Zoonotic intestinal protozoan of the wild boars, Sus scrofa, in Persian Gulf's coastal area (Bushehr province), Southwestern Iran.

PubMed

Yaghoobi, Kambiz; Sarkari, Bahador; Mansouri, Majid; Motazedian, Mohammad Hossein

2016-10-01

Wild boars, Sus scrofa , are potential reservoirs of many zoonotic diseases, and there are a possibility of transmission of the zoonotic diseases from these animals to humans and also domestic animals. This study aimed to evaluate the protozoan contamination of wild boars in the Persian Gulf's coastal area (Bushehr Province), southwestern Iran. A total of 25 crossbred boars were collected during a course of vertebrate pest control in Bushehr province, in 2013. Samples were collected from the gastrointestinal tracts of each boar in 5% formalin, Bouin's solution, sodium acetate-acetic acid-formalin, and polyvinyl alcohol fixatives. Fixed stool smears examined by trichrome and Ziehl-Neelsen staining. Each of the 25 wild boars was infected with at least one of the intestinal protozoans. The rate of contamination with intestinal protozoan was 64% for Balantidium coli , 76% for Iodamoeba sp., 52% for Entamoeba polecki , 44% for Blastocystis sp. and 8% for Chilomastix sp. No intestinal coccidian was detected in studied boars when the stool samples were evaluated by Ziehl-Neelsen staining method. Findings of this study demonstrated that wild boars in the Persian Gulf coastal area are contaminated by many protozoans, including zoonotic protozoan, which poses a potential risk to locals as well as the domestic animals of the area.

Techniques for the recovery and identification of Cryptosporidium oocysts from stool specimens.

PubMed

Garcia, L S; Bruckner, D A; Brewer, T C; Shimizu, R Y

1983-07-01

Due to increasing numbers of patients with documented infections with Cryptosporidium and other coccidia, it is important for the physician and clinical laboratory to be aware of the appropriate diagnostic techniques necessary for organism recovery and identification. Although Cryptosporidium is found in the gastrointestinal tract, tissue biopsies may be insufficient for organism recovery; the examination of stool specimens is a noninvasive procedure and will provide better overall opportunities for organism recovery. Human clinical specimens were examined from 45 patients with confirmed cryptosporidiosis or suspected of having the infection. Tissue biopsy sections, fecal wet preparations, and permanent stained smears were examined. Stool specimens were submitted in 10% Formalin, 2.5% potassium dichromate, and polyvinyl alcohol and were examined for oocysts by using 15 different methods: phase-contrast and light microscopy; Sheather's sugar flotation; Formalin concentration techniques; 10% potassium hydroxide; Giemsa; trichrome; periodic acid-Schiff; modified periodic acid-Schiff; silver methenamine; acridine orange; auramine-rhodamine; Kinyoun acid-fast; Ziehl-Neelsen carbolfuchsin; and a modified acid-fast procedure. Each technique or combination of techniques was assessed by organism quantitation, organism morphology, and ease of visual recognition. Based on these comparative studies, the modified Ziehl-Neelsen carbolfuchsin stain on 10% Formalin-preserved stool is recommended for the recovery and identification of Cryptosporidium.

Techniques for the recovery and identification of Cryptosporidium oocysts from stool specimens.

PubMed Central

Garcia, L S; Bruckner, D A; Brewer, T C; Shimizu, R Y

1983-01-01

Due to increasing numbers of patients with documented infections with Cryptosporidium and other coccidia, it is important for the physician and clinical laboratory to be aware of the appropriate diagnostic techniques necessary for organism recovery and identification. Although Cryptosporidium is found in the gastrointestinal tract, tissue biopsies may be insufficient for organism recovery; the examination of stool specimens is a noninvasive procedure and will provide better overall opportunities for organism recovery. Human clinical specimens were examined from 45 patients with confirmed cryptosporidiosis or suspected of having the infection. Tissue biopsy sections, fecal wet preparations, and permanent stained smears were examined. Stool specimens were submitted in 10% Formalin, 2.5% potassium dichromate, and polyvinyl alcohol and were examined for oocysts by using 15 different methods: phase-contrast and light microscopy; Sheather's sugar flotation; Formalin concentration techniques; 10% potassium hydroxide; Giemsa; trichrome; periodic acid-Schiff; modified periodic acid-Schiff; silver methenamine; acridine orange; auramine-rhodamine; Kinyoun acid-fast; Ziehl-Neelsen carbolfuchsin; and a modified acid-fast procedure. Each technique or combination of techniques was assessed by organism quantitation, organism morphology, and ease of visual recognition. Based on these comparative studies, the modified Ziehl-Neelsen carbolfuchsin stain on 10% Formalin-preserved stool is recommended for the recovery and identification of Cryptosporidium. Images PMID:6193138

Subcutaneous aspergillosis with coexisting atypical mycobacterial infection.

PubMed

Duraipandian, Jeyakumari; Rengasamy, Gopal; Madasamy, Balamurugan; Kulanthaivelu, Ambedkarraj; Subramanian, Girija

2010-01-01

A 60-year-old woman, a known diabetic and asthmatic, was admitted for acute exacerbation of chronic obstructive pulmonary disease. Physical examination revealed two soft nodules in the left infra axillary region. Fine needle aspiration cytology (FNAC) showed fungal granulomatous reaction suggestive of fungal infection. Periodic acid Schiff stain (PAS stain) revealed PAS positive, acutely branching, septate fungal hyphae. Wet mount of the aspirate revealed plenty of pus cells and branching septate hyphae. Ziehl-Neelsen (ZN) stain showed moderate numbers of acid fast bacilli. Culture yielded Aspergillus flavus and Mycobacterium fortuitum.

Case of Mycobacterium tuberculosis meningitis: Gram staining as a useful initial diagnostic clue for tuberculous meningitis.

PubMed

Kawakami, Sayoko; Kawamura, Yasuyosi; Nishiyama, Kyouhei; Hatanaka, Hiroki; Fujisaki, Ryuichi; Ono, Yasuo; Miyazawa, Yukihisa; Nishiya, Hajime

2012-12-01

A 32-year-old man was admitted to our hospital because of fever, headache, and loss of consciousness. Four days before admission, he had had difficulty speaking. On the day of admission, his colleague had found him to be unconscious and lying on his back. He was admitted to our hospital. The temperature at the eardrum was 35.2°C. Neurologic evaluation was negative. Computed tomography (CT) scan of the brain showed slight ventricular enlargement bilaterally. An X-ray film of the chest showed no abnormality. On the second hospital day, neck stiffness was noted. The cerebrospinal fluid (CSF) contained 870 white cells/μl, most of which were neutrophils; the glucose level in the CSF was 10 mg/dl, and the protein level was 140 mg/dl. Stained smears of the CSF, including Gram staining and India-ink preparations, disclosed no microorganisms. Capsular antigen tests for several bacteria were negative. Antimicrobial agents were started. However, by changing the microscope focus slightly while viewing Gram stains of the CSF, we could see brightened and Gram-positive bacilli that had been phagocytosed by neutrophils. This finding suggested the presence of Mycobacterium tuberculosis. Ziehl-Neelsen staining of the CSF and gastric juice revealed anti-acid bacilli. Polymerase chain reaction for M. tuberculosis in the gastric juice was positive. This case showed that Gram staining could be useful as an initial adjunct for the diagnosis of tuberculous meningitis, particularly when the CSF shows predominantly neutrocytic pleocytosis, but no other evidence of bacterial meningitis.

GeneXpert MTB/RIF dans le dépistage de la tuberculose pulmonaire à l’Hôpital Provincial Général de Référence de Bukavu, à l’Est de la République Démocratique du Congo: quelles leçons tirées après 10 mois d’utilisation?

PubMed Central

Lupande, David; Kaishusha, David; Mihigo, Carine; Itongwa, Moise; Yenga, Gustave; Katchunga, Philippe

2017-01-01

Introduction En Afrique subsaharienne, les méthodes de diagnostic de la tuberculose sont insuffisantes et reposent essentiellement sur la microscopie. Elles constituent un réel frein pour le contrôle de la tuberculose. La présente étude voudrait évaluer les performances du GeneXpert MTB/RIF vis à vis de la microscopie classique de Ziehl-Neelsen à l’Hôpital Provincial Général de Référence de Bukavu, à l’Est de la République Démocratique du Congo après 10 mois d’utilisation. Méthodes Les résultats de la coloration au Ziehl-Neelsen et de la biologie moléculaire sur GeneXpert MTB/RIF de 452 patients suspects de tuberculose ont été colligés. La validité d’un test par rapport à l’autre dans la détection de la tuberculose a été étudiée. Résultats Dans le groupe entier, la fréquence de la tuberculose pulmonaire était de 16.3%. La positivité était significativement plus élevée pour le GeneXpert MTB/RIF que pour le Ziehl-Neelsen dans le groupe entier (15.9% vs 9.3%, p= 0.03) et chez les séropositifs pour le VIH (52.0% vs 24.0%; p = 0.007). Cependant, la sensibilité de GeneXpert MTB/RIF comparé au Ziehl-Neelsen n’était pas maximale (95.2%). Enfin, GeneXpert MTB/RIF a détecté 20.8% de résistance à la rifampicine. Conclusion La présente étude confirme la supériorité de GeneXpert MTB/RIF sur la coloration de Ziehl-Neelsen dans la détection de la tuberculose et dans la prédiction de la multi résistance. Son utilisation systématique couplée au Ziehl-Neelsen permettrait de mieux contrôler la tuberculose en Afrique subSaharienne. PMID:29187929

Detection of Cryptosporidium oocysts in fresh and frozen cattle faeces: comparison of three methods.

PubMed

Brook, E J; Christley, R M; French, N P; Hart, C A

2008-01-01

The aim of this study was to compare the performance of three commonly used screening tests for Cryptosporidium oocysts in fresh and frozen cattle faeces. Twenty-nine freshly voided faecal samples were collected from calves from three farms in the northwest of England. Three diagnostic tests for Cryptosporidium were carried out on each sample both before and after freezing - the modified Ziehl-Neelsen (MZN) and auramine phenol (APh) stains and a commercial enzyme immunoassay (EIA) kit, the ProSpecT Cryptosporidium Microplate assay (Remel, Lenexa, KS). Twelve samples were deemed positive by the reference standard (polymerase chain reaction, PCR). There were some discrepancies between the results of the screening tests and the levels of agreement were quantified. The sensitivity and specificity of each method was determined, with PCR as the gold standard. Sensitivity and specificity of the MZN stain was optimized when samples with fewer than two oocyst-like bodies were classified as negative. All three screening methods used were effective in detecting Cryptosporidium infection in both fresh and frozen calf faeces. This study has highlighted the value of determining characteristics of tests used for diagnosis and epidemiological studies.

Gastrointestinal parasites of owls (Strigiformes) kept in captivity in the Southern region of Brazil.

PubMed

da Silva, Aleksandro S; Zanette, Régis A; Lara, Valéria M; Gressler, Luciane T; Carregaro, Adriano B; Santurio, Janio M; Monteiro, Silvia G

2009-01-01

The aim of this research was to study the gastrointestinal parasitism in 12 adult owls kept in captivity in the Southern region of Brazil. Cloacal contents of the species Rhinoptynx clamator, Tyto alba, Athene cunicularia, Megascops spp., and Bubo virginianus were evaluated. Feces and urine were collected and analyzed by the zinc sulfate centrifugal-flotation method and stained by the modified Ziehl-Neelsen technique. Eggs of Capillaria spp. and Strongylida, oocysts of Cryptosporidium spp., Eimeria spp., and Isospora spp. were observed. The birds showed no clinical signs, probably due to the mild nature of the infection.

Comparison of different diagnostic techniques for the detection of cryptosporidiosis in bovines

PubMed Central

Rekha, K. M. H.; Puttalakshmamma, G. C.; D’Souza, Placid E.

2016-01-01

Aim: Aim of the present study was to compare different methods, viz., Sheather's sugar flotation (SSF), Ziehl-Neelsen (ZN), Kinyoun's acid-fast method (KAF), safranin-methylene blue staining (SMB), and negative staining techniques such as nigrosin staining, light green staining, and malachite green staining for the detection of Cryptosporidium spp. oocysts in bovines. Materials and Methods: A total of 455 fecal samples from bovines were collected from private, government farms and from the clinical cases presented to Department of Medicine, Veterinary College, Bengaluru. They were subjected for SSF, ZN, KAF, SMB and negative staining methods. Results: Out of 455 animal fecal samples screened 5.71% were found positive for Cryptosporidium spp. oocysts. The species were identified as Cryptosporidium parvum in calves and Cryptosporidium andersoni in adults based on the morphological characterization and micrometry of the oocysts. Conclusions: Of all the techniques, fecal flotation with sheather's was found to be more specific and sensitive method for the detection of Cryptosporidium spp. oocysts. Among the conventional staining methods, the SMB gives better differentiation between oocysts and yeast. Among the three negative staining methods, malachite green was found sensitive over the other methods. PMID:27051211

Opportunistic parasites among immunosuppressed children in Minia District, Egypt.

PubMed

Abdel-Hafeez, Ekhlas H; Ahmad, Azza K; Ali, Basma A; Moslam, Fadia A

2012-03-01

A total of 450 stool samples were collected from inpatient and outpatient clinics of Pediatric Department, Minia University Hospital, Minia District, Egypt. Two groups of patients were studied, including 200 immunosuppressed and 250 immunocompetent children. Stool samples were subjected to wet saline and iodine mounts. A concentration technique (formol-ether sedimentation method) was carried out for stool samples diagnosed negative by wet saline and iodine mounts. Samples were stained by 2 different methods; acid fast stain (modified Ziehl-Neelsen stain) and Giemsa stain. Total 188 cases (94%) were diagnosed positive for parasitic infections among immunosuppressed children, whereas 150 cases (60%) were positive in immunocompetent children (P<0.0001). The most common protozoan infection in immunosuppressed group was Cryptosporidium parvum (60.2%), followed by Blastocystis hominis (12.1%), Isospora belli (9.7%), and Cyclospora caytenensis (7.8%). On the other hand, Entamoeba histolytica (24.6%) and Giardia lamblia (17.6%) were more common than other protozoans in immunocompetent children.

Opportunistic Parasites among Immunosuppressed Children in Minia District, Egypt

PubMed Central

Ahmad, Azza K.; Ali, Basma A.; Moslam, Fadia A.

2012-01-01

A total of 450 stool samples were collected from inpatient and outpatient clinics of Pediatric Department, Minia University Hospital, Minia District, Egypt. Two groups of patients were studied, including 200 immunosuppressed and 250 immunocompetent children. Stool samples were subjected to wet saline and iodine mounts. A concentration technique (formol-ether sedimentation method) was carried out for stool samples diagnosed negative by wet saline and iodine mounts. Samples were stained by 2 different methods; acid fast stain (modified Ziehl-Neelsen stain) and Giemsa stain. Total 188 cases (94%) were diagnosed positive for parasitic infections among immunosuppressed children, whereas 150 cases (60%) were positive in immunocompetent children (P<0.0001). The most common protozoan infection in immunosuppressed group was Cryptosporidium parvum (60.2%), followed by Blastocystis hominis (12.1%), Isospora belli (9.7%), and Cyclospora caytenensis (7.8%). On the other hand, Entamoeba histolytica (24.6%) and Giardia lamblia (17.6%) were more common than other protozoans in immunocompetent children. PMID:22451735

Colour segmentation of multi variants tuberculosis sputum images using self organizing map

NASA Astrophysics Data System (ADS)

Rulaningtyas, Riries; Suksmono, Andriyan B.; Mengko, Tati L. R.; Saptawati, Putri

2017-05-01

Lung tuberculosis detection is still identified from Ziehl-Neelsen sputum smear images in low and middle countries. The clinicians decide the grade of this disease by counting manually the amount of tuberculosis bacilli. It is very tedious for clinicians with a lot number of patient and without standardization for sputum staining. The tuberculosis sputum images have multi variant characterizations in colour, because of no standardization in staining. The sputum has more variants colour and they are difficult to be identified. For helping the clinicians, this research examined the Self Organizing Map method for colouring image segmentation in sputum images based on colour clustering. This method has better performance than k-means clustering which also tried in this research. The Self Organizing Map could segment the sputum images with y good result and cluster the colours adaptively.

Immunohistochemical findings of the granulomatous reaction associated with tuberculosis.

PubMed

Karimi, Shirin; Shamaei, Masoud; Pourabdollah, Mihan; Sadr, Makan; Karbasi, Mehrdad; Kiani, Arda; Bahadori, Moslem

2016-12-01

The histological diagnosis of Mycobacterium tuberculosis (MTB) has long been a diagnostic challenge in the anatomical pathology field despite availability of different laboratory methods. Immunohistochemistry (IHC) could not only confirm granulomatous tissue involvement but also demonstrate MTB antigen immunolocalization. This study tries to clarify the details of IHC staining for MTB with pAbBCG. A total of 50 patients undergoing simultaneous biopsy and tissue culture with positive tissue culture for MTB during 2005-2009 were selected from the MRC Department at Masih Daneshvari Hospital, Tehran, Iran. Using the archives of the Pathology Department of this hospital, which is a referral center for pathological lung lesions, hematoxylin and eosin slides of the selected patients were evaluated. Twenty-three confirmed TB granulomatous tissue samples with adequate tissue and number of granulomas were chosen and studied by Ziehl-Neelsen and IHC staining with pAbBCG. A total of 23 cases were evaluated, of which 17 (73.9%) were males. The types of tissue obtained from study cases were as follows: pleura (9 cases, 39.1%), lymph node (cervical, axillary, and thoracic [9 cases, 39.1%]), and lung tissues (5 cases, 21.7%). IHC staining was positive in all samples, whereas Ziehl-Neelsen staining was positive in nine cases of 23 (39.1%). IHC showed positive coarse granular cytoplasmic and round, fragmented bacillary staining. In this study, epithelioid cells clearly showed more positive staining at the periphery rather than at the center of granuloma. There is also positive staining in endothelial cells, fibroblasts, plasma cells, macrophages, and lymphocytes outside the granuloma. Detection of TB in tissue slides is still based on the histological pattern of the granuloma, which has several differential diagnoses with different treatments. Presence of mycobacterial antigens and tissue morphology can be evaluated using the IHC technique. Considering the criteria of positive IHC

Identification and application of ssDNA aptamers against H₃₇Rv in the detection of Mycobacterium tuberculosis.

PubMed

Aimaiti, Rusitanmujiang; Qin, Lianhua; Cao, Ting; Yang, Hua; Wang, Jie; Lu, Junmei; Huang, Xiaochen; Hu, Zhongyi

2015-11-01

Microscopy of direct smear with the Ziehl-Neelsen stain is still broadly used in tuberculosis diagnosis. However, this method suffers from low specificity and is difficult to distinguish Mycobacterium tuberculosis (MTB) from nontuberculosis mycobacterial (NTM), since all mycobacterial species are positive in Ziehl-Neelsen stain. In this study, we utilized whole cell SELEX to obtain species-specific aptamers for increasing the specificity of MTB detection. Whole cell SELEX was performed in MTB reference strain H37Rv by two selection processes based on enzyme-linked plate or Eppendorf tube, respectively. To increase success rate of generating aptamers, the selection processes were systematically monitored to understand the dynamic evolution of aptamers against complex structure of target bacteria. Two preponderant groups and ten high-affinity aptamers were obtained by analyzing the dynamic evolution. Preponderant aptamer MA1 from group I showed relatively high binding affinity with apparent dissociation constant (KD value) of 12.02 nM. Sandwich ELISA assay revealed five aptamer combinations effectively bound MTB strains in preliminary evaluation, especially the combination based on aptamer MA2 (another preponderant aptamer from group II) and MA1. Further evaluated in many other strains, MA2/MA1 combination effectively identified MTB from NTM or other pathogenic bacteria, and displayed the high specificity and sensitivity. Binding analysis of aptamer MA1 or MA2 by fluorescence microscopy observation showed high binding reactivity with H37Rv, low apparent cross-reactivity with M. marinum, and no apparent cross-reactivity with Enterobacter cloacae. Taken together, this study provides attractive candidate species-specific aptamers to effectively capture or discriminate MTB strains.

Application of modern microbiological diagnostic methods for tuberculosis in Macha, Zambia.

PubMed

Verweij, K E; Kamerik, A R; van Ingen, J; van Dijk, J H; Sikwangala, P; Thuma, P; Nouwen, J L; van Soolingen, D

2010-09-01

Macha, Zambia. To assess the benefits of auramine-O staining fluorescence microscopy and Mycobacterial Growth Indicator Tube (MGIT) liquid culture with molecular identification in tuberculosis (TB) diagnostics. One hundred patients suspected of TB were subjected to three sputum sample examinations applying Ziehl-Neelsen (ZN) and auramine-O staining and MGIT culture. Positive cultures were identified using the GenoType CM assay; cultures identified as Mycobacterium tuberculosis complex were the gold standard for a diagnosis of TB. The 100 patients produced 271 sputum samples; of these, 30 patients had positive cultures. M. tuberculosis complex bacilli were isolated in 17 (56.7%) patients, non-tuberculous mycobacteria (NTM) in 11 (36.7%) and other acid-fast bacilli in two. Forty-eight samples (17.7%) were contaminated. Auramine-O detected 16 (57.1%) patients culture-positive for mycobacteria and 12 patients with culture-proven TB, vs. respectively 8 (28.6%, P = 0.008) and 7 (41.2%, P = 0.044) for ZN. Three of eight auramine-positive/ZN-negative patients were culture-positive for NTM only. The auramine-O method significantly increases sensitivity, although the higher NTM detection rate implies that this does not in itself lead to a more accurate diagnosis of TB. MGIT culture is highly sensitive, although contamination rates were a drawback; the high frequency of NTM isolation warrants a robust identification method.

Detection of BCG bacteria using a magnetoresistive biosensor: A step towards a fully electronic platform for tuberculosis point-of-care detection.

PubMed

Barroso, Teresa G; Martins, Rui C; Fernandes, Elisabete; Cardoso, Susana; Rivas, José; Freitas, Paulo P

2018-02-15

Tuberculosis is one of the major public health concerns. This highly contagious disease affects more than 10.4 million people, being a leading cause of morbidity by infection. Tuberculosis is diagnosed at the point-of-care by the Ziehl-Neelsen sputum smear microscopy test. Ziehl-Neelsen is laborious, prone to human error and infection risk, with a limit of detection of 10 4 cells/mL. In resource-poor nations, a more practical test, with lower detection limit, is paramount. This work uses a magnetoresistive biosensor to detect BCG bacteria for tuberculosis diagnosis. Herein we report: i) nanoparticle assembly method and specificity for tuberculosis detection; ii) demonstration of proportionality between BCG cell concentration and magnetoresistive voltage signal; iii) application of multiplicative signal correction for systematic effects removal; iv) investigation of calibration effectiveness using chemometrics methods; and v) comparison with state-of-the-art point-of-care tuberculosis biosensors. Results present a clear correspondence between voltage signal and cell concentration. Multiplicative signal correction removes baseline shifts within and between biochip sensors, allowing accurate and precise voltage signal between different biochips. The corrected signal was used for multivariate regression models, which significantly decreased the calibration standard error from 0.50 to 0.03log 10 (cells/mL). Results show that Ziehl-Neelsen detection limits and below are achievable with the magnetoresistive biochip, when pre-processing and chemometrics are used. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection of Mycobacterium avium in pet birds

PubMed Central

Godoy, Silvia Neri; Sakamoto, Sidnei Miyoshi; de Paula, Cátia Dejuste; Catão-Dias, José Luiz; Matushima, Eliana Reiko

2009-01-01

The present study is a report on the presence of Mycobacterium avium in four birds of the psittaciform order kept as pets. Anatomopathological diagnosis showed lesions suggestive of the agent and presence of alcohol-acid resistant bacilli (AARB) shown by the Ziehl-Neelsen staining. The identification of Mycobacterium avium was performed by means of PRA (PCR Restriction Analysis). DNA was directly extracted from tissue of the lesions and blocked in paraffin. The role of this agent in pet bird infection is discussed, as well as its zoonotic potential. PMID:24031356

Meningoencephalitis tuberculosa in a holstein cow.

PubMed

Oruç, E

2005-11-01

The gross and histopathologic lesions of meningoencephalitis tuberculosa in a 4-year-old Holstein cow showing clinical signs compatible with bovine spongiform encephalopathy are described in this report. Grossly, numerous gray to yellow, firm and caseous nodules were seen on the ventral surfaces of the brain and in the lateral and fourth ventricles. Histopathologically, foci of caseation and dystrophic mineralization were surrounded by multinucleated giant cells, epitheloid macrophages, plasma cells, lymphocytes and fibrous proliferation. Ziehl-Neelsen stains of the lesions revealed masses of slender acid-fast bacilli in the necrotic centers of lesions and within surrounding giant cells.

Detection of mycobacteria in aquarium fish in Slovenia by culture and molecular methods.

PubMed

Pate, M; Jencic, V; Zolnir-Dovc, M; Ocepek, M

2005-04-06

Thirty-five aquarium fish were investigated for the presence of mycobacteria by culture and molecular methods. The following species were examined: goldfish Carassius auratus auratus, guppy Poecilia reticulata, 4 three-spot gourami Trichogaster trichopterus, dwarf gourami Colisa lalia, Siamese fighting fish Betta splendens, freshwater angelfish Pterophyllum scalare, African cichlid fish Cichlidae spp., cichlid fish Microgeophagus altispinosus, cichlid fish Pseudotropheus lombardoi, blue streak hap Labidochromis caeruleus, sterlet Acipenser ruthenus, southern platyfish Xiphophorus maculatus, and catfish Corydoras spp. Isolates of mycobacteria were obtained in 29 cases (82.9%). Two specimens were positive using Ziehl-Neelsen (ZN) staining, but the cultivation failed. Four specimens were both ZN- and culture-negative. On the basis of GenoType Mycobacterium assay (Hain Life-science) and restriction enzyme analysis of the amplified products (PCR-RFLP), 23 isolates (79.3%) were identified: 7 as Mycobacterium fortuitum, 6 as M. gordonae, 6 as M. marinum, 3 as M. chelonae, and 1 as M. peregrinum. Five isolates remained unidentified (Mycobacterium spp.). One case probably represented a mixed infection (M. marinum/M. fortuitum). Since M. marinum infections are also detected in humans, the significance of mycobacteria in aquarium fish should not be overlooked.

Assessment of Clinically Suspected Tubercular Lymphadenopathy by Real-Time PCR Compared to Non-Molecular Methods on Lymph Node Aspirates.

PubMed

Gupta, Vivek; Bhake, Arvind

2018-01-01

The diagnosis of tubercular lymphadenitis (TBLN) is challenging. This study assesses the role of diagnostic intervention with real-time PCR in clinically suspected tubercular lymphadenopathy in relation to cytology and microbiological methods. The cross-sectional study involved 214 patients, and PCR, cytology, and Ziehl-Neelsen (ZN) staining was performed on aspirates. The findings were compared with culture on Lowenstein-Jensen medium. The overall concordance of cytology and PCR, both individually and combined, was calculated. χ2 and Phi values were assessed between cytology, PCR, and culture. A cytological diagnosis of tuberculosis (TB), reactive lymphoid hyperplasia, and suppurative lymphadenitis was made in 71, 112, and 6 patients, respectively. PCR and culture were positive in 40% of the cases. Among the TBLN patients, PCR showed higher positivity in necrosis and culture showed higher positivity in necrotizing granuloma. Positive ZN staining was observed in 29.6% of the TBLN cases, with an overall positivity of 11%. PCR could additionally detect 82 cases missed by ZN staining. The overall concordance rate for either diagnostic modality, i.e., PCR or cytology, was highest (75%), and for PCR alone was 74%. Phi values were observed to be 0.47 between PCR and culture. Real-time PCR for Mycobacterium tuberculosis complex on aspirates offers a definitive and comparable diagnosis of TBLN. Including this approach as the primary investigation in the work-up of TBLN could reduce the burden of TB. © 2017 S. Karger AG, Basel.

Methods for chromosome-specific staining

DOEpatents

Gray, Joe W.; Pinkel, Daniel

1995-01-01

Methods and compositions for chromosome-specific staining are provided. Compositions comprise heterogenous mixtures of labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA for which their associated staining reagent is specific. Methods include methods for making the chromosome-specific staining compositions of the invention, and methods for applying the staining compositions to chromosomes.

Discriminative staining methods for the nervous system: luxol fast blue--periodic acid-Schiff--hematoxylin triple stain and subsidiary staining methods.

PubMed

Goto, N

1987-09-01

This paper describes a new series of staining methods which can discriminatively demonstrate every structure of the nervous system, including axons and capillaries, in animal and human materials. Methods described in this paper consist of one primary stain, luxol fast blue-periodic acid Schiff-hematoxylin (LPH) and six different subsidiary staining methods. The LPH triple stain can precisely differentiate the following structures: neurons (Nissl bodies, cytoplasm, nuclear membrane and nucleolus), various kinds of nuclei (glia, ependyma, endothelium, leucocyte, connective tissue, etc.), myelin sheaths, neuronal processes (axons and dendrites), reacted glial cell bodies (protoplasmic astrocytes, foamy cells, etc.), blood vessels (arteries, veins and capillaries), meninges, intervening connective tissue, erythrocytes, lipofuscin granules, amyloid bodies, and others. Subsidiary staining methods are also described briefly. Applications are discussed in the context of staining technology and neuromorphological research.

Methods for chromosome-specific staining

DOEpatents

Gray, J.W.; Pinkel, D.

1995-09-05

Methods and compositions for chromosome-specific staining are provided. Compositions comprise heterogeneous mixtures of labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA for which their associated staining reagent is specific. Methods include ways for making the chromosome-specific staining compositions of the invention, and methods for applying the staining compositions to chromosomes. 3 figs.

Value of the polymerase chain reaction method for detecting tuberculosis in the bronchial tissue involved by anthracosis.

PubMed

Mirsadraee, Majid; Shafahie, Ahmad; Reza Khakzad, Mohammad; Sankian, Mojtaba

2014-04-01

Anthracofibrosis is the black discoloration of the bronchial mucosa with deformity and obstruction. Association of this disease with tuberculosis (TB) was approved. The objective of this study was to find the additional benefit of assessment of TB by the polymerase chain reaction (PCR) method. Bronchoscopy was performed on 103 subjects (54 anthracofibrosis and 49 control subjects) who required bronchoscopy for their pulmonary problems. According to bronchoscopic findings, participants were classified to anthracofibrosis and nonanthracotic groups. They were examined for TB with traditional methods such as direct smear (Ziehl-Neelsen staining), Löwenstein-Jensen culture, and histopathology and the new method "PCR" for Mycobacterium tuberculosis genome (IS6110). Age, sex, smoking, and clinical findings were not significantly different in the TB and the non-TB groups. Acid-fast bacilli could be detected by a direct smear in 12 (25%) of the anthracofibrosis subjects, and adding the results of culture and histopathology traditional tests indicated TB in 27 (31%) of the cases. Mycobacterium tuberculosis was diagnosed by PCR in 18 (33%) patients, but the difference was not significant. Detection of acid-fast bacilli in control nonanthracosis subjects was significantly lower (3, 6%), but PCR (20, 40%) and accumulation of results from all traditional methods (22, 44%) showed a nonsignificant difference. The PCR method showed a result equal to traditional methods including accumulation of smear, culture, and histopathology.

Study of Colour Model for Segmenting Mycobacterium Tuberculosis in Sputum Images

NASA Astrophysics Data System (ADS)

Kurniawardhani, A.; Kurniawan, R.; Muhimmah, I.; Kusumadewi, S.

