeology for diffusion progression history reconstruction.

PubMed

Sefer, Emre; Kingsford, Carl

2016-11-01

Diffusion through graphs can be used to model many real-world processes, such as the spread of diseases, social network memes, computer viruses, or water contaminants. Often, a real-world diffusion cannot be directly observed while it is occurring - perhaps it is not noticed until some time has passed, continuous monitoring is too costly, or privacy concerns limit data access. This leads to the need to reconstruct how the present state of the diffusion came to be from partial diffusion data. Here, we tackle the problem of reconstructing a diffusion history from one or more snapshots of the diffusion state. This ability can be invaluable to learn when certain computer nodes are infected or which people are the initial disease spreaders to control future diffusions. We formulate this problem over discrete-time SEIRS-type diffusion models in terms of maximum likelihood. We design methods that are based on submodularity and a novel prize-collecting dominating-set vertex cover (PCDSVC) relaxation that can identify likely diffusion steps with some provable performance guarantees. Our methods are the first to be able to reconstruct complete diffusion histories accurately in real and simulated situations. As a special case, they can also identify the initial spreaders better than the existing methods for that problem. Our results for both meme and contaminant diffusion show that the partial diffusion data problem can be overcome with proper modeling and methods, and that hidden temporal characteristics of diffusion can be predicted from limited data.

Phosphate-Catalyzed Hydrogen Peroxide Formation from Agar, Gellan, and κ-Carrageenan and Recovery of Microbial Cultivability via Catalase and Pyruvate.

PubMed

Kawasaki, Kosei; Kamagata, Yoichi

2017-11-01

Previously, we reported that when agar is autoclaved with phosphate buffer, hydrogen peroxide (H 2 O 2 ) is formed in the resulting medium (PT medium), and the colony count on the medium inoculated with environmental samples becomes much lower than that on a medium in which agar and phosphate are autoclaved separately (PS medium) (T. Tanaka et al., Appl Environ Microbiol 80:7659-7666, 2014, https://doi.org/10.1128/AEM.02741-14). However, the physicochemical mechanisms underlying this observation remain largely unknown. Here, we determined the factors affecting H 2 O 2 formation in agar. The H 2 O 2 formation was pH dependent: H 2 O 2 was formed at high concentrations in an alkaline or neutral phosphate buffer but not in an acidic buffer. Ammonium ions enhanced H 2 O 2 formation, implying the involvement of the Maillard reaction catalyzed by phosphate. We found that other gelling agents (e.g., gellan and κ-carrageenan) also produced H 2 O 2 after being autoclaved with phosphate. We then examined the cultivability of microorganisms from a fresh-water sample to test whether catalase and pyruvate, known as H 2 O 2 scavengers, are effective in yielding high colony counts. The colony count on PT medium was only 5.7% of that on PS medium. Catalase treatment effectively restored the colony count of PT medium (to 106% of that on PS medium). In contrast, pyruvate was not as effective as catalase: the colony count on sodium pyruvate-supplemented PT medium was 58% of that on PS medium. Given that both catalase and pyruvate can remove H 2 O 2 from PT medium, these observations indicate that although H 2 O 2 is the main cause of reduced colony count on PT medium, other unknown growth-inhibiting substances that cannot be removed by pyruvate (but can be by catalase) may also be involved. IMPORTANCE The majority of bacteria in natural environments are recalcitrant to laboratory culture techniques. Previously, we demonstrated that one reason for this is the formation of high H 2 O

Phosphate-Catalyzed Hydrogen Peroxide Formation from Agar, Gellan, and κ-Carrageenan and Recovery of Microbial Cultivability via Catalase and Pyruvate

PubMed Central

Kamagata, Yoichi

2017-01-01

ABSTRACT Previously, we reported that when agar is autoclaved with phosphate buffer, hydrogen peroxide (H2O2) is formed in the resulting medium (PT medium), and the colony count on the medium inoculated with environmental samples becomes much lower than that on a medium in which agar and phosphate are autoclaved separately (PS medium) (T. Tanaka et al., Appl Environ Microbiol 80:7659–7666, 2014, https://doi.org/10.1128/AEM.02741-14). However, the physicochemical mechanisms underlying this observation remain largely unknown. Here, we determined the factors affecting H2O2 formation in agar. The H2O2 formation was pH dependent: H2O2 was formed at high concentrations in an alkaline or neutral phosphate buffer but not in an acidic buffer. Ammonium ions enhanced H2O2 formation, implying the involvement of the Maillard reaction catalyzed by phosphate. We found that other gelling agents (e.g., gellan and κ-carrageenan) also produced H2O2 after being autoclaved with phosphate. We then examined the cultivability of microorganisms from a fresh-water sample to test whether catalase and pyruvate, known as H2O2 scavengers, are effective in yielding high colony counts. The colony count on PT medium was only 5.7% of that on PS medium. Catalase treatment effectively restored the colony count of PT medium (to 106% of that on PS medium). In contrast, pyruvate was not as effective as catalase: the colony count on sodium pyruvate-supplemented PT medium was 58% of that on PS medium. Given that both catalase and pyruvate can remove H2O2 from PT medium, these observations indicate that although H2O2 is the main cause of reduced colony count on PT medium, other unknown growth-inhibiting substances that cannot be removed by pyruvate (but can be by catalase) may also be involved. IMPORTANCE The majority of bacteria in natural environments are recalcitrant to laboratory culture techniques. Previously, we demonstrated that one reason for this is the formation of high H2O2 levels in media