2018-03-01

One of method to diagnose Tuberculosis (TB) disease is sputum test. The presence and number of Mycobacterium tuberculosis (MTB) in sputum are identified. The presence of MTB can be seen under light microscope. Before investigating through stained light microscope, the sputum samples are stained using Ziehl-Neelsen (ZN) stain technique. Because there is no standard procedure in staining, the appearance of sputum samples may vary either in background colour or contrast level. It increases the difficulty in segmentation stage of automatic MTB identification. Thus, this study investigated the colour models to look for colour channels of colour model that can segment MTB well in different stained conditions. The colour models will be investigated are each channel in RGB, HSV, CIELAB, YCbCr, and C-Y colour model and the clustering algorithm used is k-Means. The sputum image dataset used in this study is obtained from community health clinic in a district in Indonesia. The size of each image was set to 1600x1200 pixels which is having variation in number of MTB, background colour, and contrast level. The experiment result indicates that in all image conditions, blue, hue, Cr, and Ry colour channel can be used to segment MTB in one cluster well.

Two Immigrants with Tuberculosis of the Ear, Nose, and Throat Region with Skull Base and Cranial Nerve Involvement

PubMed Central

Richardus, Renate A.; Jansen, Jeroen C.; Steens, Stefan C. A.; Arend, Sandra M.

2011-01-01

We report two immigrants with tuberculosis of the skull base and a review of the literature. A Somalian man presented with bilateral otitis media, hearing loss, and facial and abducens palsy. Imaging showed involvement of both mastoid and petrous bones, extending via the skull base to the nasopharynx, suggesting tuberculosis which was confirmed by characteristic histology and positive auramine staining, while Ziehl-Neelsen staining and PCR were negative. A Sudanese man presented with torticollis and deviation of the uvula due to paresis of N. IX and XI. Imaging showed a retropharyngeal abscess and lysis of the clivus. Histology, acid-fast staining, and PCR were negative. Both patients had a positive Quantiferon TB Gold in-tube result and improved rapidly after empiric treatment for tuberculosis. Cultures eventually yielded M. tuberculosis. These unusual cases exemplify the many faces of tuberculosis and the importance to include tuberculosis in the differential diagnosis of unexplained problems. PMID:21541186

[Case of polyparasitism with long-term abdominal pain in a patient].

PubMed

Doğan, Nihal; Koçman, Nazmiye Ulkü

2013-01-01

It is known that infections caused by intestinal protozoa and helminths affect over 3.5 million people worldwide. In this case report, a patient with complaints of stomach ache for a long time who received thermal treatment is presented. During this thermal treatment, diarrhoea occurred and multiparasitism was diagnosed with two helminths; pseudoparasitism and multiprotozoa, simultaneously. Stool samples were collected from the patient on three consecutive days and one day after the treatment. All of the samples were prepared with formalin-ether sedimentation techniques after macroscopic and direct microscopic investigation. Cellophane-tape method for Enterobius vermicularis and Taenia spp. and Erlich-Ziehl-Neelsen staining method for coccidian parasites were used. At least four preparations were performed for each sample and serum physiologic, lugol' solution and trichrome stain were used for microscopic investigations.The motile segment she brought was investigated microscopically with Indian ink and identified as Taenia saginata. Under direct microscopy, Blastocystis hominis, Endolimax nana and Fasciola hepatica were seen. By formalin-ether sedimentation techniques, Ascaris lumbricoides, Fasciola hepatica, Blastocystis hominis, Endolimax nana and Entamoeba coli were identified. In recent years, intestinal parasitism is rarely seen in our city; therefore, multiparasitism in an adult and immunocompetent patient is interesting.

Prevalence of Cryptosporidium-like infection in one-humped camels (Camelus dromedarius) of northwestern Iran

PubMed Central

Yakhchali, M.; Moradi, T.

2012-01-01

Cryptosporidium is a ubiquitous enteropathogen protozoan infection affecting livestock worldwide. The present study was carried out to determine the prevalence of Cryptosporidium infection in different age groups of dromedary camels in northwestern Iran from November 2009 to July 2010. A total number of 170 fecal samples were collected and examined using modified Ziehl-Neelsen (MZN) staining under light microscope. Examination of stained fecal smears revealed that 17 camels (10%) were positive for Cryptosporidium-like. The prevalence of Cryptosporidium-like was significantly higher in camel calves (< 1 years old) (20%) than other age groups, in which the diarrhoeic calves had the prevalence of 16%. In adult camels the prevalence was 6.5%. There was no significant difference in the prevalence of Cryptosporidium-like between male and female camels. It is concluded that Cryptosporidium infection is a problem in camel husbandry and could be of public health concern in the region. PMID:22314242

Prevalence of Cryptosporidium-like infection in one-humped camels (Camelus dromedarius) of northwestern Iran.

PubMed

Yakhchali, M; Moradi, T

2012-02-01

Cryptosporidium is a ubiquitous enteropathogen protozoan infection affecting livestock worldwide. The present study was carried out to determine the prevalence of Cryptosporidium infection in different age groups of dromedary camels in northwestern Iran from November 2009 to July 2010. A total number of 170 fecal samples were collected and examined using modified Ziehl-Neelsen (MZN) staining under light microscope. Examination of stained fecal smears revealed that 17 camels (10%) were positive for Cryptosporidium-like. The prevalence of Cryptosporidium-like was significantly higher in camel calves (< 1 years old) (20%) than other age groups, in which the diarrhoeic calves had the prevalence of 16%. In adult camels the prevalence was 6.5%. There was no significant difference in the prevalence of Cryptosporidium-like between male and female camels. It is concluded that Cryptosporidium infection is a problem in camel husbandry and could be of public health concern in the region.

Safer staining method for acid fast bacilli.

PubMed Central

Ellis, R C; Zabrowarny, L A

1993-01-01

To develop a method for staining acid fast bacilli which excluded highly toxic phenol from the staining solution. A lipophilic agent, a liquid organic detergent, LOC High Studs, distributed by Amway, was substituted. The acid fast bacilli stained red; nuclei, cytoplasm, and cytoplasmic elements stained blue on a clear background. These results compare very favourably with acid fast bacilli stained by the traditional method. Detergents are efficient lipophilic agents and safer to handle than phenol. The method described here stains acid fast bacilli as efficiently as traditional carbol fuchsin methods. LOC High Suds is considerably cheaper than phenol. Images PMID:7687254

Safer staining method for acid fast bacilli.

PubMed

Ellis, R C; Zabrowarny, L A

1993-06-01

To develop a method for staining acid fast bacilli which excluded highly toxic phenol from the staining solution. A lipophilic agent, a liquid organic detergent, LOC High Studs, distributed by Amway, was substituted. The acid fast bacilli stained red; nuclei, cytoplasm, and cytoplasmic elements stained blue on a clear background. These results compare very favourably with acid fast bacilli stained by the traditional method. Detergents are efficient lipophilic agents and safer to handle than phenol. The method described here stains acid fast bacilli as efficiently as traditional carbol fuchsin methods. LOC High Suds is considerably cheaper than phenol.

Methods and compositions for chromosome-specific staining

DOEpatents

Gray, Joe W.; Pinkel, Daniel

2003-07-22

Methods and compositions for chromosome-specific staining are provided. Compositions comprise heterogenous mixtures of labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA for which their associated staining reagent is specific. Methods include methods for making the chromosome-specific staining compositions of the invention, and methods for applying the staining compositions to chromosomes.

Disseminated Mycobacterium intracellulare infection in a broad-snouted caiman Caiman latirostris.

PubMed

Kik, Marja J L

2013-11-25

A 10 yr old broad-snouted caiman Caiman latirostris from a small Dutch animal park was presented with long-term variable periods of anorexia and weight loss. Blood chemistry showed slightly elevated uric acid levels and low ionised calcium concentration. Ultrasonographical thickening of the intestinal wall in the region of the duodenum was evident. Pathological changes were a thickening of the wall of 90% of the small intestines, enlarged spleen with multifocal white foci and an enlarged light-brown liver. Histopathological lesions consisted of disseminated granulomas in the intestinal wall, the liver and the spleen. Multinucleated giant cells and epitheloid macrophages were abundant. Ziehl-Neelsen staining showed numerous intralesional acid-fast bacteria. Polymerase chain reaction for Mycobacterium intracellulare was positive.

Comparative Study of GeneXpert with ZN Stain and Culture in Samples of Suspected Pulmonary Tuberculosis

PubMed Central

Bajaj, Ashish; Bhatia, Vinay; Dutt, Sarjana

2016-01-01

Introduction Tuberculosis remains one of the deadliest communicable diseases. There are number of tests available for the diagnosis of tuberculosis but conventional microscopy has low sensitivity and culture although gold standard, but takes longer time for positivity. On the other side, Nucleic acid amplification techniques due to its rapidity and sensitivity not only help in early diagnosis and management of tuberculosis especially in patients with high clinical suspicion like immunocompromised patients, history of contact with active tuberculosis patient etc., but also curtail the transmission of the disease. Aim To evaluate the sensitivity, specificity, positive predictive value and negative predictive value of Nucleic acid amplification assay (GeneXpert) using respiratory samples in patients with suspected pulmonary tuberculosis and compare with AFB smear microscopy (Ziehl Neelsen stain) and Acid Fast Bacilli (AFB) culture. Materials and Methods We retrospectively reviewed the respiratory samples of suspected pulmonary tuberculosis (including Bronchoalveolar lavage and sputum) of 170 patients from Jan 2015 to Nov 2015 for ZN stain, culture and GeneXpert (Xpert® MTB/Rif assay). The sensitivity, specificity, PPV and NPV of GeneXpert and ZN microscopy were calculated using Liquid culture of Mycobacterium tuberculosis as gold standard. Results A total of 170 patient samples were evaluated in final analysis. Of these, 14 samples were positive by all three methods used in our study. The overall sensitivity, specificity, PPV and NPV of GeneXpert were 86.8%, 93.1%, 78.5% and 96% respectively and for BAL sample, 81.4%, 93.4%, 73.3% and 95.7% respectively. The overall sensitivity and specificity of AFB smear microscopy were 22.2%, % and 78.5% respectively and for BAL sample 22.2% and 100% respectively. For AFB negative samples sensitivity and specificity were 79.1% and 93.1% respectively. Conclusion GeneXpert has a higher sensitivity than AFB smear microscopy in

Studies on Rheomelanins. VI. The Apparent Lipofuscin Characteristics of Rheomelanins,

DTIC Science & Technology

1980-05-01

of aldchydes formed by the action of periodic acid on non carbohydrate conit-tining unsaturated phosphatides . At present we know only that in the o...A ’mV -5 1. Periodic Acid -Schiff Test 2. Long Ziehl-Neelsen Test for Acid Fast lilpofuscii 3. Alternative Nile Blue Method 4. Schmorl Test 5. Sudan...Black B in Alcohol 6. Chrome Alum Hematoxylin Test 7. Protein Determination 8. Specific Miucoprotein Test 9. Phosphoric Acid -Vanillin Lipid

Evaluation of Microscopic Observation Drug Susceptibility (MODS) and the string test for rapid diagnosis of pulmonary tuberculosis in HIV/AIDS patients in Bolivia.

PubMed

Lora, Meredith H; Reimer-McAtee, Melissa J; Gilman, Robert H; Lozano, Daniel; Saravia, Ruth; Pajuelo, Monica; Bern, Caryn; Castro, Rosario; Espinoza, Magaly; Vallejo, Maya; Solano, Marco; Challapa, Roxana; Torrico, Faustino

2015-06-06

Tuberculosis (TB) is the most common opportunistic infection and the leading cause of death in HIV-positive people worldwide. Diagnosing TB is difficult, and is more challenging in resource-scarce settings where culture-based diagnostic methods rely on poorly sensitive smear microscopy by Ziehl-Neelsen stain (ZN). We performed a cross-sectional study examining the diagnostic utility of Microscopic Observation Drug Susceptibility liquid culture (MODS) versus traditional Ziehl-Neelsen staining (ZN) and Lowenstein Jensen culture (LJ) of pulmonary tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB) in HIV-infected patients in Bolivia. For sputum scarce individuals we assessed the value of the string test and induced sputum for TB diagnosis. The presence of Mycobacterium tuberculosis (Mtb) in the sputum of 107 HIV-positive patients was evaluated by ZN, LJ, and MODS. Gastric secretion samples obtained by the string test were evaluated by MODS in 102 patients. The TB-HIV co-infection rate of HIV patients with respiratory symptoms by sputum sample was 45 % (48/107); 46/48 (96 %) were positive by MODS, 38/48 (79 %) by LJ, and 30/48 (63 %) by ZN. The rate of MDRTB was 9 % (4/48). Median time to positive culture was 10 days by MODS versus 34 days by LJ (p < 0.0001). In smear-negative patients, MODS detected TB in 17/18 patients, compared to 11/18 by LJ (94.4 % vs 61.0 %, p = 0.03 %). In patients unable to produce a sputum sample without induction, the string test cultured by MODS yielded Mtb in of 9/11 (82 %) TB positive patients compared to 11/11 (100 %) with induced sputum. Of the 10 patients unable to produce a sputum sample, 4 were TB-positive by string test. MODS was faster and had a higher Mtb detection yield compared to LJ, with a greater difference in yield between the two in smear-negative patients. The string test is a valuable diagnostic technique for HIV sputum-scarce or sputum-absent patients, and should be considered as an alternative test to induced

Exploring the links between quality assurance and laboratory resources. An audit-based study.

PubMed

Singh, Navjeevan; Panwar, Aru; Masih, Vipin Fazal; Arora, Vinod K; Bhatia, Arati

2003-01-01

To investigate and rectify the problems related to Ziehl-Neelsen (Z-N) staining in a cytology laboratory in the context of quality assurance. An audit based quality assurance study of 1,421 patients with clinical diagnoses of tubercular lymphadenopathy who underwent fine needle aspiration cytology. Data from 8 months were audited (group 1). Laboratory practices related to selection of smears for Z-N staining were studied. A 2-step corrective measure based on results of the audit was introduced for 2 months (group 2). Results were subjected to statistical analysis using the chi 2 test. Of 1,172 patients in group 1,368 had diagnoses other than tuberculosis. Overall acid-fast bacillus (AFB) positivity was 42%. AFB positivity in 249 patients in group 2 was 89% (P < .0001). Several issues in the laboratory are linked to quality assurance. Solving everyday problems can have far-reaching benefits for the performance of laboratory personnel, resources and work flow.

Cytological diagnosis of tuberculous cervicitis: A case report with review of literature.

PubMed

Kalyani, R; Sheela, Sr; Rajini, M

2012-01-01

Tuberculosis of cervix is a rare disease. Tuberculosis usually affects women of childbearing age, indicating hormone dependence of infection. The patient presents with menstrual irregularities, infertility or vaginal discharge. Cervical lesions presents as papillary/vegetative growth or ulceration mimicking cervical cancer. Cervical Papanicolaou (Pap) smear plays an important role in diagnosing the disease by non-invasive technique in which the presence of epithelioid cells and Langhan's type of giant cells is diagnostic. However, other causes of granulomatous cervicitis should be considered and ruled out. Ziehl-Neelsen (ZN) stain for acid fast bacilli, fluorescent technique, biopsy and culture help in confirming the disease. We present the case of a 45-year-old female, who presented with vaginal discharge, dysfunctional uterine bleeding, first degree uterine descent with grade II cystocele and rectocele and cervical ulcer. Pap smear revealed epithelioid cells and Langhan's type of giant cells, confirmed by ZN stain of cervical smear, fluorescent technique and culture.

SURVEY OF HOUSE RAT INTESTINAL PARASITES FROM SURABAYA DISTRICT, EAST JAVA, INDONESIA THAT CAN CAUSE OPPORTUNISTIC INFECTIONS IN HUMANS.

PubMed

Prasetyo, R H

2016-03-01

The purpose of this study was to investigate the prevalence of house rat zoonotic intestinal parasites from Surabaya District, East Java, Indonesia that have the potential to cause opportunistic infection in humans. House rat fecal samples were collected from an area of Surabaya District with a dense rat population during May 2015. Intestinal parasites were detected microscopically using direct smear of feces stained with Lugol's iodine and modified Ziehl-Neelsen stains. The fecal samples were also cultured for Strongyloides stercoralis. Ninety-eight house rat fecal samples were examined. The potential opportunistic infection parasite densities found in those samples were Strongyloides stercoralis in 53%, Hymenolepis nana in 42%, Cryptosporidium spp in 33%, and Blastocystis spp in 6%. This is the first report of this kind in Surabaya District. Measures need to be taken to control the house rat population in the study area to reduce the risk of the public health problem. Keywords: zoonotic intestinal parasites, opportunistic infection, house rat, densely populated area, Indonesia

Centrifuge-operated specimen staining method and apparatus

NASA Technical Reports Server (NTRS)

Feeback, Daniel L. (Inventor); Clarke, Mark S. F. (Inventor)

1999-01-01

A method of staining preselected, mounted specimens of either biological or nonbiological material enclosed within a staining chamber where the liquid staining reagents are applied and removed from the staining chamber using hypergravity as the propelling force. In the preferred embodiment, a spacecraft-operated centrifuge and method of diagnosing biological specimens while in orbit, characterized by hermetically sealing a shell assembly. The assembly contains slide stain apparatus with computer control therefor, the operative effect of which is to overcome microgravity, for example on board an International Space Station.

Optimized protocols for Mycobacterium leprae strain management: frozen stock preservation and maintenance in athymic nude mice.

PubMed

Trombone, Ana Paula Fávaro; Pedrini, Sílvia Cristina Barbosa; Diório, Suzana Madeira; Belone, Andréa de Faria Fernandes; Fachin, Luciana Raquel Vicenzi; do Nascimento, Dejair Caitano; Rosa, Patricia Sammarco

2014-03-23

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1(nu)). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.

Marked seasonality of Cyclospora cayetanensis infections: ten-year observation of hospital cases, Honduras.

PubMed

Kaminsky, Rina Girard; Lagos, Javier; Raudales Santos, Gabriela; Urrutia, Samuel

2016-02-04

Document seasonality occurrence and epidemiologic characteristics of Cyclospora cayetanensis infections during a 10-year period from patients consulting at the University Hospital, Honduras. Retrospective non interventional hospital-based study analyzed laboratory results from the period 2002 to 2011 of fresh and Ziehl-Nielsen carbolfuchsin stained routine stool samples received for parasitologic examination. Sporadically a sample with numerous oocysts was allowed to sporulate in 2.5 % potassium dichromate confirming the presence of bi-cystic bi-zoic oocysts. A total of 35,157 fecal samples were examined during a ten-year span, of which a third (28.4 %) was stained by the Ziehl-Neelsen carbolfuchsin method diagnosing a total of 125 (1.3 %) C.cayetanensis infections. A statistically significant apparent seasonality was observed most years during May to August (range p < 0.036-0.001), with 83.3 % of 125 cases occurring in those rainy months. All C. cayetanensis cases came from urban poor neighborhoods; male/female relation was 1:1 except in 2006, when all patients were females (p = 0.05; r(2) = 22,448). Forty four point eight percent of the stool samples were diarrheic or liquid and 65.6 % infections were identified in children 10 years old or less. Enteric helminths and protozoa co-infected Cyclospora positive patients in 52 instances.: 8 % Ascaris lumbricoides, 8 % Giardia duodenalis, 23.2 % Blastocystis spp. and less frequently Entamoeba histolytica/E. dispar, Strongyloides stercoralis, and Trichuris trichiura. Results suggest a seasonal pattern for Cyclospora infections diagnosed in a clinical setting during the rainy months in Tegucigalpa and surrounding areas. Community studies should be conducted to support or dispute these observations.

Evaluation of a rapid culture method for tuberculosis diagnosis: a Latin American multi-center study.

PubMed

Robledo, J A; Mejía, G I; Morcillo, N; Chacón, L; Camacho, M; Luna, J; Zurita, J; Bodon, A; Velasco, M; Palomino, J C; Martin, A; Portaels, F

2006-06-01

Tuberculosis (TB) diagnostic laboratories in Latin America. Evaluation of thin-layer agar (TLA) compared to Löwenstein-Jensen (LJ) culture for the diagnosis of TB. Phase II prospective study in six laboratories. Samples included sputum and extra-pulmonary specimens from patients with a clinical diagnosis of TB. Respiratory samples were decontaminated using NaOH/ NALC; all samples were centrifuged, stained with Ziehl-Neelsen for acid-fast bacilli (AFB), cultured on LJ and TLA and identified according to recommended procedures. Sensitivity and likelihood ratios (LR), growth detection time and contamination rate were calculated for both media. A total of 1118 clinical specimens were studied. Cultures detected Mycobacterium tuberculosis in all AFB-positive samples, whereas for AFB-negative specimens LJ detected 3.2% and TLA 4.4%. Sensitivity was 92.6% (95%CI 87.9-95.9) and 84.7% (95%CI 78.8-89.0) for TLA and LJ, respectively. Positive and negative LRs were similar. Contamination was 5.1% for TLA and 3.0% for LJ. Median time to detection of a positive culture was 11.5 days (95%CI 9.3-15.0) for TLA and 30.5 days (95%CI 26.9-39.0) for LJ (P < 0.0001). Difference in the characteristics of the participating laboratories, the disease prevalence and the number and type of specimens processed did not affect the overall performance of TLA as compared to LJ, supporting the robustness of the method and its feasibility in different laboratory settings.

Methods And Compositions For Chromosome-Specific Staining

DOEpatents

Gray, Joe W.; Pinkel, Daniel

2003-08-19

Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods are provided to disable the hybridization capacity of shared, high copy repetitive sequences and/or remove such sequences to provide for useful contrast. Still further methods are provided to produce chromosome-specific staining reagents which are made specific to the targeted chromosomal material, which can be one or more whole chromosomes, one or more regions on one or more chromosomes, subsets of chromosomes and/or the entire genome. Probes and test kits are provided for use in tumor cytogenetics, in the detection of disease related loci, in analysis of structural abnormalities, such as translocations, and for biological dosimetry. Further, methods and prenatal test kits are provided to stain targeted chromosomal material of fetal cells, including fetal cells obtained from maternal blood. Still further, the invention provides for automated means to detect and analyse chromosomal abnormalities.

Opportunistic and non-opportunistic intestinal parasites in HIV/ AIDS patients in relation to their clinical and epidemiological status in a specialized medical service in Goiás, Brazil.

PubMed

Barcelos, Natane Barbosa; Silva, Lorena de Freitas E; Dias, Regyane Ferreira Guimarães; Menezes Filho, Hélio Ranes de; Rodrigues, Rosângela Maria

2018-03-08

Patients infected with the Human Immunodeficiency Virus (HIV) often have opportunistic infections, among which strongyloidiasis and coccidiosis are the most common parasitic infections that aggravate their health status. This study examined the prevalence of intestinal parasites, particularly of Strongyloides stercoralis and intestinal coccidia in patients with the Human Immunodeficiency Virus (HIV)/ Acquired Immunodeficiency Syndrome (AIDS) who were treated at the Specialized Assistance Service (SAE) of Jataí, State of Goiás, Brazil, and analyzed its correlation with clinical, laboratory, and socio-epidemiological parameters. A total of 270 stool samples were analyzed by the Lutz technique, Rugai's method, Agar Plate Culture, Ritchie's method and specific staining, Ziehl-Neelsen modified technique, Kinyoun's method and the rapid safranin method. The prevalence of intestinal parasites was 28.88% including 3.8% of S. stercoralis, Cryptosporidium sp. and Cystoisospora belli. There was a significant positive correlation between intestinal parasites and the clinical status and the use of antiretroviral therapy (ART), smoking, CD4+ lymphocyte counts and sexual orientation. In conclusion, the widespread use of antiretroviral therapy and health assistance contributed to the low prevalence of S. stercoralis and coccidiosis in patients with HIV/ AIDS who were followed up at the SAE.

Outbreak of Avian Tuberculosis in Commercial Domestic Pekin Ducks ( Anas platyrhynchos domestica).

PubMed

Zhu, De-Kang; Song, Xiao-Heng; Wang, Jiang-Bo; Zhou, Wang-Shu; Ou, Xu-Ming; Chen, Hong-Xi; Liu, Ma-Feng; Wang, Ming-Shu; Jia, Ren-Yong; Chen, Shun; Sun, Kun-Feng; Yang, Qiao; Wu, Ying; Chen, Xiao-Yue; Cheng, An-Chun

2016-09-01

Avian tuberculosis is a contagious disease affecting various domestic and wild bird species, and is caused by Mycobacterium avium . It is reported extremely rarely in commercial poultry flocks and has not been reported in commercial domestic ducks to date, with domestic ducks reported to be moderately resistant to M. avium infection. Here, we report the outbreak of avian tuberculosis in commercial Pekin duck ( Anas platyrhynchos domestica) flocks. Postmortem and histopathologic findings included nodules presenting in the visceral organs of ducks, and granulomas with central caseous necrosis surrounded by infiltrating lymphocytes. The M. avium pathogen was isolated and further identified by Ziehl-Neelsen staining and PCR based on insert sequence IS901 and the 16S rRNA gene. We highlight that avian tuberculosis not only has economic significance for the duck industry, but also presents a potential zoonotic hazard to humans.

[Experimental transmission of Cryptosporidium baileyi (Apicomplexa: Cryptosporidiidae) isolated of broiler chicken to Japanese quail (Coturnix japonica)].

PubMed

Cardozo, Sergian V; Teixeira Filho, Walter L; Lopes, Carlos Wilson G

2005-01-01

In this work, oocysts of Cryptosporidium baileyi were isolated and identified in broiler chickens from three different Municipalities of the State of Rio de Janeiro, where they were isolated and identified by using the centrifuge- flotation technique associated to bright-field. Staining techniques, such as: modified Ziehl-Neelsen and safranin-methylene blue, were carried out to confirm natural infection. Oocysts of C. baileyi from broiler chickens were able to infect Coccidia-free Japanese quails, by observation of endogenous stages at histological sections, and the elimination of oocysts in the feces with prepatent period of seven days and patent period of 21 days after infection. Oocysts of C. baileyi from broiler chickens and Japanese quails were similar on bright-field microscopy. With respect to the staining techniques used in this research, all of them left to significant changes in length and width of oocysts, but shape indexes were maintained. Bright-field microscopy was the best technique for oocysts comparison shed by broiler chickens and Japanese quail because of no different among oocysts were observed.

Pyogranulomatous pleuropneumonia and mediastinitis in ferrets (Mustela putorius furo) associated with Pseudomonas luteola Infection.

PubMed

Martínez, J; Martorell, J; Abarca, M L; Olvera, A; Ramis, A; Woods, L; Cheville, N; Juan-Sallés, C; Moya, A; Riera, A; Soto, S

2012-01-01

Between 2008 and 2009, three pet ferrets from different sources presented with acute episode of dyspnoea. Cytological examination of pleural exudates revealed severe purulent inflammation with abundant clusters of rod-shaped microorganisms with a clear surrounding halo. Treatment was ineffective and the ferrets died 2-5 days later. Two ferrets were subjected to necropsy examination, which revealed pyothorax, mediastinal lymphadenopathy and multiple white nodules (1-2mm) in the lungs. Microscopical examination showed multifocal necrotizing-pyogranulomatous pleuropneumonia and lymphadenitis with aggregates of encapsulated microorganisms, some of which were positively stained by periodic acid-Schiff and alcian blue. In-situ hybridization for Pneumocystis spp., Ziehl-Neelsen staining and immunohistochemistry for distemper, coronavirus and influenza antigen were negative in all cases. Electron microscopically, the bacteria were 2-3 μm long with a thick electron-lucent capsule. Microbiology from one ferret yielded a pure culture of gram-negative bacteria identified phenotypically as Pseudomonas luteola. This speciation was later confirmed by 16S RNA gene amplification. Copyright © 2011. Published by Elsevier Ltd.

First molecular investigation of Cryptosporidium spp. in young calves in Algeria.

PubMed

Benhouda, Djahida; Hakem, Ahcène; Sannella, Anna Rosa; Benhouda, Afaf; Cacciò, Simone M

2017-01-01

To date, no information is available on the prevalence and genetic identity of Cryptosporidium spp. in cattle in Algeria. In this study, 17 dairy farms in the province of Batna, located in the northeast of the country, were visited to collect 132 fecal samples from young calves (< 8 weeks old). Samples were examined microscopically using the modified Ziehl-Neelsen acid-fast staining method, and at least one sample per farm was submitted for molecular analysis. Amplification of a fragment of the small subunit ribosomal RNA gene was positive for 24 of the 61 samples (40%), and sequence analysis identified three species, namely Cryptosporidium bovis (n = 14), C. ryanae (n = 6), and C. parvum (n = 4). The C. parvum IIaA13G2R1 subtype, an uncommon zoonotic subtype, was identified in two isolates from a single farm by sequencing a fragment of the GP60 gene. This is the first report about genotyping and subtyping of Cryptosporidium in calves in Algeria. © D. Benhouda et al., published by EDP Sciences, 2017.

First molecular investigation of Cryptosporidium spp. in young calves in Algeria

PubMed Central

Benhouda, Djahida; Hakem, Ahcène; Sannella, Anna Rosa; Benhouda, Afaf; Cacciò, Simone M.

2017-01-01

To date, no information is available on the prevalence and genetic identity of Cryptosporidium spp. in cattle in Algeria. In this study, 17 dairy farms in the province of Batna, located in the northeast of the country, were visited to collect 132 fecal samples from young calves (< 8 weeks old). Samples were examined microscopically using the modified Ziehl-Neelsen acid-fast staining method, and at least one sample per farm was submitted for molecular analysis. Amplification of a fragment of the small subunit ribosomal RNA gene was positive for 24 of the 61 samples (40%), and sequence analysis identified three species, namely Cryptosporidium bovis (n = 14), C. ryanae (n = 6), and C. parvum (n = 4). The C. parvum IIaA13G2R1 subtype, an uncommon zoonotic subtype, was identified in two isolates from a single farm by sequencing a fragment of the GP60 gene. This is the first report about genotyping and subtyping of Cryptosporidium in calves in Algeria. PMID:28497744

Methods of biological dosimetry employing chromosome-specific staining

DOEpatents

Gray, Joe W.; Pinkel, Daniel

2000-01-01

Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods are provided to disable the hybridization capacity of shared, high copy repetitive sequences and/or remove such sequences to provide for useful contrast. Still further methods are provided to produce chromosome-specific staining reagents which are made specific to the targeted chromosomal material, which can be one or more whole chromosomes, one or more regions on one or more chromosomes, subsets of chromosomes and/or the entire genome. Probes and test kits are provided for use in tumor cytogenetics, in the detection of disease related loci, in analysis of structural abnormalities, such as translocations, and for biological dosimetry. Further, methods and prenatal test kits are provided to stain targeted chromosomal material of fetal cells, including fetal cells obtained from maternal blood. Still further, the invention provides for automated means to detect and analyse chromosomal abnormalities.

Differentiation of histoplasma and cryptococcus in cytology smears: a diagnostic dilemma in severely necrotic cases.

PubMed

Ranjan, R; Jain, D; Singh, L; Iyer, V K; Sharma, M C; Mathur, S R

2015-08-01

The correct identification of fungal organisms is important for the appropriate clinical management of patients. It becomes difficult in necrotic smears when the tissue response is not clearly discernible. It is difficult to distinguish between histoplasma and cryptococcus in severely necrotic cases, where both appear as variably sized clear refractile haloes. Four cases of adrenal necrotic histoplasma infection were studied and the morphology was compared with that of non-necrotic histoplasmosis and cases of cryptococcal infection. Eleven cases were analysed in fine needle aspiration cytology (FNAC) smears. Ziehl-Neelsen (ZN) stain was performed to exclude tuberculosis in necrotic smears. A clinical and serology correlation was performed where available. Necrotic cases of histoplasma infection revealed negative refractile clear haloes similar to those of cryptococcus. Histoplasma showed methylene blue-stained organisms in ZN stains, whereas the cryptococcus cases were negative. Similar methylene blue-stained organisms were seen in non-necrotic histoplasma infection. As a result of morphological overlap between cryptococcus and histoplasma, the distinction between the two fungi can be difficult in many cases. ZN staining appears to have a role in the differentiation of these fungi in severely necrotic cases. This observation needs to be validated on a larger number of cases with complete correlation with clinical, serology and treatment records. © 2014 John Wiley & Sons Ltd.

The use of a differential fluorescent staining method to detect bacteriuria.

PubMed

Ciancaglini, Ettore; Fazii, Paolo; Sforza, Giuseppe Riario

2004-01-01

This report describes a differential staining method which distinguishes gram-positive from gram-negative bacteria in fluorescence. Gram-positive bacteria appear yellow and gram-negative bacteria appear green. The method is based on two fluorochromes, one acting in the wavelength of red, i.e. the acridine orange, and another acting in the wavelength of green, i.e. the fluorescein, which together form a red/ green system. In this report we compared the accuracy of the differential fluorescent staining method and the Gram stain in screening for bacteriuria, as detected by conventional cultures. A total of 1487 urine samples were tested. 289 cultures were positive. 237 specimens grew a single organism at 10(5) and 10(4) CFU/ml. 224 smears were detected by the differential fluorescent staining method and 162 were detected by Gram stain. 1198 samples failed to grow organisms at 10(5) and 10(4) CFU/ml. 107 smears were falsely positive by the fluorescent staining procedure and 289 were falsely positive by the Gram stain. On the basis of the culture results, the sensitivity of the differential fluorescent staining method was 94.5% and that of the Gram stain 68.3%. The specificity of the fluorescent staining procedure was 91.6% and that of the Gram stain 75.8%. The positive predictive value and the negative predictive value of the fluorescent staining method were 67.6% and 98.8%, respectively. Those of the Gram stain were 35.9% and 92.3%, respectively. A wide range of microbiological and chemical techniques are available to identify bacteria in urine. This fluorescent staining method represents a simple, rapid, reliable method with low-running costs. The main advantage of this technique is that it enables the microbiologist to exclude the presence of bacteria in the urine within a short time after specimen receipt and to eliminate a large number of specimens for culture with significant cost saving. Another advantage of the method is that it allows to distinguish gram

[Histochemical stains for minerals by hematoxylin-lake method].

PubMed

Miyagawa, Makoto

2013-04-01

The present study was undertaken to establish the experimental animal model by histological staining methods for minerals. After intraperitoneal injections of minerals, precipitates deposited on the surface of the liver. Liver tissues were fixed in paraformaldehyde, embedded in paraffin and cut into thin sections which were used as minerals containing standard section. Several reagents for histological stains and spectrophotometry for minerals were applied in both test-tube experiments and stainings of tissue sections to test for minerals. Hematoxylin-lake was found of capable of staining minerals in tissue. A simple technique used was described for light microscopic detection of minerals.

Surgical site infections due to rapidly growing mycobacteria in puducherry, India.

PubMed

Kannaiyan, Kavitha; Ragunathan, Latha; Sakthivel, Sulochana; Sasidar, A R; Muralidaran; Venkatachalam, G K

2015-03-01

Rapidly growing Mycobacteria are increasingly recognized, nowadays as an important pathogen that can cause wide range of clinical syndromes in humans. We herein describe unrelated cases of surgical site infection caused by Rapidly growing Mycobacteria (RGM), seen during a period of 12 months. Nineteen patients underwent operations by different surgical teams located in diverse sections of Tamil Nadu, Pondicherry, Karnataka, India. All patients presented with painful, draining subcutaneous nodules at the infection sites. Purulent material specimens were sent to the microbiology laboratory. Gram stain and Ziehl-Neelsen staining methods were used for direct examination. Culture media included blood agar, chocolate agar, MacConkey agar, Sabourauds agar and Lowenstein-Jensen medium for Mycobacteria. Isolated microorganisms were identified and further tested for antimicrobial susceptibility by standard microbiologic procedures. Mycobacterium fortuitum and M.chelonae were isolated from the purulent drainage obtained from wounds by routine microbiological techniques from all the specimens. All isolates analyzed for antimicrobial susceptibility pattern were sensitive to clarithromycin, linezolid and amikacin but were variable to ciprofloxacin, rifampicin and tobramycin. Our case series highlights that a high level of clinical suspicion should be maintained for patients presenting with protracted soft tissue lesions with a history of trauma or surgery as these infections not only cause physical but also emotional distress that affects both the patients and the surgeon.

Detection of Mycobacterium tuberculosis based on H37R(v) binding peptides using surface functionalized magnetic microspheres coupled with quantum dots – a nano detection method for Mycobacterium tuberculosis.

PubMed

Yang, Hua; Qin, Lianhua; Wang, Yilong; Zhang, Bingbo; Liu, Zhonghua; Ma, Hui; Lu, Junmei; Huang, Xiaochen; Shi, Donglu; Hu, Zhongyi

2015-01-01

Despite suffering from the major disadvantage of low sensitivity, microscopy of direct smear with the Ziehl-Neelsen stain is still broadly used for detection of acid-fast bacilli and diagnosis of tuberculosis. Here, we present a unique detection method of Mycobacterium tuberculosis (MTB) using surface functionalized magnetic microspheres (MMSs) coupled with quantum dots (QDs), conjugated with various antibodies and phage display-derived peptides. The principle is based upon the conformation of the sandwich complex composed of bacterial cells, MMSs, and QDs. The complex system is tagged with QDs for providing the fluorescent signal as part of the detection while magnetic separation is achieved by MMSs. The peptide ligand H8 derived from the phage display library Ph.D.-7 is developed for MTB cells. Using the combinations of MMS-polyclonal antibody+QD-H8 and MMS-H8+QD-H8, a strong signal of 10(3) colony forming units (CFU)/mL H37R(v) was obtained with improved specificity. MS-H8+QD-H8 combination was further optimized by adjusting the concentrations of MMSs, QDs, and incubation time for the maximum detection signal. The limit of detection for MTB was found to reach 10(3) CFU/mL even for the sputum matrices. Positive sputum samples could be distinguished from control. Thus, this novel method is shown to improve the detection limit and specificity of MTB from the sputum samples, and to reduce the testing time for accurate diagnosis of tuberculosis, which needs further confirmation of more clinical samples.

Evidence of disseminated infection by Mycobacterium avium subspecies hominissuis in a pet ferret (Mustela putorius furo).

PubMed

Bezos, Javier; Álvarez-Carrión, Beatriz; Rodríguez-Bertos, Antonio; Fernández-Manzano, Álvaro; de Juan, Lucía; Huguet, Cristina; Briones, Víctor; Romero, Beatriz

2016-12-01

The infection caused by the zoonotic opportunistic pathogen Mycobacterium avium subsp. hominissuis (Mah) was reported for the first time in a pet ferret. Both owners were HIV-positive. Euthanasia of the pet was recommended due to medical reasons and as a preventive action. Disseminated and open tuberculosis lesions were observed in the gastrointestinal and respiratory systems of the ferret. Ecographic and radiographic surveys showed a severe generalized lymphadenopathy, strong thickening of the gastric wall and peritoneum layer. The histopathological findings revealed a disseminated, granulomatous, chronic inflammation affecting the gastrointestinal tract, lungs, lymphoid tissues (spleen, tonsils and lymph nodes) and liver. Ziehl-Neelsen staining displayed the presence of positive acid-fast bacilli within these granulomas. Bacteriology and sequencing of the isolates yielded Mah sequevar code 3. Ferrets can act as reservoirs of mycobacteria exposing their owners to the infection, which is of major concern in immunodeficient individuals, as those HIV-infected. Copyright © 2016 Elsevier Ltd. All rights reserved.

Atypical mycobacterial infection presenting as persistent skin lesion in a patient with ulcerative colitis.

PubMed

Bamias, Giorgos; Daikos, George L; Siakavellas, Spyros I; Kaltsa, Garyfallia; Smilakou, Stavroula; Katsogridakis, Ioannis; Vafiadis-Zouboulis, Irene; Ladas, Spiros D

2011-01-01

Immunosuppressive drugs are commonly used for the treatment of inflammatory bowel disease. Patients receiving immunosuppressants are susceptible to a variety of infections with opportunistic pathogens. We present a case of skin infection with Mycobacterium chelonae in a 60-year-old Caucasian woman with ulcerative colitis who had been treated with corticosteroids and azathioprine. The disease manifested with fever and rash involving the right leg. Infliximab was administered due to a presumptive diagnosis of pyoderma gangrenosum, leading to worsening of the clinical syndrome and admission to our hospital. Routine cultures from various sites were all negative. However, Ziehl-Neelsen staining of pus from the lesions revealed acid-fast bacilli, and culture yielded a rapidly growing mycobacterium further identified as M. chelonae. The patient responded to a clarithromycin-based regimen. Clinicians should be aware of skin lesions caused by atypical mycobacteria in immunocompromised patients with inflammatory bowel disease. Furthermore, they should be able to thoroughly investigate and promptly treat these conditions.

Morbidity pattern of hydatid disease (cystic echinococcosis) and lack of its knowledge in patients attending Mamata General Hospital, Khammam, Andhra Pradesh.

PubMed

Hemachander, S Suguna; Prasad, C Rajendra; Jessica, M

2008-01-01

There is hearsay that prevalence of hydatid disease in Khammam and Nalgonda districts of Andhra Pradesh is high. We report here a preliminary study conducted to determine the magnitude of the problem of hydatid disease and the morbidity associated with it in patients attending MGH, KMM, A.P. (rural hospital). Eleven cases were identified during the period from November 2005 to May 2006 (seven months). Pain in abdomen, mass per abdomen, loss of appetite, pregnancy complicated by cystic echinococcosis (CE), and jaundice were the main clinical symptoms and signs. Ultrasonography, detection and removal of the cysts on the operation table, microscopic examination of the aspirated hydatid fluid were confirmatory. Ziehl-Neelsen stain of the aspirated fluid revealed acid-fast scolices. Interrogation of the patients and their family members (50) revealed that there was a total lack of knowledge of dog-tapeworm-caused infection in humans. They knew 'rabies' as the only disease man gets from dogs, and tapeworms are from pork and beef.

Primary Conjunctival Tuberculosis Presenting as Dry Eye: A Rare Case Report and Review of the Literature.

PubMed

Brar, Rupinder Kaur; Singh, Ashok; Deshpande, Archana Hemant; Gargade, Chitrawati Bal; Das, Sujit

2017-11-01

Primary conjunctival tuberculosis is very rare in the developed countries. In an endemic country like India, it should be considered in the differential diagnosis of any unusual conjunctival lesion with unilaterality, chronicity, and nonresolution of symptoms after steroid use. We present the case of a 52-year-old female who presented with unilateral itching and blurring of vision for 20 days. There were irregular nodular elevated areas with shrinkage of the lower palpebral conjunctiva. A biopsy of the lesion revealed necrotizing epithelioid cell granulomas along with Langhans type of giant cells. However, no acid-fast bacilli were seen on Ziehl-Neelsen stain. Systemic examination of the patient was normal, and there was no evidence of pulmonary tuberculosis. Polymerase chain reaction of conjunctival scrapings was positive for Mycobacterium tuberculosis . The patient was started on antitubercular drugs. We present this very rare case of primary tuberculosis of the conjunctiva presenting with dryness of the eye.

[Dysphagia in a young woman from Somalia].

PubMed

Veldhuis, Suzanne; van Altena, Richard; van Steenwijk, Reindert P; Rauws, Erik A J; Eeftinck Schattenkerk, Jan Karel M

2013-01-01

Multidrug-resistant tuberculosis is increasing worldwide. The determination of possible resistance is essential for adequate treatment. Tuberculosis is common amongst immigrants from Somalia and extra-pulmonary localisation is often seen. A 21-year-old woman from Somalia presented with progressive dysphagia and severe weight loss. Endoscopy revealed two ulcers in the mid-oesophagus. A chest x-ray showed enlarged lymph nodes in the right hilar and mediastinal regions. The Ziehl-Neelsen stain and PCR for mycobacteria were negative. Sputum samples and oesophageal biopsies were cultured. Quadruple tuberculostatic therapy was started empirically. After five weeks, a sputum culture grew Mycobacterium tuberculosis, which was resistant to rifampicin and isoniazid. She was treated with second-line anti-tuberculous therapy and eventually recovered. Tuberculosis can manifest in many ways. It is important to obtain patient material for culture; not only to confirm the diagnosis but also for the determination of possible resistance which is necessary for adequate therapy.

Improved method for combination of immunocytochemistry and Nissl staining.

PubMed

Kádár, Andrea; Wittmann, Gábor; Liposits, Zsolt; Fekete, Csaba

2009-10-30

Nissl staining is a widely used method to study morphology and pathology of neural tissue. After standard immunocytochemistry, the Nissl staining labels only the nucleus of neurons and the characteristic staining of the neuronal perikarya is absent or very weak. We hypothesized that the RNA degradation during the immunocytochemical treatment results in the loss of cytoplasmic staining with Nissl-dyes. To test this hypothesis, we used RNAse-free conditions for all steps of immunostaining. To further prevent the RNA-degradation by RNAse contaminations, the RNAse inhibitor heparin was added to all antibody-containing solutions. The efficiency of Nissl staining after standard and RNAse-free double-labeling immunocytochemistry was compared using antibodies against c-Fos and neuropeptide Y (NPY) on tissues of rats refed after 3 days of fasting. After standard immunocytochemistry, the Nissl-staining labeled the nuclei of neurons and only very faintly the cytoplasm of these cells. The RNAse-free treatment did not alter the distribution of immunoreaction signal, but preserved the staining of neuronal perikarya by the Nissl-dyes. In conclusion, the RNAse-free conditions during immunocytochemistry allow the labeling of neuronal perikarya by Nissl-dyes. The described method facilitates the mapping of immunocytochemical signals and makes possible the light microscopic examination of the innervation of neurons identified by their nuclear protein content.

[Comparison of the quick Gram stain method to the B&M modified and favor methods].

PubMed

Osawa, Kayo; Kataoka, Nobumasa; Maruo, Toshio

2011-01-01

The Gram stain is an established method for bacterial identification, but the time needed to carry out this stain is 2-3 min. We attempted to shorten this time and stained a total of 70 clinical specimens isolated from using the Bartholomew & Mittwer (B&M) modified or Favor methods with a 3 s duration for washing and staining steps. Results were plotted and analyzed using a Hue Saturation Intensity (HSI) model. The range based on a plot of the two methods with the HSI model was presented as a reference interval. Our results indicated that 100% (35/35) of strains were Gram positive and 97.1% (34/35) were Gram negative for the quick B&M modified method. In the quick Favor method, 80.0% (28/35) were Gram positive and 68.6% (24/35) of strains were Gram negative. We propose that the quick B&M modified method is equivalent to the standard Gram staining method and is superior to the quick Favor method.

Detection and molecular characterization of Mycobacterium celatum as a cause of splenitis in a domestic ferret (Mustela putorius furo).

PubMed

Piseddu, E; Trotta, M; Tortoli, E; Avanzi, M; Tasca, S; Solano-Gallego, L

2011-01-01

Mycobacterium celatum is a slow growing non-tuberculous mycobacterium described mainly as occurring in human patients. Only two cases of infection with this pathogen have been reported previously in animals. A 5-year-old, neutered male ferret was presented with progressive weight loss and muscle atrophy. Pale mucous membranes, slight alopecia of the tail and splenomegaly, confirmed by abdominal ultrasound, were observed. Fine-needle aspirations of the spleen revealed extramedullary haematopoiesis and marked macrophage-dominated inflammation associated with mycobacterial infection. Ziehl-Neelsen staining demonstrated sporadic acid-fast bacilli within macrophages. These organisms were identified as M. celatum by microbiological and molecular methods. Phylogenetic analysis based on the 16S rDNA gene compared this isolate with previously reported strains and demonstrated close relatedness to the human strains of M. celatum types 1 and 3. The ferret was treated with enrofloxacin, rifampicin and azithromycin, resulting in clinical improvement. After 40 days of treatment, the spleen was re-evaluated. Cytological evaluation revealed only extramedullary haematopoiesis without evidence of infection. Discontinuation of therapy was followed by rapid deterioration and death. Copyright © 2010 Elsevier Ltd. All rights reserved.

Evaluation of staining methods for cytologic diagnosis of oral lesions.

PubMed

Almeida, Janete Dias; Lima, Celina Faig; Brandão, Adriana Aigotti Haberbeck; Cabral, Luiz Antonio Guimarães

2008-01-01

To compare the efficacy ofPapanicolaou, hematoxylin-eosin (H-E), Leishman and periodic acid-Schiff (PAS) staining for cytologic diagnosis of oral lesions. Patients from the Discipline of Stomatology, São José dos Campos Dental School, from the wards of Hosapital Heliópolis and from the dentistry outpatient clinic of the University Hospital, University of São Paulo Medical School, with the following diseases, were selected: erythematous candidiasis (n=9), pseudomembranous candidiasis (n=10), squamous cell carcinoma (n=19), herpes simplex (n=8), paracoccidioidomycosis (n=8) and pemphigus vulgaris (n=1). The different staining methods were compared regarding the quality of definition of cytoplasmic and nuclear morphologic characteristics and the identification of bacteria, fungi, inflammatory cells and secretions. Papanicolaou and H-E staining were considered better methods. In cases of fungal infections, PAS staining is useful and should be applied as a complementary method. Within the limitations of this study, it can be concluded that the cytologic diagnosis of oral lesions along with different staining methods is a useful tool for oral diagnosis.

Evaluation the virulence of Mycobacterium bovis isolated from milk samples through histopathological study in laboratory animals.

PubMed

Al-Saqur, I M; Al-Thwani, A N; Al-Attar, I M; Al-Mashhadani, M S

2016-12-01

Mycobacterium bovis has a broad host range, and it is the principal agent responsible for tuberculosis (TB) in bovine, domestic and wild mammals. M. bovis also infects human, causing zoonotic TB through ingestion, inhalation and, less frequently by contact with mucous membranes and broken skin. Zoonotic TB was formerly an endemic disease, usually transmitted to man by consumption of raw cow's milk. It is indistinguishable clinically or pathologically from TB caused by M. tuberculosis. The aims of this study were, to isolate and identified M. bovis from raw milk samples by different methods, and evaluate the virulence of M. bovis in laboratory animals (Rabbit). To conduct the study, ninety three cow's milk samples were collected from farms around Baghdad governorate. The decontamination of milk samples was firstly carried out, then samples were subjected to routine tests which include, direct smear for Ziehl Neelsen acid fast stain, culture, each sample was cultured on Lowenstein Jensen media with Sodium pyruvite (All cultures incubated on 37°C for 4-10weeks with continuous observation), and biochemical testes as Nitrate reduction test, Niacin paper strip test and pyrazinamidase test, were employed to diagnose and identified the bacteria. Beside molecular assay was used to confirm the identification of the isolates by Polymerase Chain Reaction (PCR) using specific primers for M. bovis. The virulence of these isolates were investigated through inoculate it in group of laboratory animals consist of 8 rabbit in addition to other group of 4 animals as control (inoculate with Phosphate Buffer Saline). The animals were scarified after 6weeks of inoculation, post- mortem examination was carried out, smears were taken from lesions, and tissue samples were collected from lymph nodes and different organs. The results revealed five isolates of M. bovis in direct smear by acid fast Ziehl-Neelsen stain, while eight isolates observed by culture, the colonies appeared with

Improved method for combination of immunocytochemistry and Nissl staining

PubMed Central

Kádár, Andrea; Wittmann, Gábor; Liposits, Zsolt; Fekete, Csaba

2009-01-01

Nissl-staining is a widely used method to study morphology and pathology of neural tissue. After standard immunocytochemistry, the Nissl-staining labels only the nucleus of neurons and the characteristic staining of the neuronal perikarya is absent or very weak. We hypothesized that the RNA degradation during the immunocytochemical treatment results in the loss of cytoplasmic staining with Nissl-dyes. To test this hypothesis, we used RNAse-free conditions for all steps of immunostaining. To further prevent the RNA-degradation by RNAse contaminations, the RNAse inhibitor heparin was added to all antibody-containing solutions. The efficiency of Nissl-staining after standard and RNAse-free double-labeling immunocytochemistry was compared using antibodies against c-Fos and neuropeptide Y (NPY) on tissues of rats refed after three days of fasting. After standard immunocytochemistry, the Nissl-staining labeled the nuclei of neurons and only very faintly the cytoplasm of these cells. The RNAse-free treatment did not alter the distribution of immunoreaction signal, but preserved the staining of neuronal perikarya by the Nissl-dyes. In conclusion, the RNAse-free conditions during immunocytochemistry, allows the labeling of neuronal perikarya by Nissl-dyes. The described method facilitates the mapping of immunocytochemical signals and makes possible the light microscopic examination of the innervation of neurons identified by their nuclear protein content. PMID:19615409

Mycobacteriosis caused by Mycobacterium marinum in reared mullets: first evidence from Sardinia (Italy).

PubMed

Antuofermo, E; Pais, A; Polinas, M; Cubeddu, T; Righetti, M; Sanna, M A; Prearo, M

2017-03-01

Mycobacterium marinum is a slow-growing non-tuberculous mycobacterium, and it is considered the most common aetiologic agent of mycobacteriosis in wild and cultured fish. The diagnosis is principally made by histology when positive Ziehl-Neelsen stain granulomas are detected. The aim of this study was to investigate the occurrence of mycobacteriosis in extensively cultured Mugilidae of two lagoons (Cabras and San Teodoro) from Sardinia by the use of histology, microbiology, PCR and DNA sequencing. Nine of 106 mullets examined were affected by mycobacteriosis, and the spleen was the most affected organ. The histology detected higher rate (100%) of infection in spleen than the culture and PCR (75% and 62.5%, respectively). The sequencing of hsp65 gene identified M. marinum as the primary cause of mycobacteriosis in the mullets examined. Mullets affected by mycobacteriosis were mainly fished in the San Teodoro lagoon characterized by critical environmental conditions. Histology remains the most common method in detecting fish affected by mycobacteriosis, and PCR-based methods are essential for species identification. Our finding are worthy of attention because mycobacteriosis caused by M. marinum in reared mullets was evidenced for the first time in Sardinia, suggesting that this disease may be underestimated also in other cultured fish species. © 2016 John Wiley & Sons Ltd.

Usefulness of Leifson Staining Method in Diagnosis of Helicobacter pylori Infection

PubMed Central

Piccolomini, Raffaele; Di Bonaventura, Giovanni; Neri, Matteo; Di Girolamo, Arturo; Catamo, Giovanni; Pizzigallo, Eligio

1999-01-01

The Leifson staining method was used to diagnose Helicobacter pylori infection and was compared to histology, culture, and the rapid urease test (RUT). Histology gave the best sensitivity (98%), compared to Leifson staining (97%), culture (92%), and RUT (85%) (P < 0.005). Leifson staining is a sensitive, rapid, economical method for diagnosis of H. pylori infection in dyspeptic patients. PMID:9854090

COMPARISON OF PERMANENT STAINING METHODS FOR THE LABORATORY DIAGNOSIS OF TRICHOMONIASIS

PubMed Central

MENEZES, Camila Braz; MELLO, Mariana dos Santos; TASCA, Tiana

2016-01-01

Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease (STD) in the world. The diagnosis is based on wet mount preparation and direct microscopy on fixed and stained clinical specimens. The aim of this study was to compare the performance of different fixing and staining techniques used in the detection of T. vaginalis in urine. The smears were fixed and submitted to different methods of permanent staining and then, the morphological aspects of the parasites were analyzed and compared. The Papanicolaou staining with ethanol as the fixative solution showed to be the best method of permanent staining. Our data suggest that staining techniques in association with wet mount examination of fresh specimens contribute to increase the sensitivity in the diagnosis of trichomoniasis. PMID:26910452

Exploring methods for prevention of oxidative stain in soft maple

Treesearch

Michael C. Wiemann; Richard D. Bergman; Mark Knaebe; Scott A. Bowe

2009-01-01

Interior gray enzymatic oxidative stain for white woods such as maple has plagued the wood industry for many years because methods that have been found to reduce stain are hard to scale up to industrial levels. We examined possible alternative treatments to eliminate stain in soft maple (Acer rubrum L.), and found that exposure to sulfur dioxide gas eliminates interior...

Fine needle aspiration cytology in lymphadenopathy of HIV-positive cases.

PubMed

Saikia, U N; Dey, P; Jindal, B; Saikia, B

2001-01-01

To evaluate the role of fine needle aspiration biopsy (FNAB) material in 25 HIV-positive cases with lymphadenopathy. We selected 25 cases for the present study who were enzyme-linked immunosorbent assay positive for HIV (HIV-1). FNAB was performed as a routine, outdoor procedure with informed consent of the patient. For each case, along with routine May-Grünwald-Giemsa and hematoxylin and eosin staining, Ziehl-Neelsen staining for acid-fast bacilli and periodic acid-Schiff staining for fungi were performed wherever necessary. A total of 28 sites were aspirated from 25 HIV patients. All these patients were heterosexual, and none had a history of drug abuse. FNAB was performed under ultrasound guidance in all four cases of a retroperitoneal group of lymph nodes. The most common FNAB diagnosis was reactive lymphoid hyperplasia (10), followed by tuberculosis (8). There were three cases diagnosed as fungal infection (two, Cryptococcus; one, histoplasmosis). FNAB of a case of lymph node was suggestive of tuberculosis. There was one case each diagnosed as non-Hodgkin's lymphoma and squamous cell carcinoma (metastatic). One case of a small axillary lymph node did not yield representative material. FNAB is a relatively inexpensive initial investigative technique in the diagnosis and management of HIV-positive patients. It can obviate the need for surgical excision and enable immediate treatment of specific infections.

Touch cytology in diagnosing Helicobacter pylori: comparison of four staining methods.

PubMed

Hashemi, M R; Rahnavardi, M; Bikdeli, B; Dehghani Zahedani, M; Iranmanesh, F

2008-06-01

Helicobacter pylori (Hp), a major cause of peptic ulcer disease and an important risk factor for gastric malignancy, can be diagnosed by several methods. Touch cytology (TC) of the gastric mucosa has been noted to give good results and has been found to be very simple, inexpensive and rapid. However, evidence regarding the accuracy of different staining methods of TC is lacking. The present study aims at defining the diagnostic accuracy of four different staining methods of TC. Biopsy specimens were taken from the antral mucosa of one hundred consecutive patients referred for upper gastrointestinal endoscopy (UGIE) for various indications. TC slides were processed by four staining methods: Wright, Giemsa, Papanicolaou and Gram. Rapid urease test (RUT) and histological examination of specimens were also performed. The same experienced pathologist evaluated the coded samples. A patient's Hp status was established by minimum concordance of the three tests, including histology, RUT, and 'Touch mean'. The latter was defined positive when at least three of the four TC staining methods were positive. Forty-six patients (46%) were positive for Hp according to Hp status. TC stained by Wright had excellent agreement with both histology (kappa = 0.80, P < 0.001) and RUT (kappa = 0.84, P < 0.001). Regarding Hp status, histology was 100% sensitive and RUT was 100% specific. Wright-stained TC (88.89%) was significantly more specific than both Giemsa- (74.07%; P < 0.05) and Papanicolaou-stained (70.37%; P < 0.05) TC. RUT should still be acknowledged as the primary test in diagnosing Hp following UGIE. If RUT is negative and Hp detection is intended only, Wright-stained TC can safely substitute for histology. However, when assessment for severity of mucosal damage or cell atypias is meant, histology cannot be neglected.

Identification of mycobacterium tuberculosis in sputum smear slide using automatic scanning microscope

NASA Astrophysics Data System (ADS)

Rulaningtyas, Riries; Suksmono, Andriyan B.; Mengko, Tati L. R.; Saptawati, Putri

2015-04-01

Sputum smear observation has an important role in tuberculosis (TB) disease diagnosis, because it needs accurate identification to avoid high errors diagnosis. In development countries, sputum smear slide observation is commonly done with conventional light microscope from Ziehl-Neelsen stained tissue and it doesn't need high cost to maintain the microscope. The clinicians do manual screening process for sputum smear slide which is time consuming and needs highly training to detect the presence of TB bacilli (mycobacterium tuberculosis) accurately, especially for negative slide and slide with less number of TB bacilli. For helping the clinicians, we propose automatic scanning microscope with automatic identification of TB bacilli. The designed system modified the field movement of light microscope with stepper motor which was controlled by microcontroller. Every sputum smear field was captured by camera. After that some image processing techniques were done for the sputum smear images. The color threshold was used for background subtraction with hue canal in HSV color space. Sobel edge detection algorithm was used for TB bacilli image segmentation. We used feature extraction based on shape for bacilli analyzing and then neural network classified TB bacilli or not. The results indicated identification of TB bacilli that we have done worked well and detected TB bacilli accurately in sputum smear slide with normal staining, but not worked well in over staining and less staining tissue slide. However, overall the designed system can help the clinicians in sputum smear observation becomes more easily.

Improved Nissl method to stain formaldehyde or glutaraldehyde-fixed material.

PubMed

Böck, P

1979-05-15

Nissl staining of paraffin sections from formaldehyde- or glutaraldehyde-fixed specimens is significantly intensified when sections are kept in a 50% (w/v) aqueous solution of potassium metabisulfite before being stained by a conventional Nissl method.

Bacterial, fungal, parasitological and pathological analyses of abortions in small ruminants from 2012-2016.

PubMed

Schnydrig, P; Vidal, S; Brodard, I; Frey, C; Posthaus, H; Perreten, V; Rodriguez-Campos, S

2017-12-01

Abortion in small ruminants presents a clinical and economic problem with legal implications regarding animal health and zoonotic risk by some of the abortive pathogens. Several bacteria, fungi and parasites can cause abortion, but cost-orientated routine diagnostics only cover the most relevant epizootic agents. To cover a broad-range of common as well as underdiagnosed abortifacients, we studied 41 ovine and 36 caprine abortions by Stamp's modification of the Ziehl-Neelsen stain, culture for classical and opportunistic abortive agents, real-time PCR for C. burnetii, C. abortus, pathogenic Leptospira spp., Toxoplasma gondii and Neospora caninum. When the dam's serum was available detection of antibodies against B. melitensis, C. burnetii, C. abortus and Leptospira spp. was performed. In 37 cases sufficient placental tissue was available for pathological and histopathological examination. From the 77 cases 11 (14.3%) were positive by staining whereas real-time PCR detected C. burnetii and C. abortus in 49.3% and 32.5% of the cases. Antibodies against C. abortus and Leptospira spp. (33.3 and 26.7%) were detected. In 23.4% a bacterial culturable pathogen was isolated. Fungal abortion was confirmed in 1.3% of cases. A single abortive agent was identified in 44.2% of the cases and in 31.2% multiple possible abortifacients were present. Our study shows that the highest clarification rate can only be achieved by a combination of methods and evidences the role that multi-infections play as cause of abortion.

Efficacy of evaluation of rooster sperm morphology using different staining methods.

PubMed

Lukaszewicz, E; Jerysz, A; Partyka, A; Siudzińska, A

2008-12-01

This work focused on inexpensive methods of evaluation fowl sperm morphology, based on eosin-nigrosin smears, which can determine disorders in spermatogenesis and can be recommended for evaluating the fertilising potency and selecting males in flocks reproduced by artificial insemination. Four fowl breeds (Black Minorca, Italian Partridge, Forwerk and Greenleg Partridge) were used to determine the efficacy of sperm morphology evaluation using four eosin-nigrosin staining methods (according to Blom, Bakst and Cecil, Morisson, Jaśkowski) and three examiners of different experience (high, medium, novice). There were significant (P< or = 0.01) differences in sperm morphology between Blom's staining method and those of Bakst and Cecil, Morisson or Jaśkowski, irrespective of fowl breed and examiners experience. Blom stain caused sperm head swelling and showed a drastic reduction in the proportion of live spermatozoa with normal morphology. The staining method had a greater influence on sperm morphology evaluation than the experience of the examiners.

Cytological diagnostic of lymphadenitis tuberculosis by eosinophilic material

NASA Astrophysics Data System (ADS)

Delyuzar; Amir, Z.; Kusumawati, L.

2018-03-01

AFB sputum and chest X-ray are used to identify patients with pulmonary TB. For extrapulmonary TB, fine needle aspiration cytology is needed, even though occasionally found not atypical feature in the form of eosinophilic material with dark brown particles, suspected as TB. This research was to show that eosinophilic material with dark brown particles is accurate as new criteria for the cytological diagnosis of TB. By performing fine needle aspiration biopsy stained with Giemsa, if an eosinophilic material with dark brown particles was encountered, we continued with Ziehl-Neelsen AFB stain and confirmed with PCR. To assess accuracy, we used a diagnostic test to evaluate sensitivity and specificity of eosinophilic material with dark brown particles by using AFB and PCR as the gold standard. The sensitivity and specificity of cytological diagnosis in tuberculosis of eosinophilic material with dark brown particles were 93.65% and 70.99%, respectively if confirmed with AFB. On the other hand, if confirmed with PCR using Mycobacterium tuberculosis DNA, the sensitivity and specificity were 98.95% and 96.79%, respectively. In conclusion, eosinophilic masses with dark brown particles is accurate as new criteria of TB diagnostic cytology with high sensitivity and specificity confirmed with AFB and PCR test.

First report of birds infection by intestinal parasites in Khorramabad, west Iran.

PubMed

Badparva, Ebrahim; Ezatpour, Behrouz; Azami, Mehdi; Badparva, Masoud

2015-12-01

Parasitic infections in birds are omnipresent, even when they occur in low amounts, may result in subclinical diseases. There aren't any studies, based on Iranian data, investigating the prevalence of intestinal parasitic infections in some birds' species. We conducted a cross-sectional study between December 2011 and December 2012. The fecal samples were taken from 451 birds including hen, turkey, sparrow, pigeon and decorative birds. The samples screened for intestinal parasitic infections using direct smear, formalin-ether concentration technique, modified Ziehl-Neelsen staining, Culture in RPMI 1640 medium, sporulation with potassium dichromate and Trichrome and Giemsa staining. Out of 451 birds' species, 157 (34.8 %), were infected with one or more type of intestinal parasites. We identified two nematode, two cestoda species and five protozoan parasites species. No trematodes were found in the samples studied. The parasites identified among birds involved Raillietina spp. (4.2 %) and Eimeria spp. (7.1 %) were the most common helminthes and protozoa respectively. From total of birds study, 12 (2.7 %) and 6 (1.3 %) have two and three mixed infections respectively. Intestinal parasitic infections are common in birds in west Iran. The future studies are needed in order to determine to which extent the infections influence mortality and performance of the birds.

Staining Methods for Normal and Regenerative Myelin in the Nervous System.

PubMed

Carriel, Víctor; Campos, Antonio; Alaminos, Miguel; Raimondo, Stefania; Geuna, Stefano

2017-01-01

Histochemical techniques enable the specific identification of myelin by light microscopy. Here we describe three histochemical methods for the staining of myelin suitable for formalin-fixed and paraffin-embedded materials. The first method is conventional luxol fast blue (LFB) method which stains myelin in blue and Nissl bodies and mast cells in purple. The second method is a LBF-based method called MCOLL, which specifically stains the myelin as well the collagen fibers and cells, giving an integrated overview of the histology and myelin content of the tissue. Finally, we describe the osmium tetroxide method, which consist in the osmication of previously fixed tissues. Osmication is performed prior the embedding of tissues in paraffin giving a permanent positive reaction for myelin as well as other lipids present in the tissue.

Tuberculous brain abscess and subdural empyema in an immunocompetent child: Significance of AFB staining in aspirated pus

PubMed Central

Vijayakumar, B.; Sarin, K.; Mohan, Girija

2012-01-01

Tuberculous brain abscess and subdural empyema are extremely rare manifestations of central nervous system tuberculosis. Here, we report a case of an 11-year-old immunocompetent child who developed temporal lobe abscess and subdural empyema following chronic otitis media. A right temporal craniotomy was performed and the abscess was excised. The Ziehl Nielsen staining of the aspirated pus from the temporal lobe abscess yielded acid fast bacilli. Prompt administration of antituberculous treatment resulted in complete recovery of the child. Even though the subdural abscess was not drained, we presume that to be of tubercular aetiology. Ours is probably the first case of brain abscess and subdural empyema due to Mycobacterium tuberculosis reported in the same child. This case is being reported because of its rarity and to stress the importance of routine staining for tubercle bacilli in all cases of brain abscess, especially in endemic areas, as it is difficult to differentiate tuberculous from pyogenic abscess clinically as well as histopathologically. PMID:22566728

Tuberculous brain abscess and subdural empyema in an immunocompetent child: Significance of AFB staining in aspirated pus.

PubMed

Vijayakumar, B; Sarin, K; Mohan, Girija

2012-04-01

Tuberculous brain abscess and subdural empyema are extremely rare manifestations of central nervous system tuberculosis. Here, we report a case of an 11-year-old immunocompetent child who developed temporal lobe abscess and subdural empyema following chronic otitis media. A right temporal craniotomy was performed and the abscess was excised. The Ziehl Nielsen staining of the aspirated pus from the temporal lobe abscess yielded acid fast bacilli. Prompt administration of antituberculous treatment resulted in complete recovery of the child. Even though the subdural abscess was not drained, we presume that to be of tubercular aetiology. Ours is probably the first case of brain abscess and subdural empyema due to Mycobacterium tuberculosis reported in the same child. This case is being reported because of its rarity and to stress the importance of routine staining for tubercle bacilli in all cases of brain abscess, especially in endemic areas, as it is difficult to differentiate tuberculous from pyogenic abscess clinically as well as histopathologically.

Neurotuberculosis in cattle in southern Brazil.

PubMed

Konradt, Guilherme; Bassuino, Daniele Mariath; Bianchi, Matheus Viezzer; Bandinelli, Marcele Bettim; Driemeier, David; Pavarini, Saulo Petinatti

2016-06-01

Tuberculosis in cattle is a chronic infectious-contagious disease characterized by the development of nodular lesions (granulomas) in mainly the lungs and regional lymph nodes. It is caused by Mycobacterium tuberculosis complex, an acid-fast bacillus (AFB). Tuberculosis in the central nervous system is a rare condition in cattle. Herein, we describe the clinical and pathological findings of six neurotuberculosis cases in cattle diagnosed in Southern Brazil. The average age of the cattle affected was 12 months, and they varied in breed and sex. The clinical history ranged from 5 to 30 days and was characterized by motor incoordination, opisthotonus, blindness, and progression to recumbency. The cattle were euthanized, and grossly, the leptomeninges at the basilar brain showed marked and diffuse expansion, with nodular yellowish lesions ranging in size. On microscopic examination, there were multifocal granulomas located mainly in the meninges, though sometimes extending to adjacent neuropil or existing as isolated granulomas in neuropil. AFBs were observed in the cytoplasm of epithelioid macrophages and multinucleated giant cells through Ziehl-Neelsen histochemical staining and identified as Mycobacterium sp. through immunohistochemistry.

[Cryptosporidium sp infections and other intestinal parasites in food handlers from Zulia state, Venezuela].

PubMed

Freites, Azael; Colmenares, Deisy; Pérez, Marly; García, María; Díaz de Suárez, Odelis

2009-03-01

Cryptosporidiosis in food handlers from Venezuela is unknown, being this an important public health problem in immunosuppressed patients. To determine the prevalence of Cryptosporidium sp and other intestinal parasites in food handlers from Zulia State, one hundred nineteen fecal samples were evaluated by wet mount, concentrated according to Ritchie and modified Ziehl-Neelsen staining. Fourteen (11.8%) were positive for Cryptosporidium sp and associated with other protozoosis (P < 0.05), being most frequent Endolimax nana (42.9%). The general prevalence of the intestinal parasitism was 48.7%, emphasizing E. nana (41.2%), followed by Blastocystis hominis (38.7%) and Entamoeba coli (17.6%). The most frequent pathogenic protozoa was Giardia lamblia (13.4%), followed by the complex Entamoeba histolytica/E. dispar (9.2%). 4.1% were positive for intestinal helminthes. The infection by Cryptosporidium sp is frequent in food handlers from Zulia State. Given to the results of this investigation and the nonexistence of studies in this population, is necessary to deepen in the impact of this parasitism in food handlers and the consumers of their products.

Prevalence of Cryptosporidium sp. infection in diarrheic and non-diarrheic humans in Iran

PubMed Central

2007-01-01

For evaluation of the prevalence of Cryptosporidium sp. infection in diarrheic and non-diarrheic humans in Iran, fecal specimens from diarrheic (n = 129) and non-diarrheic humans (n = 271) were collected and examined for the presence of Cryptosporidium sp. oocysts. The presence of Cryptosporidium sp. oocysts was determined by Ziehl-Neelsen acid-fast staining. Humans were grouped according to their age as follows: younger than 15, 16-25, 26-35, 36-50, and over 51 years. The results showed that the overall prevalence of infection in all 400 samples was 10.8%, but the prevalence (25.6%) in diarrheic humans was higher than that (3.7%) in non-diarrheic humans. Oocysts of Cryptosporidium sp. were detected in the feces of 21.4%, 9.3%, 8.8%, 6.7% and 5.7% of different age groups, respectively. The intensity of oocysts was significantly higher in diarrheic humans than in non-diarrheic ones. There was a significant association between Cryptosporidium sp. infection and occurrence of diarrhea (P < 0.05). The results indicate that Cryptosporidium sp. infection is prevalent in diarrheic humans in Iran. PMID:17570977

Immunocytochemical detection of Mycobacterium Tuberculosis complex specific antigen, MPT64, improves diagnosis of tuberculous lymphadenitis and tuberculous pleuritis.

PubMed

Tadele, Agerie; Beyene, Demissew; Hussein, Jemal; Gemechu, Tuffa; Birhanu, Asaye; Mustafa, Tehmina; Tsegaye, Aster; Aseffa, Abraham; Sviland, Lisbet

2014-11-25

A rapid, sensitive and accurate laboratory diagnosis is of prime importance in suspected extrapulmonary tuberculosis (EPTB) cases. However, traditional techniques for the detection of acid-fast bacilli have limitations. The aim of the study was to evaluate the diagnostic value of immunocytochemical staining for detection of Mycobacterium tuberculosis complex specific antigen, MPT64, in aspirates from pleural effusions and lymph nodes, the most common presentations of EPTB. A cross-sectional study was conducted by including patients at Tikur Anbessa Specialized Hospital and the United Vision Medical Services from December 2011 to June 2012. Lymph node aspirates and pleural fluid samples were collected and analyzed from a total of 51 cases (26 tuberculous (TB) pleuritis and 25 TB lymphadenitis) and 67 non-TB controls. Each specimen was subjected to Ziehl-Neelsen (ZN) staining, culture on Lowenstein- Jensen (LJ) medium, cytological examination, Polymerase Chain Reaction (PCR) using IS1081gene sequence as a primer and immunocytochemistry (ICC) with polyclonal anti-MPT64 antibody. All patients were screened for HIV. ICC was positive in 38 of 51 cases and in the 7 of 67 controls giving an overall sensitivity and specificity of 74.5% and 89.5%, respectively. Using IS1081-PCR as a reference method, the sensitivity and specificity, positive and negative predictive value of ICC was 88.1%, 89.5%, 82.2% and 93.2%, respectively. The case detection rate increased from 13.7% by ZN stain to 19.6% by LJ culture, to 66.7% by cytology and 74.5% by ICC. Immunocytochemistry with anti-MPT64 antigen improved detection of TB in pleural effusion and lymph node aspirates. Further studies using monoclonal antibodies on samples from other sites of EPTB is recommended to validate this relatively simple diagnostic method for EPTB.

Assessment of microscopic and molecular tools for the diagnosis and follow-up of cryptosporidiosis in patients at risk.

PubMed

Le Govic, Y; Guyot, K; Certad, G; Deschildre, A; Novo, R; Mary, C; Sendid, B; Viscogliosi, E; Favennec, L; Dei-Cas, E; Fréalle, E; Dutoit, E

2016-01-01

Cryptosporidiosis is an important though underreported public health concern. Molecular tools might be helpful in improving its diagnosis. In this study, ZR Fecal DNA MiniPrep™ Kit (ZR) and NucliSens® easyMAG® (EM) were compared using four Cryptosporidium-seeded feces and 29 Cryptosporidium-positive stools. Thereafter, ZR was selected for prospective evaluation of Cryptosporidium detection by 18S rDNA and LAXER quantitative PCR (qPCR) in 69 stools from 56 patients after Cryptosporidium detection by glycerin, modified Ziehl-Neelsen (ZN) and auramine-phenol (AP) stainings. The combination of any of the two extraction methods with 18S qPCR yielded adequate detection of Cryptosporidium in seeded stools, but the ZR kit showed the best performance. All 29 Cryptosporidium-positive samples were positive with 18S qPCR, after both ZR and EM extraction. However, false-negative results were found with LAXER qPCR or nested PCR. Cryptosporidiosis was diagnosed in 7/56 patients. All the microscopic methods enabled the initial diagnosis, but Cryptosporidium was detected in 12, 13, and 14 samples from these seven patients after glycerin, ZN, and AP staining respectively. Among these samples, 14 and 12 were positive with 18S and LAXER qPCR respectively. In two patients, Cryptosporidium DNA loads were found to be correlated with clinical evolution. Although little known, glycerin is a sensitive method for the initial detection of Cryptosporidium. When combined with 18S qPCR, ZR extraction, which had not been evaluated so far for Cryptosporidium, was an accurate tool for detecting Cryptosporidium and estimating the oocyst shedding in the course of infection.

Intestinal parasitosis in school children of Lalitpur district of Nepal

PubMed Central

2013-01-01

Background Enteric parasites are the most common cause of parasitic diseases and cause significant morbidity and mortality, particularly in developing countries like Nepal. The objective of this study was to estimate the prevalence and risk factors of intestinal parasitic infections among school going children of Lalitpur district of Nepal. Methods A total of 1392 stool samples were collected from school children of two government, two private and two community schools of the same district. The stool samples were examined for evidence of parasitic infections by direct microscopy and confirmed by concentration methods (formal ether sedimentation technique or floatation technique by using Sheather’s sugar solution). Modified Ziehl-Neelsen (ZN) staining was performed for the detection of coccidian parasites. Results Prevalence of intestinal parasitosis was found to be 16.7%. The highest prevalence rate was seen with Giardia lamblia (7.4%) followed by Entamoeba histolytica (3.4%) and Cyclospora cayetanensis (1.6%). Children aged 11–15 years and the ones belonging to family of agriculture workers were most commonly affected. Hand washing practice and type of drinking water also showed significant difference. Conclusions The burden of parasitic infections among the school children, coupled with the poor sanitary conditions in the schools, should be regarded as an issue of public health priority and demands for effective school health programs involving periodic health education and screening. PMID:24207086

A robust, efficient and flexible method for staining myelinated axons in blocks of brain tissue.

PubMed

Wahlsten, Douglas; Colbourne, Frederick; Pleus, Richard

2003-03-15

Previous studies have demonstrated the utility of the gold chloride method for en bloc staining of a bisected brain in mice and rats. The present study explores several variations in the method, assesses its reliability, and extends the limits of its application. We conclude that the method is very efficient, highly robust, sufficiently accurate for most purposes, and adaptable to many morphometric measures. We obtained acceptable staining of commissures in every brain, despite a wide variety of fixation methods. One-half could be stained 24 h after the brain was extracted and the other half could be stained months later. When staining failed because of an exhausted solution, the brain could be stained successfully in fresh solution. Relatively small changes were found in the sizes of commissures several weeks after initial fixation or staining. A half brain stained to reveal the mid-sagittal section could then be sectioned coronally and stained again in either gold chloride for myelin or cresyl violet for Nissl substance. Uncertainty, arising from pixelation of digitized images was far less than errors arising from human judgments about the histological limits of major commissures. Useful data for morphometric analysis were obtained by scanning the surface of a gold chloride stained block of brain with an inexpensive flatbed scanner.

Modified Field's staining--a rapid stain for Trichomonas vaginalis.

PubMed

Afzan, M Yusuf; Sivanandam, S; Kumar, G Suresh

2010-10-01

Trichomonas vaginalis, a flagellate protozoan parasite commonly found in the human genitourinary tract, is transmitted primarily by sexual intercourse. Diagnosis is usually by in vitro culture method and staining with Giemsa stain. There are laboratories that use Gram stain as well. We compared the use of modified Field's (MF), Giemsa, and Gram stains on 2 axenic and xenic isolates of T. vaginalis, respectively. Three smears from every sediment of spun cultures of all 4 isolates were stained, respectively, with each of the stains. We showed that MF staining, apart from being a rapid stain (20 s), confers sharper staining contrast, which differentiates the nucleus and the cytoplasm of the organism when compared to Giemsa and Gram staining especially on parasites from spiked urine samples. The alternative staining procedure offers in a diagnostic setting a rapid stain that can easily visualize the parasite with sharp contrasting characteristics between organelles especially the nucleus and cytoplasm. Vacuoles are more clearly visible in parasites stained with MF than when stained with Giemsa. Copyright © 2010 Elsevier Inc. All rights reserved.

Prevalence and risk factors of intestinal protozoan infections: a population-based study in rural areas of Boyer-Ahmad district, Southwestern Iran.

PubMed

Sarkari, Bahador; Hosseini, Ghasem; Motazedian, Mohammad Hossein; Fararouei, Mohammad; Moshfe, Abdolali

2016-11-25

Parasitic infections are still a significant health problem in rural areas in developing countries including Iran. There is no recent population-based data about the prevalence of human intestinal parasites in most rural areas of Iran. The current study aimed to determine the prevalence of intestinal protozoan infection in inhabitants of rural areas of Boyer-Ahmad district, Southwestern Iran. A total of 1025 stool samples were collected from the inhabitant of 50 randomly selected villages in Boyer-Ahmad Township. The stool samples were evaluated by parasitological methods including, direct wet-mounting, formalin ethyl acetate concentration, zinc sulfate floatation, and Trichrome permanent stain for detection of protozoan infections. Diarrheic samples were further evaluated with a modified Ziehl-Neelsen staining method for detection of coccidian parasites. The prevalence of both pathogenic and nonpathogenic intestinal parasites in the population was 37.5% (385 out of 1025 cases), some individual with multiple infections. Giardia lamblia was detected in 179 (17.46%), Blastocystis hominis in 182 (17.76%), Entamoeba histolytica/dispar in 9 (0.87%), Endolimax nana in 216 (21.07%), Entamoeba coli in 151 (14.73%), Ioedamoeba butschlii in 45 (4.39%), Chillomastix mesnili in 22 (2.14%), Trichomonas hominis in 2 (0.19%) and Dientamoeba fragillis in 2 (0.19%) of cases. Multivariate logistic regression revealed significant associations between protozoan infection (pathogenic protozoa) and contact with animals (OR yes/no = 2.22, p < 0.001) and educational status (OR higher/illiterate = 0.40, P = 0.01). Findings of this study demonstrated that protozoan infection rate in rural areas of southwestern Iran is still high and remained as a challenging health problem in these areas.

Cryptosporidiosis and other intestinal parasitic infections in patients with chronic diarrhea.

PubMed

Mahdi, Nadham K; Ali, Naeel H

2004-09-01

To consider the relationship of the parasitic infections including cryptosporidium with chronic diarrhea. Also the effect of chronic disease as pulmonary tuberculosis (TB) and nosocomial infection on the occurrence rate of parasites in cases of chronic diarrhea. Stool samples were collected from 205 patients in teaching, general, child and maternity hospitals in Basrah, Iraq, suffering from chronic diarrhea during 2000. Out of these patients, there were 40 patients with pulmonary TB and 50 inpatients with nosocomial infection. Also 175 apparently healthy individuals who have no episodes of diarrhea for at least 2-months were served as a control group. Direct smear method and then formalin ether sedimentation method were carried out for stool samples to detect intestinal parasites. Fecal smears were prepared from the sediment and stained by the modified Ziehl Neelsen stain for the recovery of red pink oocysts of cryptosporidium. Out of the 205 examined patients, cryptosporidium oocysts were found to be excreted in 20 (9.7%) patients in comparing to 1.1% of the control group. The difference is statistically significant. There were 109 (53.2%) patients found to be positive for intestinal parasitic infections compared to 26 (14.8%) of the control group. The difference is also statistically significant. Out of the 40 TB patients, 2 (5%) were found to excrete cryptosporidium oocysts and also 27 (67.3%) were positive for intestinal parasites. In addition, there were 4 (8%) excreting cryptosporidium oocysts and 23 (46%) infecting by intestinal parasites among the in patients with nosocomial infection. Both acid and non-acid fast parasites should be considered in the differential diagnosis of undiagnosed chronic diarrhea especially among patients with pulmonary TB or nosocomial infection.

Use of Fine Needle Aspirate from Peripheral Nerves of Pure-neural Leprosy for Cytology and Polymerase Chain Reaction to Confirm the Diagnosis: A Follow-up Study of 4 Years.

PubMed

De, Abhishek; Hasanoor Reja, Abu Hena; Aggarwal, Ishad; Sen, Sumit; Sil, Amrita; Bhattacharya, Basudev; Sharma, Nidhi; Ansari, Asad; Sarda, Aarti; Chatterjee, Gobinda; Das, Sudip

2017-01-01

Pure neural leprosy (PNL) still remains a diagnostic challenge because of the absence of sine qua non skin lesions of leprosy and a confirmatory diagnostic method. The authors had earlier described a simple yet objective technique of combining fine needle aspiration cytology (FNAC) coupled with a multiplex polymerase chain reaction (PCR) in a pilot study, wherein the technique showed promise of a reliable diagnostic tool. In the pursuit of further evidence, the authors carried out a 4-year study with PNL cases to find the efficacy and reliability of the said method in a larger sample size. This study was conducted to find the efficacy, reliability, and reproducibility of FNAC coupled with multiplex PCR and Ziehl-Neelsen (ZN) staining in identifying the cases of PNL. All cases that were suspected to be suffering from PNL, following evaluation by two independent observers were included in the study and were subjected to FNAC from the affected nerve, and the aspirates were evaluated for cytology, ZN staining, and multiplex PCR for Mycobacterium leprae genome. In addition, serum anti-PGL1 levels were also performed in all the study subjects. Fifteen non-PNL cases were also included in the control arm. A total of 47 cases were included in the test arm and subjected to FNAC. Conventional ZN staining could demonstrate acid-fast bacilli (AFB) in only 15 out of 47 cases (31.91%) while M. leprae DNA could be elicited in 37 (78.72%) cases by the multiplex PCR. Only 13 (27.65%) out of 47 cases showed anti-PGLI-1 antibody positivity. On cytological examination of the nerve aspirates, only 11 (23.40%) cases showed epithelioid cells whereas nonspecific inflammation was seen in 26 (75.60%) cases. The results of this study conducted over a larger sample size corroborate with the findings of our pilot study. In a resource poor set up, FNAC in combination with ZN staining and multiplex PCR is a rapid, simple, and easily performed test, which can give a reproducible and objective

Microscopic quantification of bacterial invasion by a novel antibody-independent staining method.

PubMed

Agerer, Franziska; Waeckerle, Stephanie; Hauck, Christof R

2004-10-01

Microscopic discrimination between extracellular and invasive, intracellular bacteria is a valuable technique in microbiology and immunology. We describe a novel fluorescence staining protocol, called FITC-biotin-avidin (FBA) staining, which allows the differentiation between extracellular and intracellular bacteria and is independent of specific antibodies directed against the microorganisms. FBA staining of eukaryotic cells infected with Gram-negative bacteria of the genus Neisseria or the Gram-positive pathogen Staphylococcus aureus are employed to validate the novel technique. The quantitative evaluation of intracellular pathogens by the FBA staining protocol yields identical results compared to parallel samples stained with conventional, antibody-dependent methods. FBA staining eliminates the need for cell permeabilization resulting in robust and rapid detection of invasive microbes. Taken together, FBA staining provides a reliable and convenient alternative for the differential detection of intracellular and extracellular bacteria and should be a valuable technical tool for the quantitative analysis of the invasive properties of pathogenic bacteria and other microorganisms.

Differential fluorescent staining method for detection of bacteria in blood cultures, cerebrospinal fluid and other clinical specimens.

PubMed

Fazii, P; Ciancaglini, E; Riario Sforza, G

2002-05-01

The aim of this study was to evaluate a differential staining method to distinguish gram-positive from gram-negative bacteria in fluorescence. The method is based on two fluorochromes, one acting in the wavelength of red, i.e. the acridine orange, and another acting in the wavelength of green, i.e. the fluorescein. With this method, gram-positive bacteria appear yellow and gram-negative bacteria appear green. In view of the importance of a rapid aetiological diagnosis in cases of septicaemia, the differential staining method in fluorescence was compared with Gram stain for the detection of bacteria in blood. Of 5,820 blood cultures entered into the study and identified by the Bactec 9120 fluorescent series instrument (Becton Dickinson Europe, France), 774 were positive. Of the 774 positive cultures, 689 yielded only a single organism. The differential staining method in fluorescence detected 626 of the 689 cultures, while Gram stain detected 468. On the basis of these results, the sensitivity of the differential staining method in fluorescence was 90.9%, while that of Gram stain was 67.9%. The difference between the two methods was statistically significant ( P<0.001). The differential fluorescent staining method was more sensitive than Gram stain in the detection of bacteria in blood cultures during the incubation period. This technique provides a rapid, simple and highly sensitive staining method that can be used in conjunction with subculture methods. Whereas subculture requires an incubation period of 18-24 h, the fluorescent staining technique can detect bacteria on the same day that smears are prepared and examined. The differential fluorescent staining method was also evaluated for its ability to detect microorganisms in cerebrospinal fluid and other clinical specimens. The microorganisms were easily detected, even when bacterial counts in the specimens were low.

Methods of staining target chromosomal DNA employing high complexity nucleic acid probes

DOEpatents

Gray, Joe W.; Pinkel, Daniel; Kallioniemi, Ol'li-Pekka; Kallioniemi, Anne; Sakamoto, Masaru

2006-10-03

Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML), retinoblastoma, ovarian and uterine cancers, and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy†

PubMed Central

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-soo; Torelli, Marco D.; Hamers, Robert J.; Murhpy, Catherine J.; Orr, Galya

2015-01-01

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate eficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells. PMID:24816810

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

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

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localizationmore » patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.« less

Gram staining.

PubMed

Coico, Richard

2005-10-01

Named after Hans Christian Gram who developed the method in 1884, the Gram stain allows one to distinguish between Gram-positive and Gram-negative bacteria on the basis of differential staining with a crystal violet-iodine complex and a safranin counterstain. The cell walls of Gram-positive organisms retain this complex after treatment with alcohol and appear purple, whereas gram-negative organisms decolorize following such treatment and appear pink. The method described here is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures.

Gram staining.

PubMed

Coico, R

2001-05-01

Named after Hans Christian Gram who developed the method in 1884, the Gram stain allows one to distinguish between Gram-positive and Gram-negative bacteria on the basis of differential staining with a crystal violet-iodine complex and a safranin counterstain. The cell walls of Gram-positive organisms retain this complex after treatment with alcohol and appear purple, whereas gram-negative organisms decolorize following such treatment and appear pink. The method described here is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures.

[REASONS OF DELAYED DIAGNOSIS OF BLADDER TUBERCULOSIS].

PubMed

Kul'chavenja, E V; Holtobin, D P

2015-01-01

The fourth, terminal, stage of bladder tuberculosis (BT) manifests itself in irreversible changes and requires surgical treatment. To identify the reasons for delayed diagnosis of this urogenital tuberculosis complication. Medical history of 26 urogenital tuberculosis patients with a complicated form of stage 4 BT, referred to the Novosibirsk TB Research Institute for reconstructive surgery were analysed. In 22 patients, bladder volume ranged from 55 to 100 ml, 4 patients previously underwent cystostomy due to extremely small bladder volume. Average duration of BT hidden in the guise of "urogenital infection" was 6.2 years. Patients were treated with norfloxacin (a total of 104 courses), ciprofloxacin (86 courses), amikacin (43 courses), nitroxoline (27 courses), third generation cephalosporins (32 courses), lomefloxacin (17 courses), levofloxacin (11 courses), Amoxicillin clavulanate (4 courses), ampicillin (2 courses). It was demonstrated that all cases of BT stage 4 were iatrogenic. Irreversible debilitating complications occurred due to suboptimal therapy, primarily due to administration of amikacin and fluoroquinolones for urogenital infections, which was tuberculosis in disguise. Absence of M. tuberculosis growth does not exclude tuberculosis; pathological specimens must be further examined at least by PCR. Interventional material must be mandatory examined histologically and stained by Ziehl-Neelsen method to identify M. tuberculosis. Effective and not masking tuberculosis, optimal therapy for urogenital infections includes fosfomycin, furazidin (nitrofurantoin), gentamicin, III generation cephalosporins (in outpatient settings dispersible form of efixime should be preferable).

Prevalence of opportunistic intestinal parasitic infection among HIV infected patients who are taking antiretroviral treatment at Jimma Health Center, Jimma, Ethiopia.

PubMed

Zeynudin, A; Hemalatha, K; Kannan, S

2013-02-01

One of the major health problems among HIV sero-positive patients are superimposed infections due to the deficient immunity. Furthermore, intestinal parasitic (IP) infections, which are also one of the basic health problems in tropical regions, are common in these patients. Infection by opportunistic pathogens, including various forms of intestinal parasites has been the hall mark of HIV since the beginning of the epidemic. To study the prevalence of opportunistic intestinal parasitic infection among HIV patients who are taking antiretroviral treatment (ART) in Jimma, Ethiopia. Patient samples were diagnosed by examination of single stool specimen which was examined as fresh wet mounts, formal-ether concentration technique and modified Ziehl-Neelsen staining technique. Data was obtained from 91 study subjects selected by convenience sampling method. The overall prevalence of intestinal parasitic infections was found to be 39.56%. Eight types of intestinal parasites was identified, the most dominant being, Ascaris lumbricoides, 21.67%, Entamoeba histolytica, 15% and Cryptosporidium parvum 13.33%. The prevalence of opportunistic parasite was 15.38%, the prevalence of non-opportunistic parasite was 20.87% and the prevalence of both opportunistic and non opportunistic was 3.29%. The study indicated that intestinal parasites were still a problem in the study area. Data also showed that among the predisposing factors, habit of hand washing before meal, usage of latrine and duration after treatment was statistically associated with intestinal parasitic infections.

Intestinal parasitosis in school children of Lalitpur district of Nepal.

PubMed

Tandukar, Sarmila; Ansari, Shamshul; Adhikari, Nabaraj; Shrestha, Anisha; Gautam, Jyotshana; Sharma, Binita; Rajbhandari, Deepak; Gautam, Shikshya; Nepal, Hari Prasad; Sherchand, Jeevan B

2013-11-09

Enteric parasites are the most common cause of parasitic diseases and cause significant morbidity and mortality, particularly in developing countries like Nepal. The objective of this study was to estimate the prevalence and risk factors of intestinal parasitic infections among school going children of Lalitpur district of Nepal. A total of 1392 stool samples were collected from school children of two government, two private and two community schools of the same district. The stool samples were examined for evidence of parasitic infections by direct microscopy and confirmed by concentration methods (formal ether sedimentation technique or floatation technique by using Sheather's sugar solution). Modified Ziehl-Neelsen (ZN) staining was performed for the detection of coccidian parasites. Prevalence of intestinal parasitosis was found to be 16.7%. The highest prevalence rate was seen with Giardia lamblia (7.4%) followed by Entamoeba histolytica (3.4%) and Cyclospora cayetanensis (1.6%). Children aged 11-15 years and the ones belonging to family of agriculture workers were most commonly affected. Hand washing practice and type of drinking water also showed significant difference. The burden of parasitic infections among the school children, coupled with the poor sanitary conditions in the schools, should be regarded as an issue of public health priority and demands for effective school health programs involving periodic health education and screening.

Practical parasitology courses and infection with intestinal parasites in students.

PubMed

Fallahi, Sh; Rostami, A; Mohammadi, M; Ebrahimzadeh, F; Pournia, Y

2016-01-01

Students who are working in research or educational laboratories of parasitology, as well as health care workers providing care for patients, are at the risk of becoming infected with parasites through accidental exposure. The main purpose of this study was to identify potential positive cases of intestinal parasitic infections among students who took practical parasitology courses compared with students who did not take any practical parasitology courses in Lorestan University of Medical Sciences, Khorramabad, Iran, in 2013-2014. A total of 310 subjects from various majors were invited to voluntarily participate in the study. Various demographic data were collected using questionnaires. Three stool samples were collected from each individual on alternate days. Saline wet mounts (SWM), formalin-ether sedimentation test (FEST), Sheather floatation test (SHFT) and trichrome and modified Ziehl-Neelsen staining methods were used to diagnose the presence of intestinal parasites. The prevalence rate of intestinal parasites (IPs) among the students was 11.93%. There was a significant difference between majors in the infection with IPs (P<0.05). The most frequently observed IPs were Blastocystis hominis (4.51%) and Giardia intestinalis (3.54%). The results of this study showed that the transmission of pathogenic parasites in the educational course of practical parasitology could occur and must be taken into careful consideration. Copyright © 2015 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

Use of two conventional staining methods to assess the acrosomal status of stallion spermatozoa.

PubMed

Runcan, E E; Pozor, M A; Zambrano, G L; Benson, S; Macpherson, M L

2014-07-01

The acrosome is a highly specialised region of the spermatozoon that is essential for fertilisation. Defects or dysfunction of this structure have been associated with fertility problems in man and various domestic species including stallions. Current methods of evaluating the acrosome of stallion spermatozoa are time consuming and require specialised equipment, which is cost prohibitive to the average practitioner. To evaluate 2 conventional stains (Dip Quick and Spermac) and determine their usefulness in assessing acrosome integrity in stallions as compared with specific acrosomal labelling with a fluorescein-conjugated lectin - a method that has been validated for acrosome status evaluation in stallions. In vivo experimental design. Semen from 6 mature Miniature horse stallions of known fertility was collected on 5 separate occasions. To increase the number of reacted acrosomes, portions of each ejaculate were incubated with the calcium ionophore, A23187. Ejaculates were divided and semen samples were processed according to recommendations for fluorescein-conjugated peanut lectin, Pisum sativum agglutin, Dip Quick, and Spermac staining methods. Slides were evaluated independently by 2 separate investigators. Spermatozoa were classified as having intact, reacting, reacted or defective acrosomes. All parameters obtained by both investigators, using all 3 staining methods were highly correlated (P<0.001). There was no statistical difference (P>0.05) between investigators or staining method for the percentages of intact or reacted acrosomes. However, there was a significant difference between investigators and staining methods for determining reacting acrosome percentages (P<0.05). Dip Quick and Spermac stains are useful for determining intact vs. reacted acrosomes for stallion spermatozoa. © 2013 EVJ Ltd.

Tuberculous Otitis with Proteus mirabilis Co-Infection: An Unsuspected Presentation Encountered in Clinical Practice.

PubMed

Rajesh Gandham, Nageswari; Sardar, Moumita; Jadhav, Savita Vivek; Vyawahare, Chanda; Misra, Rabindranath

2014-05-01

Tuberculosis, a contagious bacterial disease which is caused by Mycobacterium tuberculosis, primarily involves the lungs.Though Pulmonary tuberculosis (PTB) is the commonest clinical presentation, there is a need for alertness towards uncommon presentations which involve other organs. Tuberculous otitis media (TOM) is one such rare presentation seen in paediatric practice. It is characterized by painless otorrhoea which fails to respond to the routine antibacterial treatment. TOM usually occurs secondary to PTB. Here is a case of tuberculous otitis media with Proteus mirabilis co-infection, with no evidence of PTB. In the sample of ear discharge obtained from the patient, acid fast bacilli were demonstrated on direct microscopy after Ziehl-Neelsen staining. Culture done on Lowenstein-Jensen medium demonstrated slow-growing Mycobacterium. Bacteriological culture and identification helped in isolating Proteus mirabilis. PCR, followed by Line- Probe Assay for early identification and susceptibility testing to primary drugs, was done. Further, patient tested negative for the Mantoux test. Patient was enrolled in National Tuberculosis programme- RNTCP. This case emphasizes on one of the less common presentations of a common disease. A high clinical suspicion and laboratory confirmation are required for appropriate patient management.

Tuberculous Otitis with Proteus mirabilis Co-Infection: An Unsuspected Presentation Encountered in Clinical Practice

PubMed Central

Sardar, Moumita; Jadhav, Savita Vivek; Vyawahare, Chanda; Misra, Rabindranath

2014-01-01

Tuberculosis, a contagious bacterial disease which is caused by Mycobacterium tuberculosis, primarily involves the lungs.Though Pulmonary tuberculosis (PTB) is the commonest clinical presentation, there is a need for alertness towards uncommon presentations which involve other organs. Tuberculous otitis media (TOM) is one such rare presentation seen in paediatric practice. It is characterized by painless otorrhoea which fails to respond to the routine antibacterial treatment. TOM usually occurs secondary to PTB. Here is a case of tuberculous otitis media with Proteus mirabilis co-infection, with no evidence of PTB. In the sample of ear discharge obtained from the patient, acid fast bacilli were demonstrated on direct microscopy after Ziehl-Neelsen staining. Culture done on Lowenstein-Jensen medium demonstrated slow-growing Mycobacterium. Bacteriological culture and identification helped in isolating Proteus mirabilis. PCR, followed by Line- Probe Assay for early identification and susceptibility testing to primary drugs, was done. Further, patient tested negative for the Mantoux test. Patient was enrolled in National Tuberculosis programme- RNTCP. This case emphasizes on one of the less common presentations of a common disease. A high clinical suspicion and laboratory confirmation are required for appropriate patient management. PMID:24995225

Granulomatous encephalomyelitis and intestinal ganglionitis in a spectacled Amazon parrot (Amazona albifrons) infected with Mycobacterium genavense.

PubMed

Gomez, G; Saggese, M D; Weeks, B R; Hoppes, S M; Porter, B F

2011-01-01

An approximately 30-year-old male spectacled Amazon parrot (Amazona albifrons) was presented with a 2-week history of ataxia, head shaking, weight loss and seizures. Gross findings on necropsy examination included atrophy of the musculature, ruffled feathers and minimal epicardial and abdominal fat. Microscopically, there were perivascular cuffs of macrophages with fewer lymphocytes in the grey and white matter of the brain and spinal cord. These lesions were accompanied by gliosis and mild vacuolation of the white matter. In the small intestine, up to 70% of the intestinal ganglia were effaced by infiltrates of macrophages and fewer lymphocytes. The intestinal lamina propria contained multiple inflammatory aggregates of a similar nature. Ziehl-Neelsen staining revealed the presence of numerous bacilli within the cytoplasm of macrophages in the central nervous system (CNS) and enteric ganglia. Amplification of the DNAJ gene confirmed a mycobacterial infection and subsequent polymerase chain reaction (PCR) using a species-specific primer confirmed the aetiology as Mycobacterium genavense. Infection of the CNS with Mycobacterium spp. is uncommon and has not been previously reported in a parrot. This case is unusual in that the organism exhibited tropism for neural tissue. Copyright © 2010 Elsevier Ltd. All rights reserved.

Point prevalence of Cryptosporidium oocyst in calves grazing along River Rima bank in Sokoto, Nigeria.

PubMed

Faleke, O O; Yabo, Y A; Olaleye, A O; Dabai, Y U; Ibitoye, E B

2014-02-01

The present study was conducted to investigate the point prevalence of Cryptosporidium oocysts infection in calves grazing along the bank of Rima River Sokoto in October 2011. The river bank is a converging zone for domestic animals reared in different quarters of the town and the surrounding settlements. A total number of 2,959 cattle were enumerated out of which 147 (4.97%) were calves. Faecal samples were collected from 100 (68.02%) calves by convenient sampling technique. Formol-Ether sedimentation and modified Ziehl-Neelsen staining techniques were used to identify the Cryptosporidium oocysts in the faecal samples. Faecal consistency was also used to identify diarrhoeic and non-diarrhoeic calves. Cryptosporidium oocysts were identified in 33 (33.0%) of the calves examined. The detection rate was higher among the male calves (38.46%) than females while the Rahaji breed had the highest prevalence of 62.5%. A total of 6 (18.18%) among the positive cases were diarrhoeic. The differences in prevalence based on sex, breeds and presence of diarrhoea were not statistically significant. Calves may become sources of Cryptosporidia infection to man and other animals in the study area through unrestricted movements and interactions with the environment.

Sporotrichoid Mycobacterium marinum infection of the face following a cat scratch.

PubMed

Phan, Tai Anh; Relic, John

2010-02-01

Mycobacterium marinum infections in humans uncommonly affect the face and are not known to be associated with cat scratches. We describe a 24-year-old woman who presented with a 3-month history of multiple tender, occasionally discharging cystic nodules involving the left side of her face in a sporotrichoid distribution. She had suffered a cat scratch to her left lower eyelid 3 weeks before the onset of the eruption and owned multiple tropical fish tanks. She was systemically well and had no lymphadenopathy. She had a background history of a 4.5-mm-thick nodular melanoma of her temple treated by wide local excision and negative sentinel lymph node biopsy 4 years prior. Skin biopsies showed multiple variably sized granulomas surrounded by thick cuffs of lymphocytes involving the superficial and deep dermis with no organisms seen on Ziehl-Neelsen, peroidic acid-Schiff and methenamine silver stains. Laboratory investigations showed a mildly raised erythrocyte sedimentation rate but normal full blood count and C-reactive protein. Fluid from the left cheek grew an acid-fast bacillus identified as Mycobacterium marinum. The skin eruption cleared after 5-month treatment with oral clarithromycin 500 mg twice daily and rifampicin 600 mg daily.

Myxobolus sp. and Henneguya sp. (Cnidaria: Myxobolidae) natural co-infection in the kidney of Piaractus mesopotamicus (Characiformes: Serrasalmidae).

PubMed

Manrique, Wilson Gómez; Figueiredo, Mayra Araguaia Pereira; de Andrade Belo, Marco Antonio; Martins, Maurício Laterça; Molnár, Kálmán

2017-10-01

This study evaluated the myxozoan infection and histopathology of the kidney of freshwater fish Piaractus mesopotamicus from intensive fish farming in Brazil. A total of 55 fish were examined for myxozoan infection. Infected organs were processed by usual histology and stained with hematoxylin-eosin (H&E) and Ziehl-Neelsen (ZN). From the total of 55 fish analyzed, 47 (85.45%) presented myxospores, being 9.09% (5/55) only with Myxobolus sp., 5.45% (3/55) only with Henneguya sp., and 70.91% (39/55) presenting both parasites. The presence of myxospores was associated with histological alterations in both stromal and renal parenchyma. Myxospores were found mostly in the peritubular interstitial tissue and in low intensity in the glomerulus which caused nuclear hypertrophy and loss of Bowman space. An increase in the glomerular tuft and a reduction in the lumen of the collector tubules were also observed, besides the high number of melanomacrophage cells in the glomerulus. This study reports for the first time detection of myxozoan mixed infection in one organ of pacu and discuss the possible transportation of myxospores in the circulating blood.

Low cost automated whole smear microscopy screening system for detection of acid fast bacilli.

PubMed

Law, Yan Nei; Jian, Hanbin; Lo, Norman W S; Ip, Margaret; Chan, Mia Mei Yuk; Kam, Kai Man; Wu, Xiaohua

2018-01-01

In countries with high tuberculosis (TB) burden, there is urgent need for rapid, large-scale screening to detect smear-positive patients. We developed a computer-aided whole smear screening system that focuses in real-time, captures images and provides diagnostic grading, for both bright-field and fluorescence microscopy for detection of acid-fast-bacilli (AFB) from respiratory specimens. To evaluate the performance of dual-mode screening system in AFB diagnostic algorithms on concentrated smears with auramine O (AO) staining, as well as direct smears with AO and Ziehl-Neelsen (ZN) staining, using mycobacterial culture results as gold standard. Adult patient sputum samples requesting for M. tuberculosis cultures were divided into three batches for staining: direct AO-stained, direct ZN-stained and concentrated smears AO-stained. All slides were graded by an experienced microscopist, in parallel with the automated whole smear screening system. Sensitivity and specificity of a TB diagnostic algorithm in using the screening system alone, and in combination with a microscopist, were evaluated. Of 488 direct AO-stained smears, 228 were culture positive. These yielded a sensitivity of 81.6% and specificity of 74.2%. Of 334 direct smears with ZN staining, 142 were culture positive, which gave a sensitivity of 70.4% and specificity of 76.6%. Of 505 concentrated smears with AO staining, 250 were culture positive, giving a sensitivity of 86.4% and specificity of 71.0%. To further improve performance, machine grading was confirmed by manual smear grading when the number of AFBs detected fell within an uncertainty range. These combined results gave significant improvement in specificity (AO-direct:85.4%; ZN-direct:85.4%; AO-concentrated:92.5%) and slight improvement in sensitivity while requiring only limited manual workload. Our system achieved high sensitivity without substantially compromising specificity when compared to culture results. Significant improvement in

Performance of the BacT Alert 3D System Versus Solid Media for Recovery and Drug Susceptibility Testing of Mycobacterium tuberculosis in a Tertiary Hospital in Korea.

PubMed

Kim, Seoung-Cheol; Jeon, Bo-Young; Kim, Jin-Sook; Choi, In Hwan; Kim, Jiro; Woo, Jeongim; Kim, Soojin; Lee, Hyeong Woo; Sezim, Monoldorova; Cho, Sang-Nae

2016-10-01

Tuberculosis (TB) is a major health problem, and accurate and rapid diagnosis of multidrug-resistant (MDR) and extended drug-resistant (XDR) TB is important for appropriate treatment. In this study, performances of solid and liquid culture methods were compared with respect to MDR- and XDR-TB isolate recovery and drug susceptibility testing. Sputum specimens from 304 patients were stained with Ziehl-Neelsen method. Mycobacterium tuberculosis (Mtb) isolates were tested for recovery on Löwenstein-Jensen (LJ) medium and the BacT Alert 3D system. For drug susceptibility testing of Mtb, isolates were evaluated on M-KIT plates and the BacT Alert 3D system. The recovery rates were 94.9% (206/217) and 98.2% (213/217) for LJ medium and the BacT Alert 3D system, respectively (kappa coefficient, 0.884). The rate of drug resistance was 13.4% for at least one or more drugs, 6.0% for MDR-TB and 2.3% for XDR-TB. M-KIT plate and BacT 3D Alert 3D system were comparable in drug susceptibility testing for isoniazid (97.7%; kappa coefficient, 0.905) and rifampin (98.6%; kappa coefficient, 0.907). Antibiotic resistance was observed using M-KIT plates for 24 of the total 29 Mtb isolates (82.8%). The liquid culture system showed greater reduction in the culture period, as compared with LJ medium; however, drug susceptibility testing using M-KIT plates was advantageous for simultaneous testing against multiple drug targets.

Method and apparatus for staining immobilized nucleic acids

DOEpatents

Ramsey, J. Michael; Foote, Robert S.; Jacobson, Stephen C.

2000-01-01

A method for staining immobilized nucleic acids includes the steps of affixing DNA probes to a solid substrate, moving target DNA material into proximity with the DNA probes, whereby the target DNA hybridized with specific ones of the DNA probes, and moving a fluorescent dye into proximity with the hybridized target DNA, whereby the fluorescent dye binds to the hybridized DNA to enable subsequent detection of fluorescence.

[Comparison of four different staining methods for ear cytology of dogs with otitis externa].

PubMed

Bouassiba, C; Osthold, W; Mueller, R S

2013-01-01

Cytological examination is crucial for the diagnosis and classification of canine otitis externa. Staining should reveal micro-organisms as perpetuating factors of otitis externa. The aim of the study was to compare four different staining methods (Diff-Quik®, Diff-Quik® after dipping in acetone, Gram Quick stain® and a commercial rapid stain for otitis externa) for ear cytology of dogs with otitis externa and to investigate the agreement of cytology and culture. In a study evaluating dogs with otitis externa, five ear swabs (one for culture and four for cytology) were taken from the horizontal part of the external auditory canal of 224 affected ears and compared semi-quantitatively. Diff-Quik® with and without prior dipping in acetone as well as the Gram Quick stain® displayed a high degree of agreement in the detection of micro-organisms (cocci p = 0.2366; rods p = 0.4832; yeasts p = 0.1574), while the commercial otitis rapid stain revealed significantly less micro-organisms (p < 0.001 for all comparisons). The results of the first three stains corresponded to the culture results by >  70%; the agreement was lower with the commercial otitis rapid stain. The quickest and easiest method was staining with Diff-Quik®. Diff-Quik® with or without prior dipping in acetone and the Gram Quick stain® had a high agreement in the detection of microorganisms and can thus be considered nearly equivalent for the diagnosis of otitis externa infectiosa. The commercial otitis rapid stain is less reliable. Based on this study Diff-Quik® can be recommended for the routine cytology of ear swabs. Additionally, a culture may be indicated and must be interpreted in the context of the cytology.

Standardization of fixation, processing and staining methods for the central nervous system of vertebrates.

PubMed

Aldana Marcos, H J; Ferrari, C C; Benitez, I; Affanni, J M

1996-12-01

This paper reports the standardization of methods used for processing and embedding various vertebrate brains of different size in paraffin. Other technical details developed for avoiding frequent difficulties arising during laboratory routine are also reported. Some modifications of the Nissl and Klüver-Barrera staining methods are proposed. These modifications include: 1) a Nissl stain solution with a rapid and efficient action with easier differentiation; 2) the use of a cheap microwave oven for the Klüver-Barrera stain. These procedures have the advantage of permitting Nissl and Klüver-Barrera staining of nervous tissue in about five and fifteen minutes respectively. The proposed procedures have been tested in brains obtained from fish, amphibians, reptiles and mammals of different body sizes. They are the result of our long experience in preparing slides for comparative studies. Serial sections of excellent quality were regularly obtained in all the specimens studied. These standardized methods, being simple and quick, are recommended for routine use in neurobiological laboratories.

Quantitative cytochemistry of nuclear and cytoplasmic proteins using the Naphthol Yellow S and dinitrofluorobenzene staining methods.

PubMed

Tas, J; James, J

1981-09-01

The 'total protein staining' of biological specimens with the electrostatically binding Naphthol Yellow S or the covalently binding dinitrofluorobenzene must be interpreted as methods which yield data on the specific amino acid pool of the proteins concerned. Both dyes bind to certain free amino-acid side-chains, giving different dye--protein ratios for various proteins. In the presence of DNA, dinitrofluorobenzene stains all proteins present in cell nuclei, whereas Naphthol Yellow S only stains the majority of the non-histone proteins. When protein staining methods are combined with the Feulgen--Pararosanile (SO2) procedure for DNA, decreased Feulgen--DNA contents were measured in dinitrofluorobenzene-stained isolated nuclei and lymphocytes.

Epidemiology of Intestinal Polyparasitism among Orang Asli School Children in Rural Malaysia

PubMed Central

Al-Delaimy, Ahmed K.; Al-Mekhlafi, Hesham M.; Nasr, Nabil A.; Sady, Hany; Atroosh, Wahib M.; Nashiry, Mohammed; Anuar, Tengku S.; Moktar, Norhayati; Lim, Yvonne A. L.; Mahmud, Rohela

2014-01-01

Background This cross-sectional study aimed to investigate the current prevalence and risk factors associated with intestinal polyparasitism (the concurrent infection with multiple intestinal parasite species) among Orang Asli school children in the Lipis district of Pahang state, Malaysia. Methods/Principal findings Fecal samples were collected from 498 school children (50.6% boys and 49.4% girls), and examined by using direct smear, formalin-ether sedimentation, trichrome stain, modified Ziehl Neelsen stain, Kato-Katz, and Harada Mori techniques. Demographic, socioeconomic, environmental, and personal hygiene information were collected by using a pre-tested questionnaire. Overall, 98.4% of the children were found to be infected by at least one parasite species. Of these, 71.4% had polyparasitism. The overall prevalence of Trichuris trichiura, Ascaris lumbricoides, hookworm, Giardia duodenalis, Entamoeba spp., and Cryptosporidium spp. infections were 95.6%, 47.8%, 28.3%, 28.3%, 14.1% and 5.2%, respectively. Univariate and multivariate analyses showed that using an unsafe water supply as a source for drinking water, presence of other family members infected with intestinal parasitic infections (IPI), not washing vegetables before consumption, absence of a toilet in the house, not wearing shoes when outside, not cutting nails periodically, and not washing hands before eating were significant risk factors associated with intestinal polyparasitism among these children. Conclusions/Significance Intestinal polyparasitism is highly prevalent among children in the peninsular Malaysian Aboriginal communities. Hence, effective and sustainable control measures, including school-based periodic chemotherapy, providing adequate health education focused on good personal hygiene practices and proper sanitation, as well as safe drinking water supply should be implemented to reduce the prevalence and consequences of these infections in this population. PMID:25144662

Pericardial fluid Gram stain

MedlinePlus

... stain To use the sharing features on this page, please enable JavaScript. Pericardial fluid Gram stain is a method of staining a sample of fluid taken from the pericardium. This is the sac surrounding ...

Use of Fine Needle Aspirate from Peripheral Nerves of Pure-neural Leprosy for Cytology and Polymerase Chain Reaction to Confirm the Diagnosis: A Follow-up Study of 4 Years

PubMed Central

De, Abhishek; Hasanoor Reja, Abu Hena; Aggarwal, Ishad; Sen, Sumit; Sil, Amrita; Bhattacharya, Basudev; Sharma, Nidhi; Ansari, Asad; Sarda, Aarti; Chatterjee, Gobinda; Das, Sudip

2017-01-01

Background: Pure neural leprosy (PNL) still remains a diagnostic challenge because of the absence of sine qua non skin lesions of leprosy and a confirmatory diagnostic method. The authors had earlier described a simple yet objective technique of combining fine needle aspiration cytology (FNAC) coupled with a multiplex polymerase chain reaction (PCR) in a pilot study, wherein the technique showed promise of a reliable diagnostic tool. In the pursuit of further evidence, the authors carried out a 4-year study with PNL cases to find the efficacy and reliability of the said method in a larger sample size. Aim: This study was conducted to find the efficacy, reliability, and reproducibility of FNAC coupled with multiplex PCR and Ziehl-Neelsen (ZN) staining in identifying the cases of PNL. Materials and Methods: All cases that were suspected to be suffering from PNL, following evaluation by two independent observers were included in the study and were subjected to FNAC from the affected nerve, and the aspirates were evaluated for cytology, ZN staining, and multiplex PCR for Mycobacterium leprae genome. In addition, serum anti-PGL1 levels were also performed in all the study subjects. Fifteen non-PNL cases were also included in the control arm. Results: A total of 47 cases were included in the test arm and subjected to FNAC. Conventional ZN staining could demonstrate acid-fast bacilli (AFB) in only 15 out of 47 cases (31.91%) while M. leprae DNA could be elicited in 37 (78.72%) cases by the multiplex PCR. Only 13 (27.65%) out of 47 cases showed anti-PGLI-1 antibody positivity. On cytological examination of the nerve aspirates, only 11 (23.40%) cases showed epithelioid cells whereas nonspecific inflammation was seen in 26 (75.60%) cases. Conclusion: The results of this study conducted over a larger sample size corroborate with the findings of our pilot study. In a resource poor set up, FNAC in combination with ZN staining and multiplex PCR is a rapid, simple, and easily

Concurrent Hepatic Tuberculosis and Hepatic Graft-versus-host Disease in an Allogeneic Hematopoietic Stem Cell Transplant Recipient: A Case Report.

PubMed

Zhao, Z; Leow, W Q

2017-09-01

Infection and graft-versus-host disease (GVHD) are among the most common complications after hematopoietic stem cell transplantation (HSCT). With well-known risk factors including allogeneic HSCT and GVHD, tuberculosis (TB) has a higher incidence and shorter survival rate in HSCT recipients than in the general population. A 55-year-old Indonesian female with a history of latent TB was found to have acute myeloid leukemia 3 months after allogeneic HSCT. She presented with fever, abdominal pain, and predominant cholestatic-type liver function tests derangement. Computed tomography scans showed a relatively unremarkable liver. Liver biopsy specimens revealed multiple necrotizing granulomas with numerous acid-fast bacilli shown using Ziehl-Neelsen histochemical stain. No fungal organisms are detected by Grocott's methenamine silver and periodic acid-Schiff stains. There was also mild portal hepatitis with prominent bile duct injury and scattered apoptotic bodies, compatible with GVHD. In addition, the patient was also discovered to have cutaneous and intestinal TB as well as cutaneous and colonic GVHD during investigation. She was started on anti-TB treatment and adjusted immunosuppression scheme accordingly. Unfortunately, our patient died of spontaneous intracranial haemorrhage approximately 2 months after the diagnosis of post-transplantation TB and GVHD. We report a case of concurrent hepatic TB and GVHD in an allogeneic HSCT recipient. Recognition of the dual pathology in the biopsy results aids proper treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

[Prevalence of microsporidia and other intestinal parasites in patients with HIV infection, Bogota, 2001].

PubMed

Flórez, Astrid Carolina; García, Dabeiba Adriana; Moncada, Ligia; Beltrán, Mauricio

2003-09-01

Opportunistic intestinal parasites are a common cause of diarrhea in HIV-infected patients. To determine the prevalence of microsporidia and other opportunistic parasites infecting HIV patients in Bogotá, Colombia, 115 patients were examined for these infections during the year 2001. The institution and the sample percent from each are as follows: Santa Clara Hospital, 33.0%; San Pedro Claver, 20.0%; Simón Bolívar Hospital, 14.8%; San José Hospital, 13.9%; Central de la Policía Hospital, 6.1%; Compensar, 5.2%; Colombian League against AIDS, 2.6%; San Ignacio Hospital, 2.6%, and the Military Hospital, 1.7%. The average patient age was 36 years, with a range from 18 to 71 years. Patients with complaint of gastrointestinal symptoms were asked to provide two consecutive stool samples. The samples were concentrated in formalin-ether and examined microscopically for intestinal coccidian parasites by direct wet slide mounts. The prevalence of intestinal opportunistic parasites was 10.4% for Cryptosporidium sp. Initially, 29% of the samples were found to be positive for microsporidian spores using a modified Ziehl Neelsen chromotrope stain, but only 3.5% of them were confirmed as positive when a calcofluor/Gram chromotrope stain was used. The general prevalence of intestinal parasites was 59.1%. The most frequently found pathogens were Blastocystis hominis, 25.2%, and Entamoeba histolytica, 13%. In other studies with HIV patients in Colombia, lower prevalences of Cryptosporidium sp. infection were observed.

A method for acetylcholinesterase staining of brain sections previously processed for receptor autoradiography.

PubMed

Lim, M M; Hammock, E A D; Young, L J

2004-02-01

Receptor autoradiography using selective radiolabeled ligands allows visualization of brain receptor distribution and density on film. The resolution of specific brain regions on the film often can be difficult to discern owing to the general spread of the radioactive label and the lack of neuroanatomical landmarks on film. Receptor binding is a chemically harsh protocol that can render the tissue virtually unstainable by Nissl and other conventional stains used to delineate neuroanatomical boundaries of brain regions. We describe a method for acetylcholinesterase (AChE) staining of slides previously processed for receptor binding. AChE staining is a useful tool for delineating major brain nuclei and tracts. AChE staining on sections that have been processed for receptor autoradiography provides a direct comparison of brain regions for more precise neuroanatomical description. We report a detailed thiocholine protocol that is a modification of the Koelle-Friedenwald method to amplify the AChE signal in brain sections previously processed for autoradiography. We also describe several temporal and experimental factors that can affect the density and clarity of the AChE signal when using this protocol.

A rapid method combining Golgi and Nissl staining to study neuronal morphology and cytoarchitecture.

PubMed

Pilati, Nadia; Barker, Matthew; Panteleimonitis, Sofoklis; Donga, Revers; Hamann, Martine

2008-06-01

The Golgi silver impregnation technique gives detailed information on neuronal morphology of the few neurons it labels, whereas the majority remain unstained. In contrast, the Nissl staining technique allows for consistent labeling of the whole neuronal population but gives very limited information on neuronal morphology. Most studies characterizing neuronal cell types in the context of their distribution within the tissue slice tend to use the Golgi silver impregnation technique for neuronal morphology followed by deimpregnation as a prerequisite for showing that neuron's histological location by subsequent Nissl staining. Here, we describe a rapid method combining Golgi silver impregnation with cresyl violet staining that provides a useful and simple approach to combining cellular morphology with cytoarchitecture without the need for deimpregnating the tissue. Our method allowed us to identify neurons of the facial nucleus and the supratrigeminal nucleus, as well as assessing cellular distribution within layers of the dorsal cochlear nucleus. With this method, we also have been able to directly compare morphological characteristics of neuronal somata at the dorsal cochlear nucleus when labeled with cresyl violet with those obtained with the Golgi method, and we found that cresyl violet-labeled cell bodies appear smaller at high cellular densities. Our observation suggests that cresyl violet staining is inadequate to quantify differences in soma sizes.

Identification by real-time PCR with SYBR Green of Leishmania spp. and Serratia marcescens in canine 'sterile' cutaneous nodular lesions.

PubMed

Cornegliani, Luisa; Corona, Antonio; Vercelli, Antonella; Roccabianca, Paola

2015-06-01

Noninfectious, non-neoplastic, nodular to diffuse, so-called 'sterile' granulomatous/pyogranulomatous skin lesions (SGPSLs) are infrequently identified in dogs and may represent a diagnostic challenge. Their correct identification is based on history, histopathology and absence of intralesional foreign bodies and micro-organisms. The aim of this study was to investigate the presence of Leishmania spp., Mycobacterium spp., Serratia marcescens and Nocardia spp. by real-time PCR in canine nodular skin lesions histologically diagnosed as putatively sterile. Formalin-fixed skin biopsies were collected from 40 dogs. All samples were associated with an SGPSL diagnosis characterized by multifocal, nodular to diffuse, periadnexal and perifollicular pyogranulomas/granulomas. Neither micro-organisms nor foreign bodies were detected with haematoxylin and eosin staining, under polarized light. Further analyses included periodic acid Schiff, Ziehl-Neelsen, Fite Faraco, Giemsa and Gram histochemical stains; anti-Bacillus Calmette-Guérin (BCG) and Leishmania spp. immunohistochemistry; and real-time PCR analysis for Leishmania spp., Mycobacterium spp., S. marcescens and Nocardia spp. Special stains and BCG/immunohistochemistry were negative in all samples. Real-time PCR was positive for Leishmania spp. in four of 40 biopsies and for S. marcescens in two of 40 samples. Real-time PCR for Mycobacterium spp. and Nocardia spp. was negative. No correlation between real-time PCR positivity and a specific histological pattern was identified. Leishmania spp. have been previously identified as possible agents of certain SGPSLs, while the involvement of S. marcescens has not been investigated previously. According to our findings, Serratia spp. should be included in the list of agents possibly associated with a subgroup of granulomatous/pyogranulomatous skin lesions in dogs. © 2015 ESVD and ACVD.

New visible and selective DNA staining method in gels with tetrazolium salts.

PubMed

Paredes, Aaron J; Naranjo-Palma, Tatiana; Alfaro-Valdés, Hilda M; Barriga, Andrés; Babul, Jorge; Wilson, Christian A M

2017-01-15

DNA staining in gels has historically been carried out using silver staining and fluorescent dyes like ethidium bromide and SYBR Green I (SGI). Using fluorescent dyes allows recovery of the analyte, but requires instruments such as a transilluminator or fluorimeter to visualize the DNA. Here we described a new and simple method that allows DNA visualization to the naked eye by generating a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in SGI and nitro blue tetrazolium (NBT) solution that, when exposed to sunlight, produces a purple insoluble formazan precipitate that remains in the gel after exposure to light. A calibration curve made with a DNA standard established a detection limit of approximately 180 pg/band at 500 bp. Selectivity of this assay was determined using different biomolecules, demonstrating a high selectivity for DNA. Integrity and functionality of the DNA recovered from gels was determined by enzymatic cutting with a restriction enzyme and by transforming competent cells after the different staining methods, respectively. Our method showed the best performance among the dyes employed. Based on its specificity, low cost and its adequacy for field work, this new methodology has enormous potential benefits to research and industry. Copyright © 2016 Elsevier Inc. All rights reserved.

Port-wine stain

MedlinePlus

... Laser therapy is most successful in removing port-wine stains. It is the only method that can destroy the tiny blood vessels in the skin without causing much damage to the skin. The exact type ... on the person's age, skin type, and particular port-wine stain.

Automated single-slide staining system

NASA Technical Reports Server (NTRS)

Mills, S. M.; Wilkins, J. R.

1974-01-01

Apparatus developed to Gram-stain single slides automatically is flexible enough to accommodate other types of staining procedures. Method frees operator and eliminates necessity for subjective evaluations as to length of staining or decolorizing time.

Intestinal parasitic infections in different groups of immunocompromised patients in Kashan and Qom cities, central Iran.

PubMed

Rasti, Sima; Hassanzadeh, Malihe; Hooshyar, Hossein; Momen-Heravi, Mansooreh; Mousavi, Seyed Gholam Abbas; Abdoli, Amir

Intestinal parasitic infections (IPIs) are important causes of morbidity and mortality in patients with immunocompromising conditions. The aim of this study was to determine the prevalence of IPIs in different groups of immunocompromised patients, including hemodialysis patients (HD), renal transplant recipients (RTR), cancer and HIV/AIDS patients in comparison with healthy individuals in two central cities of Iran (Kashan and Qom). In this case-control study, the stool samples of 135 HD, 50 RTR, 60 cancer patients, 20 HIV/AIDS patients and 120 healthy subjects were tested using direct-smear, formol-ether concentration, Ziehl-Neelsen staining and Agar plate method. The overall infection rate was 11.7% (31/265) in patient groups and 0% (0/120) in the control group. The frequency of parasites was 25% in HIV/AIDS patients, 11.9% (16/135) in HD, 12.0% (6/50) in RTR and 6.7% (4/60) in cancer patients. Blastocystis hominis (4.2%) and Giardia lamblia (3.0%) were the most prevalent parasites in patient groups. The infection rate was significantly higher in male (17.6%) than female (5.4%) patients (p = .002), but no statistically significant association was observed according to the age and educational levels. This study showed a high prevalence of IPIs in immunocompromised patients. The results of this study suggest that periodic stool examinations for screening of IPIs should be included as a part of routine medical care in these patients.

[Histology of liver lesions due to Schistosoma mekongi. About six cases with severe portal hypertension operated in Cambodia].

PubMed

Monchy, D; Dumurgier, C; Heng, T K; Hong, K; Khun, H; Hou, S V; Sok, K E; Huerre, M R

2006-12-01

Schistosomiasis mekongi was shown to be endemic, along the Mekong River, in northern Cambodia, affecting many patients with portal hypertension. Surgical procedures were proposed to some patients with digestive haemorrhage history to avoid fatal recurrence. The aim of our study was to evaluate the intensity of the liver fibrosis among these patients. During surgical treatment, liver biopsies were collected, fixed in Bouin or in formalin and processed at the Institut Pasteur of Cambodia. Sections were stained by H&E, Masson's trichrome, PAS, Ziehl-Neelsen's method and Congo Red. A total of six biopsies from patients aged 16-36 were analysed. There was complete disorganization of hepatic architecture with fibrous enlargement of portal tracts and some portal-portal bridging fibrosis, but there was no cirrhosis. In portal areas, there was blood vessel congestion and thrombosis with inflammation. Bile ducts were normal. In the parenchyma, congestion of sinusoidal capillaries was combined with focal mononuclear inflammatory infiltrate. There was no steatosis, no necrosis, no cholestasis, no iron accumulation and no amyloidosis. Numerous eggs of Schistosoma mekongi were observed in five cases, mostly in fibrous areas and more rarely in the parenchyma. Eggs were round or oval, measuring 60 x 40 microns with an acid-fast thin hyaline wall. Some eggs were surrounded by epithelioid and giant cell reaction. In conclusion, our findings illustrated a surprisingly high degree of fibrosis among young adults which contrasts with other schistosomiasis.

Prevalence of Tuberculosis among Veterans, Military Personnel and their Families in East Azerbaijan Province Violators of the last 15 Years.

PubMed

Azad Aminjan, Maboud; Moaddab, Seyyed Reza; Hosseini Ravandi, Mohammad; Kazemi Haki, Behzad

2015-10-01

Nowadays in the world, tuberculosis is the second largest killer of adults after HIV. Due to the location of presidios that is mostly located in hazardous zones soldiers and army personnel are considered high risk, therefore we decided to determine the prevalence of tuberculosis status in this group of people. This was a cross-sectional descriptive research that studied the prevalence of pulmonary tuberculosis in soldiers and military personnel in the last 15 years in tuberculosis and lung disease research center at Tabriz University of Medical Sciences. The statistical population consisted of all the soldiers and military personnel. The detection method in this study was based on microscopic examination following Ziehl-Neelsen Stain and in Leuven Stein Johnson culturing. Descriptive statistics was used for statistical analysis and statistical values less than 0.05 were considered significant. By review information in this center since the 1988-2013 with 72 military personnel suffering from tuberculosis, it was revealed that among them 30 women, 42 men, 14 soldiers, 29 family members, and 29 military personnel are pointed. A significant correlation was found between TB rates among military personnel and their families. Although in recent years, the national statistics indicate a decline of tuberculosis, but the results of our study showed that TB is still a serious disease that must comply with the first symptoms of tuberculosis in military personnel and their families that should be diagnosed as soon as possible.

A Rapid Method Combining Golgi and Nissl Staining to Study Neuronal Morphology and Cytoarchitecture

PubMed Central

Pilati, Nadia; Barker, Matthew; Panteleimonitis, Sofoklis; Donga, Revers; Hamann, Martine

2008-01-01

The Golgi silver impregnation technique gives detailed information on neuronal morphology of the few neurons it labels, whereas the majority remain unstained. In contrast, the Nissl staining technique allows for consistent labeling of the whole neuronal population but gives very limited information on neuronal morphology. Most studies characterizing neuronal cell types in the context of their distribution within the tissue slice tend to use the Golgi silver impregnation technique for neuronal morphology followed by deimpregnation as a prerequisite for showing that neuron's histological location by subsequent Nissl staining. Here, we describe a rapid method combining Golgi silver impregnation with cresyl violet staining that provides a useful and simple approach to combining cellular morphology with cytoarchitecture without the need for deimpregnating the tissue. Our method allowed us to identify neurons of the facial nucleus and the supratrigeminal nucleus, as well as assessing cellular distribution within layers of the dorsal cochlear nucleus. With this method, we also have been able to directly compare morphological characteristics of neuronal somata at the dorsal cochlear nucleus when labeled with cresyl violet with those obtained with the Golgi method, and we found that cresyl violet–labeled cell bodies appear smaller at high cellular densities. Our observation suggests that cresyl violet staining is inadequate to quantify differences in soma sizes. (J Histochem Cytochem 56:539–550, 2008) PMID:18285350

Spectral feature characterization methods for blood stain detection in crime scene backgrounds

NASA Astrophysics Data System (ADS)

Yang, Jie; Mathew, Jobin J.; Dube, Roger R.; Messinger, David W.

2016-05-01

Blood stains are one of the most important types of evidence for forensic investigation. They contain valuable DNA information, and the pattern of the stains can suggest specifics about the nature of the violence that transpired at the scene. Blood spectral signatures containing unique reflectance or absorption features are important both for forensic on-site investigation and laboratory testing. They can be used for target detection and identification applied to crime scene hyperspectral imagery, and also be utilized to analyze the spectral variation of blood on various backgrounds. Non-blood stains often mislead the detection and can generate false alarms at a real crime scene, especially for dark and red backgrounds. This paper measured the reflectance of liquid blood and 9 kinds of non-blood samples in the range of 350 nm - 2500 nm in various crime scene backgrounds, such as pure samples contained in petri dish with various thicknesses, mixed samples with different colors and materials of fabrics, and mixed samples with wood, all of which are examined to provide sub-visual evidence for detecting and recognizing blood from non-blood samples in a realistic crime scene. The spectral difference between blood and non-blood samples are examined and spectral features such as "peaks" and "depths" of reflectance are selected. Two blood stain detection methods are proposed in this paper. The first method uses index to denote the ratio of "depth" minus "peak" over"depth" add"peak" within a wavelength range of the reflectance spectrum. The second method uses relative band depth of the selected wavelength ranges of the reflectance spectrum. Results show that the index method is able to discriminate blood from non-blood samples in most tested crime scene backgrounds, but is not able to detect it from black felt. Whereas the relative band depth method is able to discriminate blood from non-blood samples on all of the tested background material types and colors.

Use of fine needle aspirate from peripheral nerves of pure-neural leprosy for cytology and PCR to confirm the diagnosis: a pilot study.

PubMed

Reja, Abu Hena Hasanoor; De, Abhishek; Biswas, Supratik; Chattopadhyay, Amitabha; Chatterjee, Gobinda; Bhattacharya, Basudev; Sarda, Aarti; Aggarwal, Ishad

2013-01-01

The diagnosis of pure neural leprosy (PNL) remained subjective because of over-dependence of clinical expertise and a lack of simple yet reliable diagnostic tool. The criteria for diagnosis, proposed by Jardim et al., are not routinely done by clinicians in developing country as it involves invasive nerve biopsy and sophisticated anti-PGL-1 detection. We conducted a study using fine needle aspiration cytology (FNAC) coupled with Ziehl Neelsen staining (ZN staining) and Multiplex-Polymerase Chain Reaction (PCR) specific for M. leprae for an objective diagnosis of pure neural leprosy (PNL), which may be simpler and yet reliable. The aim of the study is to couple FNAC with ZN staining and multiplex PCR to diagnose pure neural leprosy patients rapidly, in simpler and yet reliable way. Thirteen patients of PNL as diagnosed by two independent consultants were included as case, and 5 patients other than PNL were taken as control in the study. Fine needle aspiration was done on the affected nerve, and aspirates were evaluated for cytology, ZN staining and multiplex-PCR. Out of the 13 cases where fine needle aspiration was done, M. leprae could be elicited in the nerve tissue aspirates in 5 cases (38.4%) with the help of conventional acid-fast staining and 11 cases (84.6%) with the help of multiplex PCR. On cytological examination of the aspirates, only 3 (23%) cases showed specific epithelioid cells, whereas 8 (61.5%) cases showed non-specific inflammation, and 2 (15.3%) cases had no inflammatory cells. Our study demonstrates that in the field of laboratory diagnosis of PNL cases, FNAC in combination with ZN staining for acid-fast bacilli (AFB) and Multiplex-PCR can provide a rapid and definitive diagnosis for the majority of PNL cases. FNAC is a less-invasive, outdoor-based and simpler technique than invasive nerve biopsy procedure. Thus, this study may enlighten the future path for easy and reliable diagnosis of PNL.

Phenotypic and genotypic characteristics of drug resistance in Mycobacterium tuberculosis isolates from pediatric population of Chennai, India.

PubMed

Therese, K Lily; Gayathri, R; Balasubramanian, S; Natrajan, S; Madhavan, H N

2012-01-01

Multidrug-resistant TB (MDR-TB) has been reported in almost all parts of the world. Childhood TB is accorded low priority by national TB control programs. Probable reasons include diagnostic difficulties, limited resources, misplaced faith in BCG and lack of data on treatment. Good data on the burden of all forms of TB among children in India are not available. To study the drug sensitivity pattern of tuberculosis in children aged from 3 months to 18 years and the outcome of drug-resistant tuberculosis by BACTEC culture system and PCR-based DNA sequencing technique. This is a retrospective study. One hundred and fifty-nine clinical specimens were processed for Ziehl-Neelsen stain, Mycobacterial culture by BACTEC method, phenotypic DST for first-line drugs for Mycobacterium tuberculosis (M. tuberculosis) isolates and PCR-based DNA sequencing was performed for the M. tuberculosis isolates targeting rpoB, katG, inhA, oxyR-ahpC, rpsL, rrs and pncA. Out of the 159 Mycobacterial cultures performed during the study period, 17 clinical specimens (10.7%) were culture positive for M. tuberculosis. Among the 17 M. tuberculosis isolates, 2 were multidrug-resistant TB. PCR-based DNA sequencing revealed the presence of many novel mutations targeting katG, inhA, oxyR-ahpC and pncA and the most commonly reported mutation Ser531Leu in the rpoB gene. This study underlines the urgent need to take efforts to develop methods for rapid detection and drug susceptibility of tubercle bacilli in the pediatric population.

Airborne Mycobacterium avium infection in a group of red-shanked douc langurs (Pygathrix nemaeus nemaeus).

PubMed

Plesker, Roland; Teschner, Katja; Behlert, Olaf; Prenger-Berninghoff, Ellen; Hillemann, Doris

2010-04-01

This report describes an airborne Mycobacterium avium (MA)-infection in two red-shanked douc langurs (Pygathrix nemaeus nemaeus) from Cologne zoo. The two individuals and their tissues were investigated clinically (including x-rays), in pathology, in pathohistology, in classical bacteriology and by polymerase chain reaction (PCR). Clinically, one individual displayed emaciation and a positive reaction in an intrapalpebral testing for M. bovis/MA. The other individual was without any symptoms and did not show any reaction in the intrapalpebral test. In x-ray photos of the lungs, calcified nodules were detected. In pathology, calcified and necrotic nodules were observed within the lungs and the bronchial lymph nodes. In pathohistology, both classical tuberculous granulomas, and few acid fast rods were seen in Ziehl-Neelsen-stain. However, classical bacteriology could not demonstrate mycobacteria. In PCR, MA-infection could be confirmed in one individual using the bronchial lymph nodes. It was an airborne infection; however, the definite source of infection in these cases remained unclear. Animals in contact to the langurs (house sparrows and mice) as well as water used in the building are the most promising candidates. The risk for a zoonotic transmission in these cases has been calculated to be low.

Rapid diagnosis of tuberculosis in dromedary camel (Camelus dromedarius) using lateral flow assay-based kit.

PubMed

Ranjan, Rakesh; Narnaware, Shirish D; Nath, Kashi; Sawal, R K; Patil, N V

2018-04-01

Accurate early antemortem diagnosis of tuberculosis in dromedary camels is difficult due to the lack of reliable diagnostic test. The present study aimed to evaluate a lateral flow assay-based kit (rapid assay kit) in tuberculosis diagnosis that employs immuno-chromatographic detection of antibodies in serum, plasma, or whole blood. In a dromedary camel herd comprising 337 animals located at Bikaner, Rajasthan, India, 50 adult weak camels (11 males and 39 females) were tested by applying a single intradermal tuberculin test (SIDT) and rapid assay kit. A total of 14 animals (2 males, 12 females) were found positive in rapid assay. In SIDT, four animals revealed a positive reaction in the neck region and seven animals in the tail base. Another male animal was found SIDT positive but negative in rapid assay; it died after 12 months. Nine rapid assay positive animals died asymptomatically in 1- to 11-month period revealing postmortem tuberculosis lesions that were confirmed by Ziehl-Neelsen staining and histopathology. No tuberculous lesion was evident in the animal found positive in SIDT alone. Results of the present study indicated that serological tests like rapid assay kit can serve as a reliable test for antemortem diagnosis of tuberculosis in dromedary camel.

[Diagnostic strategies in the Tuberculosis Clinic of the Hospital General La Raza National Medical Center].

PubMed

Flores-Ibarra, Alberto Alejandro; Ochoa-Vázquez, María Dolores; Sánchez-Tec, Georgina Alejandra

2016-01-01

In order to diagnose TB infection, tuberculin skin test and interferon gamma release assay are available. The tuberculin test has a sensitivity of 99 % and a specificity of 95 %. For the detection of interferon gamma in blood there are currently two tests available: TBGold QuantiFERON-In-Tube (with a sensitivity of 0.70 and a specificity of 0.90), and T-SPOT-TB (sensitivity 0.90 and specificity 0.93). To diagnose the disease, a microscopy of direct smears for acid-fast bacilli is used if the physician is facing an extensive cavitary lung disease due to M. tuberculosis (this test has a high sensitivity: 80-90 %). The most common staining techniques used are Ziehl-Neelsen and Kinyoun, and the fluorescent technique, auramine-rhodamine. The culture is the gold standard and it has a sensitivity of 80 % and a specificity over 90 %, but the results take weeks. The nucleic acid amplification test has an overall sensitivity and specificity of 0.85 and 0.97, respectively. In the presence of a pleural effusion is necessary to perform a pleural biopsy for culture with a sensitivity of 85 % if it is percutaneous and 98 % if it was taken by thoracoscopy. The adenosine deaminase can be determined in pleural fluid with a sensitivity and specificity of 95 %.

The External Quality Assessment Scheme (EQAS): Experiences of a medium sized accredited laboratory.

PubMed

Bhat, Vivek; Chavan, Preeti; Naresh, Chital; Poladia, Pratik

2015-06-15

We put forth our experiences of EQAS, analyzed the result discrepancies, reviewed the corrective actions and also put forth strategies for risk identification and prevention of potential errors in a medical laboratory. For hematology, EQAS samples - blood, peripheral and reticulocyte smears - were received quarterly every year. All the blood samples were processed on HMX hematology analyzer by Beckman-Coulter. For clinical chemistry, lyophilized samples were received and were processed on Siemens Dimension Xpand and RXL analyzers. For microbiology, EQAS samples were received quarterly every year as lyophilized strains along with smears and serological samples. In hematology no outliers were noted for reticulocyte and peripheral smear examination. Only one outlier was noted for CBC. In clinical chemistry outliers (SDI ≥ 2) were noted in 7 samples (23 parameters) out of total 36 samples (756 parameters) processed. Thirteen of these parameters were analyzed as random errors, 3 as transcriptional errors and seven instances of systemic error were noted. In microbiology, one discrepancy was noted in isolate identification and in the grading of smears for AFB by Ziehl Neelsen stain. EQAS along with IQC is a very important tool for maintaining optimal quality of services. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification of Cryptosporidium from Dairy Cattle in Pahang, Malaysia

PubMed Central

Hisamuddin, Nur Hazirah; Hashim, Najat; Soffian, Sharmeen Nellisa; Amin, Mohd Hishammfariz Mohd; Wahab, Ridhwan Abdul; Mohammad, Mardhiah; Isa, Muhammad Lokman Md; Yusof, Afzan Mat

2016-01-01

Cryptosporidium, a protozoan parasite, can cause cryptosporidiosis which is a gastrointestinal disease that can infect humans and livestock. Cattle are the most common livestock that can be infected with this protozoan. This study was carried out to determine the prevalence of Cryptosporidium infection in cattle in Kuantan, Pahang, Malaysia and to find out the association between the occurrence of infection and 3 different ages of cattle (calves less than 1 year, yearling, and adult cattle). The samples were processed by using formol-ether concentration technique and stained by modified Ziehl Neelsen. The results showed that 15.9% (24/151) of cattle were positive for Cryptosporidium oocysts. The occurrence of Cryptosporidium in calves less than 1 year was the highest with the percentage of 20.0% (11/55) followed by yearling and adult cattle, with the percentage occurrence of 15.6 % (7/45) and 11.8% (6/51), respectively. There was no significant association between the occurrence and age of cattle and presence of diarrhea. Good management practices and proper hygiene management must be taken in order to reduce the infection. It is highly important to control the infection since infected cattle may serve as potential reservoirs of the infection to other animals and humans, especially animal handlers. PMID:27180579

Endoparasites of rodents from the Chittagong Hill Tracts in Southeastern Bangladesh.

PubMed

Fuehrer, Hans-Peter; Baumann, Timo A; Riedl, Julia; Treiber, Moritz; Igel, Petra; Swoboda, Paul; Joachim, Anja; Noedl, Harald

2012-11-01

Rodents are a key mammalian group highly successful in adapting to a variety of environments throughout the world and play an important role in many zoonotic cycles. Within this project, the gastrointestinal and extraintestinal parasite fauna of 76 rodents (Muroidea and Sciuridae) was determined in the District of Bandarban (Chittagong Hill Tracts) in Southeastern Bangladesh. Gastrointestinal and extraintestinal parasites were examined with macro- and microscopical tools (e.g. Ziehl-Neelsen Staining) at a field site in Bandarban. A wide variety of parasites were found in rodent hosts, including protozoa-Giardia sp. (n = 8), Cryptosporidium sp. (n = 1), Entamoeba sp. (n = 8), Trichomonadida (n = 4), Isospora sp. (n = 1), trematodes (Echinostoma sp.; n = 3), cestodes-Hymenolepis nana (n = 1), Hymenolepis diminuta (n = 3), Hymenolepis sp. (n = 2), Taenia taeniaeformis-Larven (n = 4), Catenotaenia sp. (n = 1), Taenia sp. (n = 1), nematodes-Heterakis spumosa (n = 4), Heterakis sp. (n = 1), Aspiculuris tetraptera (n = 2), Capillaria hepatica (n = 2), Capillaria sp. (n = 3), Syphacia sp. (n = 2), Strongyloides sp. (n = 10), Trichostrongylus sp. (n = 2) and Trichuris sp. (n = 1)-and acanthocephalans (Moniliformis moniliformis; n = 2). Several of the examined parasites are of zoonotic importance via direct or indirect transmission (e.g. C. hepatica) and may affect humans.

Gram stain of tissue biopsy

MedlinePlus

... biopsy To use the sharing features on this page, please enable JavaScript. Gram stain of tissue biopsy test involves using crystal violet stain to test a sample of tissue taken from a biopsy . The Gram stain method can ...

[Effect of staining method and sintering temperature on the color of porcelain-fused-to-metal restorations].

PubMed

Wang, Yan; Yan, Wei-hao; Zhang, Xin-chun; Huang, Yue; Shen, Lin-han

2004-12-01

To investigate the effect of staining method and sintering temperature on the color of porcelain-fused-metal restorations. 40 cylindrical stained porcelain-metal specimens of 15 mm in diameter and 6 mm in height were fabricated with customized mould, consisting of 2 mm Ni-Cr metal, 1 mm opaque porcelain, 2 mm dentine porcelain and 1 mm enamel porcelain. The specimens were prepared by 5 techniques, 8 for each group. Group A: internal staining, Group B: external staining, 900 degrees centigrade sintering temperature was used in both A and B group; Group C to E: external staining, with the sintering temperature of 880 degrees centigrade, 900 degrees centigrade and 920 degrees centigrade respectively. Sofu A2 porcelain and Sofu 44 stain system were used for the study. Using standard white plate as a reference, colors (L*,a*,b* coordinates) of the specimens were measured with a computerized colorimeter. Student's t test and one way ANOVA were used to analyze the data. DeltaE, b* and Delta C(ab)* of Group B (external staining) and group A (internal staining) were 43.72 +/- 2.99/26.51 +/- 1.64/31.31 +/- 2.48 and 39.71 +/- 1.78/23.69 +/- 0.36/26.55 +/- 2.16, respectively. The values of the former group were significantly higher than that of the latter (P < 0.05); For Group C to E, there were no significant differences in all the color parameters. Staining method has a significant effect on the color parameters of porcelain-fused-to-metal restorations; For external staining, within the clinically-used range, changing the sintering temperature does not have an obvious effect on the color.

Comparison of culture-based, vital stain and PMA-qPCR methods for the quantitative detection of viable hookworm ova.

PubMed

Gyawali, P; Sidhu, J P S; Ahmed, W; Jagals, P; Toze, S

2017-06-01

Accurate quantitative measurement of viable hookworm ova from environmental samples is the key to controlling hookworm re-infections in the endemic regions. In this study, the accuracy of three quantitative detection methods [culture-based, vital stain and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR)] was evaluated by enumerating 1,000 ± 50 Ancylostoma caninum ova in the laboratory. The culture-based method was able to quantify an average of 397 ± 59 viable hookworm ova. Similarly, vital stain and PMA-qPCR methods quantified 644 ± 87 and 587 ± 91 viable ova, respectively. The numbers of viable ova estimated by the culture-based method were significantly (P < 0.05) lower than vital stain and PMA-qPCR methods. Therefore, both PMA-qPCR and vital stain methods appear to be suitable for the quantitative detection of viable hookworm ova. However, PMA-qPCR would be preferable over the vital stain method in scenarios where ova speciation is needed.

Photometric Application of the Gram Stain Method To Characterize Natural Bacterial Populations in Aquatic Environments

PubMed Central

Saida, H.; Ytow, N.; Seki, H.

1998-01-01

The Gram stain method was applied to the photometric characterization of aquatic bacterial populations with a charge-coupled device camera and an image analyzer. Escherichia coli and Bacillus subtilis were used as standards of typical gram-negative and gram-positive bacteria, respectively. A mounting agent to obtain clear images of Gram-stained bacteria on Nuclepore membrane filters was developed. The bacterial stainability by the Gram stain was indicated by the Gram stain index (GSI), which was applicable not only to the dichotomous classification of bacteria but also to the characterization of cell wall structure. The GSI spectra of natural bacterial populations in water with various levels of eutrophication showed a distinct profile, suggesting possible staining specificity that indicates the presence of a particular bacterial population in the aquatic environment. PMID:9464416

INTESTINAL PARASITES IN DIABETIC PATIENTS IN SOHAG UNIVERSITY HOSPITALS, EGYPT.

PubMed

Elnadi, Nada A; Hassanien, Hassan A; Ahmad, Amal M; Abd Ellah, Asmaa K

2015-08-01

Intestinal parasites usually create benign diseases, though they may induce complications with high morbidity and mortality to the immunocompromised, including diabetic patients. The study detected the prevalence of intestinal parasitic infections in diabetic patients, comparing to non-diabetic controls and other parameters. A total of 100 fecal samples were collected from diabetic patients at the outpatient clinic of Sohag University Hospitals and another 100 from cross matched controls. The samples were examined macroscopically and microscopically by direct smear and different concentration methods then stained by Modified Ziehl-Neelsen Acid fast stain. Glycated hemoglobin (Hb Alc) was measured to detect DM controlled patients. The data were organized, tabulated, and statistically analyzed. Intestinal parasites were found in 25 (25%) cases out of 100 patients in diabetic group and 7(7%) cases out of 100 controls with high significance (P<0.001)). In the diabetic group, Giardia lamblia was detected in 22 cases (22%) and 5 (5%) among controls, Entamoeba histolytica in 7 cases (7%) and 3 (3%) among controls, Hymenolypis nana in 5 cases (5%) and 3 (3%) among controls, Entamoeba coli in 8 patients (8%), Entamoeba hartmanni in 3 cases (3%), Dientamoeba fragilis in a case (1%), Cryptosporidium parvum in 5 cases (5%) and microsporidia in 3 cases (3%). But, E. coli, E. hartmanni, D. fragilis and C. parvum nor microsporidia were detected in controls. The rate of G. lamblia in DM patients compared to controls was high significant (P<0.001). Hymenolepis nana was 5% (5 cases) in diabetic patients compared to 3% (3 cases) in controls. Residence and sex differences were not significant, while age, >10 years showed the highest prevalence (P< 0.003), type I infection rate was significantly higher than type II (P<0.001). DM control was also significantly affected the infection rates (P<0.007 in type I and P< 0.01 in type II).

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA.

PubMed

Wang, Hongyang; Owens, James D; Shih, Joanna H; Li, Ming-Chung; Bonner, Robert F; Mushinski, J Frederic

2006-04-27

Gene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays. The MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases. RNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts.

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA

PubMed Central

Wang, Hongyang; Owens, James D; Shih, Joanna H; Li, Ming-Chung; Bonner, Robert F; Mushinski, J Frederic

2006-01-01

Background Gene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays. Results The MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases. Conclusion RNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts. PMID:16643667

Papanicolaou stain: Is it economical to switch to rapid, economical, acetic acid, papanicolaou stain?

PubMed

Dighe, Swati B; Ajit, Dulhan; Pathuthara, Saleem; Chinoy, Roshni

2006-01-01

To standardize an inexpensive and rapid Papanicolaou staining technique with limited ethanol usage. Smears from 200 patients were collected (2 per patient) and fixed in methanol. Half were subjected to conventional Papanicolaou and half to stain ing with rapid, economical, acetic acid Papanicolaou (REAP) stain. In REAP, pre-OG6 and post-OG6 and post-EA36 ethanol baths were replaced by 1% acetic acid and Scott's tap water with tap water. Hematoxylin was preheated to 60 degrees C. Final dehydration was with methanol. REAP smears were compared with Papanicolaou smears for optimal cytoplasmic and nuclear staining, stain preservation, cost and turnaround time. With the REAP method, cytoplasmic and nuclear staining was optimal in 181 and 192 cases, respectively. The staining time was considerably reduced, to 3 minutes, and the cost per smear was reduced to one fourth. The staining quality remained good in all the smears for > 2 years. REAP is a rapid, cost-effective alternative to Papanicolaou stain. Though low stain penetration in large cell clusters is a limitation, final interpretation was not compromised.

Compositions for chromosome-specific staining

DOEpatents

Gray, Joe W.; Pinkel, Daniel

1998-01-01

Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods are provided to disable the hybridization capacity of shared, high copy repetitive sequences and/or remove such sequences to provide for useful contrast. Still further methods are provided to produce chromosome-specific staining reagents which are made specific to the targeted chromosomal material, which can be one or more whole chromosomes, one or more regions on one or more chromosomes, subsets of chromosomes and/or the entire genome. Probes and test kits are provided for use in tumor cytogenetics, in the detection of disease related loci, in analysis of structural abnormalities, such as translocations, and for biological dosimetry. Further, methods and prenatal test kits are provided to stain targeted chromosomal material of fetal cells, including fetal cells obtained from maternal blood. Still further, the invention provides for automated means to detect and analyse chromosomal abnormalities.

Compositions for chromosome-specific staining

DOEpatents

Gray, J.W.; Pinkel, D.

1998-05-26

Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. The methods produce staining patterns that can be tailored for specific cytogenetic analyses. The probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods are provided to disable the hybridization capacity of shared, high copy repetitive sequences and/or remove such sequences to provide for useful contrast. Still further methods are provided to produce chromosome-specific staining reagents which are made specific to the targeted chromosomal material, which can be one or more whole chromosomes, one or more regions on one or more chromosomes, subsets of chromosomes and/or the entire genome. Probes and test kits are provided for use in tumor cytogenetics, in the detection of disease related loci, in analysis of structural abnormalities, such as translocations, and for biological dosimetry. Methods and prenatal test kits are provided to stain targeted chromosomal material of fetal cells, including fetal cells obtained from maternal blood. The invention provides for automated means to detect and analyze chromosomal abnormalities. 17 figs.

Operational implementation of LED fluorescence microscopy in screening tuberculosis suspects in an urban HIV clinic in Uganda.

PubMed

Albert, Heidi; Nakiyingi, Lydia; Sempa, Joseph; Mbabazi, Olive; Mukkada, Sheena; Nyesiga, Barnabas; Perkins, Mark D; Manabe, Yukari C

2013-01-01

Light emitting diode (LED) fluorescence microscopy (FM) is an affordable, technology targeted for use in resource-limited settings and recommended for widespread roll-out by the World Health Organization (WHO). We sought to compare the operational performance of three LED FM methods compared to light microscopy in a cohort of HIV-positive tuberculosis (TB) suspects at an urban clinic in a high TB burden country. Two spot specimens collected from TB suspects were included in the study. Smears were stained using auramine O method and read after blinding by three LED-based FM methods by trained laboratory technicians in the Infectious Diseases Institutelaboratory. Leftover portions of the refrigerated sputum specimens were transported to the FIND Tuberculosis Research Laboratory for Ziehl Neelsen (ZN) smear preparation and reading by experienced technologist as well as liquid and solid culture. 174 of 627 (27.8%) specimens collected yielded one or more positive mycobacterial cultures. 94.3% (164/174) were M. tuberculosis complex. LED FM was between 7.3-11.0% more sensitive compared to ZN microscopy. Of the 592 specimens examined by all microscopy methods, there was no significant difference in sensitivity between the three LED FM methods. The specificity of the LED FM methods was between 6.1% and 7.7% lower than ZN microscopy (P<0.001), although exclusion of the single poor reader resulted in over 98% specificity for all FM methods. Laboratory technicians in routine settings can be trained to use FM which is more sensitive than ZN microscopy. Despite rigorous proficiency testing, there were operator-dependent accuracy issues which highlight the critical need for intensive quality assurance procedures during LED FM implementation. The low sensitivity of FM for HIV-positive individuals particularly those with low CD4 T cell counts, will limit the number of additional patients found by LED FM in countries with high rates of HIV co-infection.

Fast nuclear staining of head hair roots as a screening method for successful STR analysis in forensics.

PubMed

Lepez, Trees; Vandewoestyne, Mado; Van Hoofstat, David; Deforce, Dieter

2014-11-01

The success rate of STR profiling of hairs found at a crime scene is quite low and negative results of hair analysis are frequently reported. To increase the success rate of DNA analysis of hairs in forensics, nuclei in hair roots can be counted after staining the hair root with DAPI. Two staining methods were tested: a longer method with two 1h incubations in respectively a DAPI- and a wash-solution, and a fast, direct staining of the hair root on microscope slides. The two staining methods were not significantly different. The results of the STR analysis for both procedures showed that 20 nuclei are necessary to obtain at least partial STR profiles. When more than 50 nuclei were counted, full STR profiles were always obtained. In 96% of the cases where no nuclei were detected, no STR profile could be obtained. However, 4% of the DAPI-negative hair roots resulted in at least partial STR profiles. Therefore, each forensic case has to be evaluated separately in function of the importance of the evidential value of the found hair. The fast staining method was applied in 36 forensic cases on 279 hairs in total. A fast screening method using DAPI can be used to increase the success rate of hair analysis in forensics. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Fatal pyogranulomatous myocarditis in 10 Boxer puppies.

PubMed

Detmer, Susan E; Bouljihad, Mostafa; Hayden, David W; Schefers, Jeremy M; Armien, Anibal; Wünschmann, Arno

2016-03-01

Over a period of 5 years, 10 pure-bred Boxer puppies, 9-16 weeks old, were presented with a history of sudden death and were diagnosed with pyogranulomatous myocarditis. The myocarditis was characterized by a mixed infiltrate composed predominantly of neutrophils and macrophages. In our retrospective study, original case records and archived materials were examined. All dogs were positive for Borrelia burgdorferi on immunohistochemistry (IHC). There was no evidence of infectious agents in formalin-fixed, paraffin-embedded (FFPE) heart tissue sections stained with hematoxylin and eosin, Ziehl-Neelsen, Gram, Grocott methenamine silver, Warthin-Starry, Von Kossa, and Steiner-Chapman stains. IHC for Chlamydia sp., Toxoplasma gondii, Neospora caninum, West Nile virus, and canine parvovirus also yielded a negative result in all dogs. Polymerase chain reaction testing for vector-borne pathogens on heart tissue from 9 of the dogs (1 frozen and 8 FFPE samples) yielded positive results for 1 dog with B. burgdorferi as well as Anaplasma phagocytophilum in another dog. Subsequently, 2 additional cases were found in a French Bulldog and a French Bulldog-Beagle mix that had identical morphology, test results, age, and seasonality to these 10 Boxer dogs. The similarities in the seasonality, signalment of the affected dogs, and the gross and microscopic lesions suggest a common etiology. Positive IHC and morphologic similarities to human Lyme carditis indicate that B. burgdorferi is likely the agent involved. An additional consideration for these cases is the possibility of a breed-specific autoimmune myocarditis or potential predisposition for cardiopathogenic agents in young Boxers. © 2016 The Author(s).

Quantitative determination of superoxide in plant leaves using a modified NBT staining method.

PubMed

Bournonville, Carlos F Grellet; Díaz-Ricci, Juan Carlos

2011-01-01

In plants, the ROS (reactive oxygen species) level is tightly regulated because their accumulation produces irreversible damage leading to cell death. However, ROS accumulation plays a key role in plant signaling under biotic or abiotic stress. Although various methods were reported to evaluate ROS accumulation, they are restricted to model plants or provide only qualitative information. Develop a simple method to quantify superoxide radicals produced in plant tissues, based on the selective extraction of the formazan produced after nitroblue tetrazolium (NBT) reduction in histochemical staining. Plant leaves were stained with a standard NBT method and the formazan precipitated in tissues was selectively extracted using chloroform. The organic phase was dried and formazan residue dissolved in dimethylsulfoxide-potassium hydroxide and quantified by spectrophotometry. The method was tested in strawberry plant leaves under different stressing conditions. Formazan extracted from leaves subjected to stress conditions showed similar absorption spectra to those obtained from standard solutions using pure formazan. Calibration curves showed a linear relationship between absorbance and formazan amounts, within the range 0.5-8 µg. Outcomes suggested that formazan was retained in the solid residue of leaf tissues. This protocol allowed us to quantify superoxide radicals produced under different stress conditions. Chloroform allowed a selective formazan extraction and removal of potential endogenous, exogenous or procedural artefacts that may interfere with the quantitative determination. This protocol can be used to quantify the superoxide produced in plant tissues using any traditional qualitative NBT histochemical staining method. Copyright © 2011 John Wiley & Sons, Ltd.

Rapid contrast evaluation method based on affinity beads and backscattered electron imaging for the screening of electron stains.

PubMed

Kaku, Hiroki; Inoue, Kanako; Muranaka, Yoshinori; Park, Pyoyun; Ikeda, Kenichi

2015-10-01

Uranyl salts are toxic and radioactive; therefore, several studies have been conducted to screen for substitutes of electron stains. In this regard, the contrast evaluation process is time consuming and the results obtained are inconsistent. In this study, we developed a novel contrast evaluation method using affinity beads and a backscattered electron image (BSEI), obtained using scanning electron microscopy. The contrast ratios of BSEI in each electron stain treatment were correlated with those of transmission electron microscopic images. The affinity beads bound to cell components independently. Protein and DNA samples were enhanced by image contrast treated with electron stains; however, this was not observed for sugars. Protein-conjugated beads showed an additive effect of image contrast when double-stained with lead. However, additive effect of double staining was not observed in DNA-conjugated beads. The varying chemical properties of oligopeptides showed differences in image contrast when treated with each electron stain. This BSEI-based evaluation method not only enables screening for alternate electron stains, but also helps analyze the underlying mechanisms of electron staining of cellular structures. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

tuberculous otitis media in a renal transplant recipient.

PubMed

Ergün, Ihsan; Keven, Kenan; Sengül, Sule; Kutlay, Sim; Sertcelik, Ayse; Ertürk, Sehsuvar; Erbay, Bülent

2004-06-01

Tuberculous otitis media is a rare cause of chronic suppurative infection of the middle ear and a very uncommon form of extrapulmonary tuberculosis. Although there have been several case reports in the nonimmunosuppressive population of tuberculous otitis media, it has never been reported in an immunosuppressed allograft recipient. We present a case of diagnosed tuberculous otitis media after recurrent chronic otitis media treated several times with empiric antibiotic treatment. After the patient developed postauricular fistula and underwent surgical removal of granulation tissue, the diagnosis was made on the basis of histopathology and growth in culture of Ziehl-Neelsen. Clinical response promptly followed institution of antituberculous treatment including isoniazid, rifampicin, ethambutol, and pyrazinamide.

Endothelium adhesion molecules ICAM-1, ICAM-2, VCAM-1 and VLA-4 expression in leprosy.

PubMed

de Sousa, Juarez; Sousa Aarão, Tinara Leila; Rodrigues de Sousa, Jorge; Hirai, Kelly Emi; Silva, Luciana Mota; Dias, Leonidas Braga; Oliveira Carneiro, Francisca Regina; Fuzii, Hellen Thais; Quaresma, Juarez Antonio Simões

2017-03-01

Leprosy triggers a complex relationship between the pathogen and host immune response. Endothelium plays an important role in this immune response by directly influencing cell migration to infected tissues. The objective of this work is to investigate the possible role of endothelium in M. leprae infection, correlating the characteristics of endothelial markers with the expression pattern of cytokines. Thirty-six skin biopsy samples were cut into 5-μm thick sections and stained with hematoxylin-eosin and Ziehl-Neelsen for morphological analysis and then submitted to immunohistochemical analysis using monoclonal antibodies against ICAM-1, ICAM-2, VCAM-1, and VLA-4. Immunostaining for ICAM-1 showed a significantly larger number of stained endothelial cells in the tuberculoid leprosy (9.92 ± 1.11 cells/mm 2 ) when compared to lepromatous samples (5.87 ± 1.01 cells/mm 2 ) and ICAM-2 revealed no significant difference in the number of endothelial cells expressing this marker between the tuberculoid (13.21 ± 1.27 cells/mm 2 ) and lepromatous leprosy (14.3 ± 1.02 cells/mm 2 ). VCAM-1-immunostained showed 18.28 ± 1.46/mm 2 cells in tuberculoid leprosy and 10.67 ± 1.25 cells/mm 2 in the lepromatous leprosy. VLA-4 exhibited 22.46 ± 1.38 cells/mm 2 in the tuberculoid leprosy 16.04 ± 1.56 cells/mm 2 in the lepromatous leprosy. Samples with characteristics of the tuberculoid leprosy exhibited a larger number of cells stained with ICAM-1, VCAM-1 and VLA-4, demonstrating the importance of these molecules in the migration and selection of cells that reach the inflamed tissue. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparison of Histochemical Staining Methods and Correlation with Transient Elastography in Acute Hepatitis.

PubMed

Cabibi, Daniela; Calvaruso, Vincenza; Giuffrida, Letizia; Ingrao, Sabrina; Balsamo, Laura; Giannone, Antonino Giulio; Petta, Salvatore; Di Marco, Vito

2015-03-06

To compare Masson's trichrome (MT), Sirius red (SR) and orcein staining in acute hepatitis (AH) and to correlate them with transient elastography (TE), a noninvasive method to assess hepatic fibrosis. We evaluated liver stiffness by TE in a cohort of 34 consecutive patients and assessed MT-, SR- and orcein-stained biopsies using the METAVIR scoring system and digital image analysis (DIA). MT and SR both showed severe fibrosis (stage III-IV, DIA = 12.7%). Orcein showed absent or mild fibrosis (stage 0-II, DIA = 4.4%; p < 0.05). In 29/34 cases (85%), stiffness values were >12.5 kPa, in keeping with SR/MT but not with orcein results. Even though in AH true elastic fibrosis is typically absent or mild, TE shows elevated stiffness values, in keeping with SR/MT evaluations. If not properly evaluated in the clinical context, these results would lead to an overestimation of fibrosis. Orcein is the only staining able to evidence the absence of true elastic fibrosis, which is a typical feature of AH. This is the first study comparing different staining procedures performed on AH biopsies by DIA versus TE. © 2015 S. Karger AG, Basel.

Novel method for immunofluorescence staining of mammalian eggs using non-contact alternating-current electric-field mixing of microdroplets

PubMed Central

Hiromitsu, Shirasawa; Jin, Kumagai; Emiko, Sato; Katsuya, Kabashima; Yukiyo, Kumazawa; Wataru, Sato; Hiroshi, Miura; Ryuta, Nakamura; Hiroshi, Nanjo; Yoshihiro, Minamiya; Yoichi, Akagami; Yukihiro, Terada

2015-01-01

Recently, a new technique was developed for non-catalytically mixing microdroplets. In this method, an alternating-current (AC) electric field is used to promote the antigen–antibody reaction within the microdroplet. Previously, this technique has only been applied to histological examinations of flat structures, such as surgical specimens. In this study, we applied this technique for the first time to immunofluorescence staining of three-dimensional structures, specifically, mammalian eggs. We diluted an antibody against microtubules from 1:1,000 to 1:16,000, and compared the chromatic degree and extent of fading across dilutions. In addition, we varied the frequency of AC electric-field mixing from 5 Hz to 46 Hz and evaluated the effect on microtubule staining. Microtubules were more strongly stained after AC electric-field mixing for only 5 minutes, even when the concentration of primary antibody was 10 times lower than in conventional methods. AC electric-field mixing also alleviated microtubule fading. At all frequencies tested, AC electric-field mixing resulted in stronger microtubule staining than in controls. There was no clear difference in a microtubule staining between frequencies. These results suggest that the novel method could reduce antibody consumption and shorten immunofluorescence staining time. PMID:26477850

Clinical and Cytological Spectrum of Granulomatous Mastitis and Utility of FNAC in Picking up Tubercular Mastitis: An Eight-Year Study

PubMed Central

Bhayekar, Pallavi; Joshi, Avinash; Pandya, Nidhi; Nasare, Anuja; Lengare, Pranoti; Narkhede, Ketan Ashok

2017-01-01

Introduction Granulomatous Mastitis (GM) is a rare, benign, inflammatory disease of the breast. It is a well known mimicker of malignancy, clinically and radiologically. Patients are often subjected to number of tests for the right diagnosis. Non-specific Granulomatous Mastitis (NGM) and Tubercular Mastitis (TBM) are chief among the various causes of GM. They are important to be diagnosed early as their treatment varies significantly. Fine Needle Aspiration Cytology (FNAC) is simple, patient friendly and primary investigation modality in cases of lump in breast. Aim To find out the utility of FNAC in differentiating NGM and TBM. Materials and Methods All cases of granulomatous mastitis diagnosed on cytology over eight years were retrospectively retrieved. The clinical and radiological history was obtained from the patient file. The slides were stained with haematoxylin and eosin stain as well as Leishman stains. Special stains like Periodic Acid Schiff (PAS) and Ziehl Neelsen (ZN) stain were used for fungus and Mycobacterium tuberculosis respectively. Histopathological correlation of the available cases was done. Clinical presentation and cytological morphology of individual cases was studied in detail. Results Twenty one cases of GM obtained, of which 16 were NGM and five were TBM. Both diseases were common among young reproductive women who presented with unilateral breast lump of varying duration. Almost 25% of NGM and 60% of TBM has clinical suspicion of malignancy. About 30% had radiological suspicion of malignancy. Nearly 62.5% of NGM patients had painful swelling and none of tubercular mastitis patients had pain. About 31% of NGM patients underwent prior abscess drainage and 40% of TBM patients gave history of tuberculosis. Almost 6.25% of NGM and 60% of TBM had axillary lymphadenopathy. Cytologically epithelioid cells were identified in 100% of patients whereas, granulomas were seen in 62.5% and 80% of NGM and TBM smears respectively. Langhans giant cells

Differential staining of bacteria: gram stain.

PubMed

Moyes, Rita B; Reynolds, Jackie; Breakwell, Donald P

2009-11-01

In 1884, Hans Christian Gram, a Danish doctor, developed a differential staining technique that is still the cornerstone of bacterial identification and taxonomic division. This multistep, sequential staining protocol separates bacteria into four groups based on cell morphology and cell wall structure: Gram-positive cocci, Gram-negative cocci, Gram-positive rods, and Gram-negative rods. The Gram stain is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures. (c) 2009 by John Wiley & Sons, Inc.

Quantification of histochemical stains using whole slide imaging: development of a method and demonstration of its usefulness in laboratory quality control.

PubMed

Gray, Allan; Wright, Alex; Jackson, Pete; Hale, Mike; Treanor, Darren

2015-03-01

Histochemical staining of tissue is a fundamental technique in tissue diagnosis and research, but it suffers from significant variability. Efforts to address this include laboratory quality controls and quality assurance schemes, but these rely on subjective interpretation of stain quality, are laborious and have low reproducibility. We aimed (1) to develop a method for histochemical stain quantification using whole slide imaging and image analysis and (2) to demonstrate its usefulness in measuring staining variation. A method to quantify the individual stain components of histochemical stains on virtual slides was developed. It was evaluated for repeatability and reproducibility, then applied to control sections of an appendix to quantify H&E staining (H/E intensities and H:E ratio) between automated staining machines and to measure differences between six regional diagnostic laboratories. The method was validated with <0.5% variation in H:E ratio measurement when using the same scanner for a batch of slides (ie, it was repeatable) but was not highly reproducible between scanners or over time, where variation of 7% was found. Application of the method showed H:E ratios between three staining machines varied from 0.69 to 0.93, H:E ratio variation over time was observed. Interlaboratory comparison demonstrated differences in H:E ratio between regional laboratories from 0.57 to 0.89. A simple method using whole slide imaging can be used to quantify and compare histochemical staining. This method could be deployed in routine quality assurance and quality control. Work is needed on whole slide imaging devices to improve reproducibility. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

The comparison of pyrosequencing molecular Gram stain, culture, and conventional Gram stain for diagnosing orthopaedic infections.

PubMed

Kobayashi, Naomi; Bauer, Thomas W; Tuohy, Marion J; Lieberman, Isador H; Krebs, Viktor; Togawa, Daisuke; Fujishiro, Takaaki; Procop, Gary W

2006-08-01

We have developed a combined real-time PCR and pyrosequencing assay that successfully differentiated the vast majority of gram-positive and gram-negative bacteria when bacterial isolates were tested. The purpose of this study was to evaluate this assay on clinical specimens obtained from orthopedic surgeries, and to prospectively compare the results of "molecular Gram stain" with culture and conventional direct Gram stain. Forty-five surgical specimens were obtained from patients who underwent orthopedic surgery procedures. The DNA was extracted and a set of broad-range PCR primers that targeted a part of the 16S rDNA gene was used for pan-bacterial PCR. The amplicons were submitted for pyrosequencing and the resulting molecular Gram stain characteristics were recorded. Culture and direct Gram staining were performed using standard methods for all cases. Surgical specimens were reviewed histologically for all cases that had a discrepancy between culture and molecular results. There was an 86.7% (39/45) agreement between the traditional and molecular methods. In 12/14 (85.7%) culture-proven cases of bacterial infection, molecular Gram stain characteristics were in agreement with the culture results, while the conventional Gram stain result was in agreement only for five cases (35.7%). In the 31 culture negative cases, 27 cases were also PCR negative, whereas 4 were PCR positive. Three of these were characterized as gram negative and one as gram positive by this molecular method. Molecular determination of the Gram stain characteristics of bacteria that cause orthopedic infections may be achieved, in most instances, by this method. Further studies are necessary to understand the clinical importance of PCR-positive/culture-negative results.

Demonstration of lipofuscin and Nissl bodies in crystal violet stained sections using a fluorescence technique or pyronin Y stain.

PubMed

Terr, L I

1986-09-01

This paper presents two simple, reliable methods for identification of lipofuscin and Nissl bodies in the same section. One method shows that lipofuscin stained with crystal violet retains its ability to fluoresce and can be observed under the fluorescence microscope after the stain has faded. Fading is accompanied by a gradual increase in the intensity of the fluorescence and is complete in about 5 min. Exciting illumination from this part of the spectrum also substantially fades staining of other autofluorescing tissue elements, such as lipids. Nonfluorescing structures, such as Nissl bodies, remain stained. By changing from transillumination with tungsten light to epifluorescent illumination and vice versa, both types of structures--Nissl bodies and lipofuscin--can be identified in the same section. The second technique uses pyronin Y for staining Nissl bodies in preparations previously stained with crystal violet. Nissl bodies are stained pink but lipofuscin remains violet. Lipofuscin in these sections also remains autofluorescent after the crystal violet stain has faded under violet or near-UV light.

Development and validation of a triplex real-time PCR for rapid detection and specific identification of M. avium sub sp. paratuberculosis in faecal samples.

PubMed

Irenge, Léonid M; Walravens, Karl; Govaerts, Marc; Godfroid, Jacques; Rosseels, Valérie; Huygen, Kris; Gala, Jean-Luc

2009-04-14

A triplex real-time (TRT-PCR) assay was developed to ensure a rapid and reliable detection of Mycobacterium avium subsp. paratuberculosis (Map) in faecal samples and to allow routine detection of Map in farmed livestock and wildlife species. The TRT-PCR assay was designed using IS900, ISMAP02 and f57 molecular targets. Specificity of TRT-PCR was first confirmed on a panel of control mycobacterial Map and non-Map strains and on faecal samples from Map-negative cows (n=35) and from Map-positive cows (n=20). The TRT-PCR assay was compared to direct examination after Ziehl-Neelsen (ZN) staining and to culture on 197 faecal samples collected serially from five calves experimentally exposed to Map over a 3-year period during the sub-clinical phase of the disease. The data showed a good agreement between culture and TRT-PCR (kappa score=0.63), with the TRT-PCR limit of detection of 2.5 x 10(2)microorganisms/g of faeces spiked with Map. ZN agreement with TRT-PCR was not good (kappa=0.02). Sequence analysis of IS900 amplicons from three single IS900 positive samples confirmed the true Map positivity of the samples. Highly specific IS900 amplification suggests therefore that each single IS900 positive sample from experimentally exposed animals was a true Map-positive specimen. In this controlled experimental setting, the TRT-PCT was rapid, specific and displayed a very high sensitivity for Map detection in faecal samples compared to conventional methods.

Performance of Xpert MTB/RIF on Ascitic Fluid Samples for Detection of Abdominal Tuberculosis.

PubMed

Rufai, Syed Beenish; Singh, Sarman; Singh, Amit; Kumar, Parveen; Singh, Jitendra; Vishal, Anand

2017-01-01

Diagnosis of abdominal tuberculosis (TB) from ascitic fluid samples using routinely available diagnostic methods is challenging due to its paucibacillary nature. Although performance of Xpert MTB/RIF assay has been evaluated extensively on pulmonary samples, its performance on extrapulmonary samples is still under evaluation. The objective of this study was to find out the performance of Xpert MTB/RIF on ascitic fluid samples obtained from suspected cases of abdominal TB. Performance was compared with Mycobacterium growth indicator tube-960 (MGIT-960) culture and in-house multiplex polymerase chain reaction (PCR). The latter detects and differentiates Mycobacterium tuberculosis and nontuberculous mycobacteria simultaneously. Sixty-seven patients suspected of probable/possible abdominal TB were included in this observational, prospective study. All samples were tested by Ziehl-Neelsen staining, MGIT-960 culture, in-house multiplex PCR, and Xpert MTB/RIF assay. All 67 samples were smear negative. Seventeen (25.4%) were MGIT-960 culture positive while 12 (17.9%) were detected positive by the Xpert MTB/RIF assay and 9 (13.4%) by in-house multiplex PCR. Sensitivity and specificity of the Xpert MTB/RIF assay compared with the MGIT-960 culture were 70.6% (95%, confidence interval [CI]: 44.1-89.7) and 100% (95%, CI: 92.8-100) and that of in-house multiplex PCR were 52.9% (95%, CI: 30.9-73.8) and 100% (95%, CI: 92.8-100), respectively. Diagnostic yield of Xpert MTB/RIF assay on ascitic fluid samples was lower than MGIT-960 culture. We thus emphasize on the need for urgent discovery of new biomarkers for paucibacillary TB.

The Diagnostic Utility of Bact/ALERT and Nested PCR in the Diagnosis of Tuberculous Meningitis.

PubMed

Sastry, Apurba Sankar; Bhat K, Sandhya; Kumudavathi

2013-01-01

The early laboratory diagnosis of Tuberculous Meningitis (TBM) is crucial, to start the antitubercular chemotherapy and to prevent its complications. However, the conventional methods are either less sensitive or time consuming. Hence, the diagnostic potentials of BacT/ALERT and Polymerase Chain Reaction (PCR) was evaluated in this study. The study group comprised of 62 cases and 33 controls. The cases were divided according to Ahuja's criteria into the confirmed (two cases), highly probable (19 cases), probable (26 cases) and the possible (15 cases) subgroups. Ziehl Neelsen's (ZN) and Auramine Phenol (AP) staining, Lowenstein Jensen (LJ) medium culture, BacT/ALERT and nested Polymerase Chain Reaction (PCR) which targeted IS6110 were carried out on all the patients. The sensitivity of the LJ culture was 3.22%. BacT/ALERT showed a sensitivity and a specificity of 25.80% and 100% and those of nested PCR were found to be 40.32% and 96.97% respectively. The mean detection time of growth of the LJ culture was 31.28 days, whereas that of BacT/ALERT was 20.68 days. The contamination rate in the LJ culture and BacT/ALERT were 7.2% and 5.8% respectively. Nested PCR was found to be more sensitive, followed by BacT/ALERT as compared to the LJ culture and smear microscopy. As both false negative and false positive results have been reported for nested PCR, so it should not be used alone as a criterion for initiating or terminating the therapy, but it should be supported by clinical, radiological, cytological and other microbiological findings.

Zoonotic intestinal parasites in Papio anubis (baboon) and Cercopithecus aethiops (vervet) from four localities in Ethiopia.

PubMed

Legesse, Mengistu; Erko, Berhanu

2004-05-01

A total of 59 faecal samples from ranging Papio anubis (baboons) and another 41 from Cercopithecus aethiops (vervet) from the Rift Valley areas of Ethiopia were microscopically examined to determine the prevalence and species of major gastro-intestinal parasites of zoonotic importance. Faecal smears were prepared from fresh faecal samples, stained using modified Ziehl-Neelsen method and microscopically examined. About 3 gm of the dropping was also preserved separately in clean and properly labelled containers containing 10% formalin. The specimens were microscopically examined after formalin-ether concentration for ova, larvae, cysts and oocyst of intestinal parasites. The results of microscopic examination of faecal samples of baboons demonstrated the presence of Trichuris sp. (27.1%), Strongyloides sp. (37.3%), Trichostrongylus sp. (8.5%), Oesophagostomum sp. (10.2%), Schistosoma mansoni (20.3%), Entamoeba coli (83.1%), Entamoeba histolytica/dispar (16.9%), Blastocystis hominis (3.3%), Cyclospora sp. (13.3%) and Cryptosporidium sp. (11.9%). Likewise, the results of microscopic examination of faecal samples of vervets demonstrated the presence of Trichuris sp. (36.6%), Oesophagostomum sp. (4.9%), E. coli (61.0%), E. histolytica/dispar (24.4%), B. hominis (34.2%), Cyclospora sp. (22.0%) and Cryptosporidium sp. (29.3%). The presence of parasitic protozoa and helminths in baboons and vervets in the study areas is a high risk to human welfare because these non-human primates use the same water sources as humans and range freely in human habitats. An implication of such parasitic infection for the control programme is discussed.

Benefits and limitations of fine needle aspiration cytology in the diagnosis and classification of leprosy in primary and secondary healthcare settings.

PubMed

Ray, R; Mondal, R K; Pathak, S

2015-08-01

The goal of the World Health Organization (WHO) is to eliminate leprosy as a public health problem. This will only be possible when all patients are detected and cured using multidrug therapy, which requires accurate diagnosis prior to treatment. The objective of this study was to evaluate the possibility of the diagnosis of leprosy lesions by fine needle aspiration cytology according to a modification of the Ridley-Jopling scale, as it can be used in primary and secondary healthcare centres, especially in low-resource settings in which leprosy is prevalent. A prospective study comprising 54 cases with cardinal features of leprosy was performed. Among the 54 cases, 27 patients consented to a histopathological biopsy procedure. The slides were stained with Giemsa, modified Ziehl-Neelsen, Papanicolaou and haematoxylin and eosin methods. Among the 54 cases, 34 were reported as tuberculoid leprosy, five as mid-borderline (BB), three as borderline lepromatous (BL) and eight as lepromatous leprosy (LL); four were unsatisfactory. Histopathological study was performed in 27 cases, which showed cyto-histological correlation in 21 cases (78%). Agreement between histological and cytological diagnosis was achieved in 12 of the 15 tuberculoid cases, one of the three BB cases, one of the two BL cases and all seven LL cases. With the implementation of the WHO classification based on patch counting, there is the possibility of the over-treatment of paucibacillary cases and under-treatment of multibacillary cases. Cytology in terms of cellular type morphology and bacteriological study can complement the WHO classification. © 2014 John Wiley & Sons Ltd.

An audit of the laboratory diagnosis of cryptosporidiosis in England and Wales.

PubMed

Chalmers, Rachel M; Atchison, Christina; Barlow, Katrina; Young, Yvonne; Roche, Anita; Manuel, Rohini

2015-07-01

To assess the level of practice consistent with UK national standards for Cryptosporidium testing, an audit was performed of 156 publicly funded clinical microbiology laboratories in England and Wales between August 2013 and April 2014. Responses were received from 85 (54 %) laboratories. First line diagnostic methods used were mainly microscopy with modified Ziehl-Neelsen (mZN) or auramine phenol (AP) staining (68/85, 80 %), enzyme immunoassays (EIAs) (16/85, 19 %) or in-house PCR (1/85, 1 %). The use of EIAs was more widespread than reported previously. Various methods were used for confirmation of positive EIA reactions and laboratories frequently resorted to sending samples to the national reference laboratory for this purpose, indicating that guidance is required for performance monitoring and confirmation of positive reactions. Laboratory positivity rates were related to the diagnostic test used, with highest median rates reported by those using PCR, EIAs or AP microscopy, and the lowest by those using mZN microscopy. One-third of responding laboratories (28/85, 33 %) routinely tested all stools for Cryptosporidium. However, 16 (19 %) laboratories used stool consistency to decide whether to test for this parasite. Other selection criteria included patient age (n = 18; 21 % laboratories), history or clinical details (n = 40; 47 %), duration of hospitalization (n = 18; 21 %) or clinician requests (n = 25; 29 %). To encourage laboratories to test all stools submitted for the investigation of diarrhoeal illness for Cryptosporidium, revision of the guidance in the national standards is under way. This will enable improved assessment of the burden of illness and ability to monitor outbreaks, and measure changes in reported cases.

Short Nissl staining for incubated cryostat sections of the brain.

PubMed

Lindroos, O F

1991-01-01

Nissl stain often binds poorly to cryostat sections which have been incubated in solutions of radiolabeled ligands. Such incubation is used in receptor autoradiography of the brain when using the in vitro method. We have developed a rapid (16 min) modification of Nissl staining for sections that bind stain poorly, e.g., incubated sections. The method stains well sections which cannot be stained with other rapid Nissl staining methods.

Determination of ABO blood grouping from human oral squamous epithelium by the highly sensitive immunohistochemical staining method EnVision+.

PubMed

Noda, Hiroshi; Yokota, Makoto; Tatsumi, Shinji; Sugiyama, Shizuyuki

2002-03-01

Using the highly sensitive immunohistochemical staining method EnVision+, which employs a dextran polymer reagent for the secondary antibody, the detection of the ABH antigens was attempted in the oral squamous epithelium. This new technique uses monoclonal antibody as a primary antibody and it takes about three hours for staining. The time is much shorter than conventional absorption-elution testing or absorption-inhibition testing for the determination of ABO blood grouping. Secretor saliva samples were stained at strong intensity by the antibody, which corresponded to its blood group and anti-H. On the one hand, nonsecretor saliva samples were stained at strong intensity only by the antibody that corresponded to its blood group, and at weak intensity only by anti-H. Since human oral squamous epithelium antigens were stained specifically by this method, we can examine the ABO blood group of saliva samples and perform cytodiagnosis at the same time. Our research suggested that the EnVision+ Method is a useful technique for ABO blood grouping of saliva in forensic cases.

Pathology and diagnosis of Mycobacterium bovis in naturally infected dromedary camels (Camelus dromedarius) in India.

PubMed

Narnaware, Shirish Dadarao; Dahiya, Shyam Singh; Tuteja, Fateh Chand; Nagarajan, Govindasamy; Nath, Kashi; Patil, Nitin Vasantrao

2015-12-01

The present study investigated the pathological features of tuberculosis (TB) caused by Mycobacterium bovis and its diagnosis in naturally infected dromedary camels from an organized farm in India. During the period of the 5-year study, a total of 18 (19.56 %) camels out of 92 examined showed gross lesions compatible with TB at post-mortem. The clinical signs and pathological lesions in these camels were studied, and the efficacy of different diagnostic tests was also assessed. On the basis of occurrence and distribution of gross TB lesions, the infected camels revealed two different lesional patterns as pulmonary (n = 15) and disseminated (n = 3) form. The histopathology of affected organs revealed typical granulomatous lesions wherein the giant cells and acid-fast bacilli were occasionally observed in pulmonary form whereas they frequently observed in disseminated form. The single intradermal tuberculin test (SIDT) detected TB in 10 (55.55 %) whereas the Ziehl-Neelsen (ZN) stain and IS6110 PCR from tissue lesions detected 13 (72.22 %) and 18 (100 %) of the infected camels, respectively. The study suggests that pulmonary form of the TB is more common in camels indicating respiratory route as the major source of exposure in camel herds. Moreover, very low sensitivity of SIDT was observed which highlights the difficulty for confirmation of TB in live camels.

Prevalence of tuberculous lymphadenitis in slaughtered cattle in Eastern Cape, South Africa.

PubMed

Bhembe, Nolwazi L; Jaja, Ishmael F; Nwodo, Uchechukwu U; Okoh, Anthony I; Green, Ezekiel

2017-08-01

To detect the prevalence of Mycobacterium tuberculosis complex (MTBC) in the lymph nodes of slaughtered cattle collected from selected abattoirs in Eastern Cape Province of South Africa. A total of 376 lymph nodes were collected from slaughtered cattle over a period of 12 months. Certain characteristics (sex, age, body condition score, and breed) were observed to be associated with MTBC among slaughtered cattle. Collected samples were cultured and tested for acid-fast bacilli (AFB). DNA was isolated, purified, and quantified using a spectrophotometer. Quantified DNA was confirmed to be MTBC by multiplex PCR targeting two genes (IS6110 and mpb64). Of the 376 collected lymph nodes, 182 were positive when tested by Ziehl-Neelsen stain and 162 were confirmed positive for MTBC by PCR. MTBC was isolated from lymph nodes with nodular lesions (72.8%, 118/162) and inflamed lymph nodes (27.1%, 44/162). All detected MTBC isolates were positive for region of deletion 1 (RD1). No isolate was detected to have Mycobacterium bovis bacille Calmette-Guérin (BCG). However, 3.1% had M. bovis and 96.9% had M. tuberculosis. The presence of live Mycobacterium strains in slaughtered cattle poses a health risk to beef consumers and abattoir workers. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Opportunistic and other intestinal parasites among HIV/AIDS patients attending Gambi higher clinic in Bahir Dar city, North West Ethiopia.

PubMed

Alemu, Abebe; Shiferaw, Yitayal; Getnet, Gebeyaw; Yalew, Aregaw; Addis, Zelalem

2011-08-01

To determine the magnitude of opportunistic and non-opportunistic intestinal parasitic infections among HIV/AIDS patients in Bahir Dar. Cross-sectional study was conducted among HIV/AIDS patients attending Gambi higher clinic from April1-May 30, 2009. Convenient sampling technique was employed to identify the study subjects and hence a total of 248 subjects were included. A pre-tested structured questionnaire was used to collect socio-demographic data of patients. Stool samples were examined by direct saline, iodine wet mount, formol-ether sedimentation concentration and modified Ziehl-Neelsen staining technique. Out of 248 enrolled in the study, 171(69.0%) (90 males and 81 females) were infected with one or more intestinal parasites. The highest rate of intestinal parasites were observed among HIV/AIDS patients (80.3%, 151/188), and the infection rate of HIV negative individuals was 33.3% (20/60). Cryptosporidum parvum (43.6%), Isospora belli (15.5%) and Blastocystis hominis (10.5%) were opportunistic parasites that were found only in HIV/AIDS patients. Opportunistic parasite infections are common health problem among HIV/AIDS patients in the study area. Therefore, early detection and treatment of these parasites are important to improve the quality of life of HIV/AIDS patients. Copyright © 2011 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

Liposarcoma in a Backyard Silkie and Retrospective Summary of Neoplasms Diagnosed in Backyard Chickens Submitted to the California Animal Health and Food Safety Laboratory System, 2008-2017.

PubMed

Blakey, Julia; Crispo, Manuela; Bickford, Arthur; Stoute, Simone

2018-03-01

Liposarcomas are a malignant neoplasm of adipocytes, and are rarely diagnosed in avian species. This case report describes the evidence supporting a diagnosis of metastatic liposarcoma in a backyard silkie chicken. On September 28, 2017, a dead 3-yr-old backyard silkie chicken, with a history of unknown skin lesions involving the entire body and severe weight loss, was submitted to California Animal Health and Food Safety Laboratory System-Turlock branch for necropsy. At necropsy, raised necrotic lesions involving the majority of the skin and multiple nodules in the liver, spleen, and bone marrow were noticed. Microscopically, stellate, spindle, and myxoid cells containing large vacuoles, which were confirmed as lipid droplets by Oil Red O, were observed infiltrating the dermis and underlying a necrotic epidermis, with metastasis to liver, spleen, bone marrow, and ovary being the most significant findings. PAS, Oil Red O, Ziehl-Neelsen, Congo red, Gram, and Von Kossa stains, along with immunohistochemistry for pan cytokeratin, vimentin, S100, CD3, pp38, and Meq were used to classify the lesions. Intensely positive vimentin immunohistochemistry, along with large quantities of Oil Red O-positive lipid droplets within the neoplastic cells, were supportive of our diagnosis of liposarcoma. The incidence of neoplastic diseases diagnosed in backyard flock submissions to CAHFS system wide from 2008 to 2017 was also reviewed.

Development of Antigen Detection Assay for Diagnosis of Tuberculosis Using Sputum Samples

PubMed Central

Pereira Arias-Bouda, Lenka M.; Nguyen, Lan N.; Ho, Ly M.; Kuijper, Sjoukje; Jansen, Henk M.; Kolk, Arend H. J.

2000-01-01

The rising incidence of tuberculosis worldwide means an increasing burden on diagnostic facilities, so tests simpler than Ziehl-Neelsen staining are needed. Such tests should be objective, reproducible, and have at least as good a detection limit as 104 bacteria/ml. A capture enzyme-linked immunosorbent assay (ELISA) was developed for detection of lipoarabinomannan (LAM) in human sputum samples. As a capture antibody, we used a murine monoclonal antibody against LAM, with rabbit antiserum against Mycobacterium tuberculosis as a source of detector antibodies. The sensitivity of the capture ELISA was evaluated by using purified LAM and M. tuberculosis whole cells. We were able to detect 1 ng of purified LAM/ml and 104 M. tuberculosis whole cells/ml. LAM could also be detected in culture filtrate of a 3-week-old culture of M. tuberculosis. The culture filtrate contained approximately 100 μg of LAM/ml. The detection limit in sputum pretreated with N-acetyl-l-cysteine and proteinase K was 104 M. tuberculosis whole cells per ml. Thirty-one (91%) of 34 sputum samples from 18 Vietnamese patients with tuberculosis (32 smear positive and 2 smear negative) were positive in the LAM detection assay. In contrast, none of the 25 sputum samples from 21 nontuberculous patients was positive. This specific and sensitive assay for the detection of LAM in sputum is potentially useful for the diagnosis of tuberculosis. PMID:10834989

Prevalence and spectrum of Johne's disease lesions in cattle slaughtered at two abattoirs in Kampala, Uganda.

PubMed

Okuni, Julius Boniface; Reinacher, Manfred; Loukopoulos, Panayiotis; Ojok, Lonzy

2013-06-01

This study was conducted to determine the prevalence and characteristics of Johne's disease (JD) lesions in Ugandan cattle slaughtered at two of the main abattoirs in Kampala. Ileocaecal junction and the associated lymph nodes of 1,022 cattle were examined for gross and microscopic lesions, followed by Ziehl Neelsen staining of the tissues bearing lesions. Confirmation of Mycobacterium avium subsp. paratuberculosis infection was done in some of the tissues using culture and IS900 PCR. The lesions were then described, characterised and tabulated. Characteristic Johne's disease granulomas were found in 4.7% of the samples examined, derived from Zebu, Ankole longhorn, Friesian breeds of cattle and their crosses. Lesions were found both in the lymph nodes and ileocaecal junction mucosa. The lesions tended to be more severe in the lymph node than in the mucosa. There were also some unique and atypical lesions found in association with Johne's disease granulomas. The diagnostic values of various gross lesions and criteria of lesion classifications and diagnosis are revisited and discussed based on the findings of this study. The prevalence of Johne's disease lesions among slaughtered cattle in Kampala's two abattoirs indicates that the disease is well established in the cattle population in the country. The diverse manifestations in lesions of JD need to be considered when making histological diagnosis in tissues where the disease is suspected.

Occurrence of Intestinal Parasitic Contamination in Select Consumed Local Raw Vegetables and Fruits in Kuantan, Pahang

PubMed Central

Yusof, Afzan Mat; Mohammad, Mardhiah; Abdullahi, Muna Abshir; Mohamed, Zeehaida; Zakaria, Robaiza; Wahab, Ridhwan Abdul

2017-01-01

Intestinal parasitic infections are one of the most common causes of human diseases that result in serious health and economic issues in many developing and developed countries. Raw vegetables and fruits play an important role in transmitting parasites to humans. Hence, the aim of this study was to investigate the parasitological contamination of select commonly consumed local leafy vegetables and fruits in Kuantan, Malaysia. One kilogram of locally consumed raw vegetables and fruits were collected randomly from the Kuantan wet market (Pasar Tani) during the monsoon season (November 2014–January 2015) and the dry season (February 2015–April 2015). A standard wet mount procedure and modified Ziehl-Neelsen staining were used for the detection of parasites. In the present study, the examination of vegetables revealed five different parasite species. The vegetable samples collected from Kuantan’s wet market were positive for both helminthes and protozoa. However, the fruits samples were negative for parasitic contamination. Pegaga was the most contaminated leafy vegetable in this study, and Strongyloides was the parasite found most frequently. Furthermore, there was a high diversity in the type of parasites observed during the dry season compared to the monsoon season. Therefore, further action should be taken to reduce the occurrence of parasitic contamination in vegetables by implementing the principles of good agricultural practice and improving water treatment efficacy. PMID:28228914

Comparison of methylene blue/gentian violet stain to Gram's stain for the rapid diagnosis of gonococcal urethritis in men.

PubMed

Taylor, Stephanie N; DiCarlo, Richard P; Martin, David H

2011-11-01

We compared a simple, one-step staining procedure using a mixture of methylene blue and gentian violet to Gram stain for the detection of gonococcal urethritis. The sensitivity and specificity of both Gram stain and methylene blue/gentian violet stain were 97.3% and 99.6%, respectively. There was a 100% correlation between the 2 methods.

Selection and application of exterior stains for wood

Treesearch

R. Sam Williams; William C. Feist

1999-01-01

Exterior stains for wood protect the wood surface from sunlight and moisture. Because stains are formulated to penetrate the wood surface, they are not prone to crack or peel as can film-forming finishes, such as paints. This publication describes the properties of stains and wood, methods for applying stains, and the expected service life of stains.

New staining methods for yeast like fungi under special consideration of human pathogenic fungi

NASA Astrophysics Data System (ADS)

Paulitsch-Fuchs, Astrid; Treiber, Fritz; Grasser, Erik; Buzina, Walter; Rosker, Christian

2010-11-01

A new method for in-cellular staining of yeast like fungi with Oregon Green and SYTOX Green is presented enabling their detection as well as the observation of cellular details via confocal laser scanning microscopy. Fluorochromes play an important role in many scientific disciplines including medicine, cell biology and botany. For the visualisation of fungal cell walls Calcofluor White is the flourochrome of choice. The necessity of an UV laser for its excitation makes it unpracticable for daily use. Safranin O, DAPI, 2NBDG, Ethidium Bromide and Acridin-orange are commonly used stains for nuclei in fugal microscopy. The attention was given to the possibility of using the differences in staining patterns to distinguish certain pathogenic yeast species e.g. Candida albicans and Candida krusei. Our results show that high quality microscopy of yeast like organisms can readily be achieved by the use of two suitable fluorochromes.

Effect of fabric mounting method and backing material on bloodstain patterns of drip stains on textiles.

PubMed

Chang, J Y M; Michielsen, S

2016-05-01

Textiles may provide valuable bloodstain evidence to help piece together events or activities at violent crime scenes. However, in spite of over 75 years of research, there are still difficulties encountered in many cases in the interpretation and identification of bloodstains on textiles. In this study, we dripped porcine blood onto three types of fabric (plain woven, single jersey knit, and denim) that are supported in four different ways (hard, taut, loose, and semi-hard, i.e., fabric laid on denim). These four mounting methods represent different ways in which a textile may be present when blood from a violent act lands on it. This study investigates how the fabric mounting method and backing material affect the appearance of drip stains on textiles. We found that bloodstain patterns formed on fabric lying flat on a hard surface were very different from when the same fabric was suspended loosely. We also found that bloodstains formed on the technical back of single jersey knit were vastly different from those on the technical face. Interestingly, some drip stains showed blood passing through the textile and leaving a stain behind it that resembled insect stains. By observing, recording, and describing how a blood stained textile is found or presented at the scene, the analyst may be able to better understand bloodstains and bloodstain patterns on textiles, which could be useful to confirm or refute a witness's account of how blood came to be where it was found after a bloodshed event.

[Newly established causes of diarrhea: the protozoan Cyclospora cayetanensis (Coccidia, Apicomplexa)].

PubMed

Lepes, T

1998-01-01

Coccidia Cyclospora cavetanesis has been considered a saprophytic or accidental infection without special medical significance until 1984. However, epidemics of diarrhea first in Peru and later in USA. Haiti and Nepal have aroused interest in pathogenic characteristics of this parasite and diseases it causes. The study first deals with biological cycle of parasite development as well as clinical picture and treatment, diagnostics and epidemiology of this parasitic disease. On average the incubation period was 2-11 days with frequent stools (3 or more times a day) without blood or mucus and with tendency for recurrence. The prodromal phase is similar to influenza. It may last several weeks especially in immunodeficient persons (especially patients with AIDS). Trimethoprim (160 mg) and sulfamethoxazole (800 mg) is the treatment of choice, twice a day during a 7 day period, whereas in immunodeficient patients greater doses are necessary during a few weeks, but in such cases individual treatment planning is required. Laboratory diagnosis is made on the basis of microscopic examination of native preparation from the stool, especially if stool concentration procedure is used due to small number of oocysts in the stool. As this method does not provide identification of species, numerous staining methods are recommended (modified Ziehl-Neelsen method, Carbol-Fuchsin and safranine). Epidemiology is still unclear. It is considered that there are two sources of infection: contaminated drinking water and fruits. The disease has a seasonal character and the highest incidence is recorded in late spring and summer months. The infection is caused only by sporozoites developed by sporulation of oocysts under environmental conditions excluding the possibility of direct transmission from one person to another. It is to be expected that further investigations will give answers to numerous questions still open.

Epidemiology of intestinal polyparasitism among Orang Asli school children in rural Malaysia.

PubMed

Al-Delaimy, Ahmed K; Al-Mekhlafi, Hesham M; Nasr, Nabil A; Sady, Hany; Atroosh, Wahib M; Nashiry, Mohammed; Anuar, Tengku S; Moktar, Norhayati; Lim, Yvonne A L; Mahmud, Rohela

2014-08-01

This cross-sectional study aimed to investigate the current prevalence and risk factors associated with intestinal polyparasitism (the concurrent infection with multiple intestinal parasite species) among Orang Asli school children in the Lipis district of Pahang state, Malaysia. Fecal samples were collected from 498 school children (50.6% boys and 49.4% girls), and examined by using direct smear, formalin-ether sedimentation, trichrome stain, modified Ziehl Neelsen stain, Kato-Katz, and Harada Mori techniques. Demographic, socioeconomic, environmental, and personal hygiene information were collected by using a pre-tested questionnaire. Overall, 98.4% of the children were found to be infected by at least one parasite species. Of these, 71.4% had polyparasitism. The overall prevalence of Trichuris trichiura, Ascaris lumbricoides, hookworm, Giardia duodenalis, Entamoeba spp., and Cryptosporidium spp. infections were 95.6%, 47.8%, 28.3%, 28.3%, 14.1% and 5.2%, respectively. Univariate and multivariate analyses showed that using an unsafe water supply as a source for drinking water, presence of other family members infected with intestinal parasitic infections (IPI), not washing vegetables before consumption, absence of a toilet in the house, not wearing shoes when outside, not cutting nails periodically, and not washing hands before eating were significant risk factors associated with intestinal polyparasitism among these children. Intestinal polyparasitism is highly prevalent among children in the peninsular Malaysian Aboriginal communities. Hence, effective and sustainable control measures, including school-based periodic chemotherapy, providing adequate health education focused on good personal hygiene practices and proper sanitation, as well as safe drinking water supply should be implemented to reduce the prevalence and consequences of these infections in this population.

Prevalence of Candida co-infection in patients with pulmonary tuberculosis.

PubMed

Kali, Arunava; Charles, Mv Pravin; Noyal, Mariya Joseph; Sivaraman, Umadevi; Kumar, Shailesh; Easow, Joshy M

2013-01-01

Candida species are emerging as a potentially pathogenic fungus in patients with broncho-pulmonary diseases. The synergistic growth promoting association of Candida and Mycobacterium tuberculosis has raised increased concern for studying the various Candida spp . and its significance in pulmonary tuberculosis patients during current years. This study was undertaken with the objective of discovering the prevalence of co-infection caused by different Candida species in patients with pulmonary tuberculosis. A total of 75 patients with pulmonary tuberculosis diagnosed by sputum Ziehl-Neelsen staining were included in the study. Candida co-infection was confirmed using the Kahanpaa et al. criteria. Candida species were identified using gram stain morphology, germ tube formation, morphology on cornmeal agar with Tween-80, sugar fermentation tests and HiCrome Candida Agar. Candida co-infection was observed in 30 (40%) of patients with pulmonary tuberculosis. Candida albicans was the most common isolate observed in 50% of the patients with co-infection, followed by C. tropicalis (20%) and C. glabrata (20%). Candida co-infection was found in 62.5% of female patients, while it was observed in only 29.4% of the male patients (P value 0.0133). Mean ± SD age of the patients with C. glabrata infection was 65.83 ± 3.19, while the mean ± SD age of the patients with other Candida infections was 43.25 ± 20.44 (P value 0.0138). Many patients with pulmonary tuberculosis have co-infection with Candida spp. The prevalence of non-albicans Candida species is increasing and may be associated with inadequate response to anti-tubercular drugs. C. glabrata infection has a strong association with old age.

Blood culture gram stain, acridine orange stain and direct sensitivity-based antimicrobial therapy of bloodstream infection in patients with trauma.

PubMed

Behera, B; Mathur, P; Gupta, B

2010-01-01

The purpose of this study was to ascertain if the simple practice of Gram stain, acridine orange stain and direct sensitivity determination of positive blood culture bottles could be used to guide early and appropriate treatment in trauma patients with clinical suspicion of sepsis. The study also aimed to evaluate the error in interpreting antimicrobial sensitivity by direct method when compared to standard method and find out if specific antibiotic-organism combination had more discrepancies. Findings from consecutive episodes of blood stream infection at an Apex Trauma centre over a 12-month period are summarized. A total of 509 consecutive positive blood cultures were subjected to Gram staining. AO staining was done in BacT/ALERT-positive Gram-stain negative blood cultures. Direct sensitivity was performed from 369 blood culture broths, showing single type of growth in Gram and acridine orange staining. Results of direct sensitivity were compared to conventional sensitivity for errors. No 'very major' discrepancy was found in this study. About 5.2 and 1.8% minor error rates were noted in gram-positive and gram-negative bacteria, respectively, while comparing the two methods. Most of the discrepancies in gram-negative bacteria were noted in beta lactam - beta lactamase inhibitor combinations. Direct sensitivity testing was not reliable for reporting of methicillin and vancomycin resistance in Staphylococci. Gram stain result together with direct sensitivity testing is required for optimizing initial antimicrobial therapy in trauma patients with clinical suspicion of sepsis. Gram staining and AO staining proved particularly helpful in the early detection of candidaemia.

Validation of a low cost computer-based method for quantification of immunohistochemistry-stained sections.

PubMed

Montgomery, Jill D; Hensler, Heather R; Jacobson, Lisa P; Jenkins, Frank J

2008-07-01

The aim of the present study was to determine if the Alpha DigiDoc RT system would be an effective method of quantifying immunohistochemical staining as compared with a manual counting method, which is considered the gold standard. Two readers were used to count 31 samples by both methods. The results obtained using the Bland-Altman for concordance deemed no statistical difference between the 2 methods. Thus, the Alpha DigiDoc RT system is an effective, low cost method to quantify immunohistochemical data.

Gram stain method shows better sensitivity than clinical criteria for detection of bacterial vaginosis in surveillance of pregnant, low-income women in a clinical setting.

PubMed Central

Tam, M T; Yungbluth, M; Myles, T

1998-01-01

OBJECTIVE: The purpose of the study is to determine whether the Gram stain method is superior to the clinical criteria for the diagnosis of bacterial vaginosis in low-income pregnant women seen in a resident clinic setting. The clinical criteria is the current diagnostic method employed to diagnose bacterial vaginosis. STUDY DESIGN: In this study, 51 pregnant women with vaginal discharge were prospectively evaluated. All were screened using the clinical criteria, Gram stain method, and culture of the discharge. The modified scoring system instituted by Nugent et al. (J Clin Microbiol 29:297-301, 1991) was employed in reading the Gram stain smears. The clinical criteria were then compared with the Gram stain method. Isolation of moderate to many Gardnerella vaginalis growth by culture was used as the confirmatory finding. RESULTS: Sensitivity of the Gram stain method (91%) was significantly higher than that of the clinical criteria (46%), (sign test P = 0.0023, < 0.01). The Gram stain method also has both a low false-negative (4%) and high negative predictive value (96%), making it an ideal diagnostic test. CONCLUSION: The Gram stain method is a rapid and cost-effective test that is also highly reproducible and readily available in many laboratories. These features make the Gram stain method a more desirable screening procedure for bacterial vaginosis in a clinic population. PMID:9894174

A method for rapid quantitative assessment of biofilms with biomolecular staining and image analysis

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

Larimer, Curtis J.; Winder, Eric M.; Jeters, Robert T.

Here, the accumulation of bacteria in surface attached biofilms, or biofouling, can be detrimental to human health, dental hygiene, and many industrial processes. A critical need in identifying and preventing the deleterious effects of biofilms is the ability to observe and quantify their development. Analytical methods capable of assessing early stage fouling are cumbersome or lab-confined, subjective, and qualitative. Herein, a novel photographic method is described that uses biomolecular staining and image analysis to enhance contrast of early stage biofouling. A robust algorithm was developed to objectively and quantitatively measure surface accumulation of Pseudomonas putida from photographs and results weremore » compared to independent measurements of cell density. Results from image analysis quantified biofilm growth intensity accurately and with approximately the same precision of the more laborious cell counting method. This simple method for early stage biofilm detection enables quantifiable measurement of surface fouling and is flexible enough to be applied from the laboratory to the field. Broad spectrum staining highlights fouling biomass, photography quickly captures a large area of interest, and image analysis rapidly quantifies fouling in the image.« less

A method for rapid quantitative assessment of biofilms with biomolecular staining and image analysis

DOE PAGES

Larimer, Curtis J.; Winder, Eric M.; Jeters, Robert T.; ...

2015-12-07

Here, the accumulation of bacteria in surface attached biofilms, or biofouling, can be detrimental to human health, dental hygiene, and many industrial processes. A critical need in identifying and preventing the deleterious effects of biofilms is the ability to observe and quantify their development. Analytical methods capable of assessing early stage fouling are cumbersome or lab-confined, subjective, and qualitative. Herein, a novel photographic method is described that uses biomolecular staining and image analysis to enhance contrast of early stage biofouling. A robust algorithm was developed to objectively and quantitatively measure surface accumulation of Pseudomonas putida from photographs and results weremore » compared to independent measurements of cell density. Results from image analysis quantified biofilm growth intensity accurately and with approximately the same precision of the more laborious cell counting method. This simple method for early stage biofilm detection enables quantifiable measurement of surface fouling and is flexible enough to be applied from the laboratory to the field. Broad spectrum staining highlights fouling biomass, photography quickly captures a large area of interest, and image analysis rapidly quantifies fouling in the image.« less

A method for rapid quantitative assessment of biofilms with biomolecular staining and image analysis.

PubMed

Larimer, Curtis; Winder, Eric; Jeters, Robert; Prowant, Matthew; Nettleship, Ian; Addleman, Raymond Shane; Bonheyo, George T

2016-01-01

The accumulation of bacteria in surface-attached biofilms can be detrimental to human health, dental hygiene, and many industrial processes. Natural biofilms are soft and often transparent, and they have heterogeneous biological composition and structure over micro- and macroscales. As a result, it is challenging to quantify the spatial distribution and overall intensity of biofilms. In this work, a new method was developed to enhance the visibility and quantification of bacterial biofilms. First, broad-spectrum biomolecular staining was used to enhance the visibility of the cells, nucleic acids, and proteins that make up biofilms. Then, an image analysis algorithm was developed to objectively and quantitatively measure biofilm accumulation from digital photographs and results were compared to independent measurements of cell density. This new method was used to quantify the growth intensity of Pseudomonas putida biofilms as they grew over time. This method is simple and fast, and can quantify biofilm growth over a large area with approximately the same precision as the more laborious cell counting method. Stained and processed images facilitate assessment of spatial heterogeneity of a biofilm across a surface. This new approach to biofilm analysis could be applied in studies of natural, industrial, and environmental biofilms.

Experimental demonstration of the possible role of Acanthamoeba polyphaga in the infection and disease progression in Buruli Ulcer (BU) using ICR mice

PubMed Central

Azumah, Bright K.; Addo, Phyllis G.; Dodoo, Alfred; Awandare, Gordon; Mosi, Lydia; Boakye, Daniel A.

2017-01-01

The transmission of Buruli ulcer (BU), caused by Mycobacterium ulcerans (MU), remains puzzling although a number of hypothesis including through bites of infected aquatic insects have been proposed. We report the results of experiments using ICR mice that give credence to our hypothesis that Acanthamoeba species may play a role in BU transmission. We cocultured MU N2 and MU 1615 which expresses red fluorescent protein (RFP) and Acanthamoeba polyphaga (AP), and confirmed infected AP by Ziehl-Neelsen (ZN) staining. We tested for viability of MU inside AP and observed strong RFP signals inside both trophozoites and cysts after 3 and 42 days of coculturing respectively. ICR mice were topically treated, either on shaved intact or shaved pinpricked rumps, with one of the following; MU N2 only (2.25 x 106 colony forming units [CFU] / ml), MU N2:AP coculture (2.96 x 104 CFU: 1.6 x 106 cells/ml), AP only (1.6 x 106 cells/ml), PYG medium and sterile distilled water. Both MU N2 only and MU N2:AP elicited reddening on day (D) 31; edema on D 45 and D 44 respectively, and ulcers on D 49 at pinpricked sites only. To ascertain infectivity and pathogenicity of MU N2 only and MU N2:AP, and compare their virulence, the standard mouse footpad inoculation method was used. MU N2:AP elicited reddening in footpads by D 3 compared to D 14 with MU N2 only of the same dose of MU N2 (2.96 x 104 CFU). ZN-stained MU were observed in both thin sectioned and homogenized lesions, and aspirates from infected sites. Viable MU N2 were recovered from cultures of the homogenates and aspirates. This study demonstrates in ICR mice MU transmission via passive infection, and shows that punctures in the skin are prerequisite for infection, and that coculturing of MU with AP enhances pathogenesis. PMID:28329001

Improving Diagnostic of Pulmonary Tuberculosis in HIV Patients by Bronchoscopy: A Cross Sectional Study.

PubMed

Santoso, Prayudi; Soeroto, Arto Y; Juniati, Rianita; Hartantri, Yovita; Wisaksana, Rudi; Alisjabana, Bachti; Nataprawira, Heda M; Parwati, Ida

2017-10-01

diagnostic of pulmonary TB in HIV patients is a problem due to non specific clinical features, or radiological appearance. HIV patients with CD4≤200 cells/mL infected with M. tuberculosis have less capacity in containing M. tuberculosis, developing granulomas, casseous necrosis, or cavities. This condition is caused by weakend inflammatory which later reduced sputum production and may cause false negative result. This study aimed to assess differences in the positivity level of acid fast bacilli (AFB) and cultures of M. tuberculosis from non-bronchoscopic sputum (spontaneous and induced sputum) compared to bronchoscopic sputum (bronchoalveolar lavage) in HIV positive patients suspected pulmonary tuberculosis with CD4<200 cells/μL. this cross sectional study was conducted in adult HIV patients treated in Hasan Sadikin Hospital with CD4≤200 cells/μL suspected with pulmonary tuberculosis by using paired comparative analytic test. All patients expelled sputum spontaneously or with sputum induction on the first day. On the next day, bronchoalveolar lavage (BAL) was performed. The two samples obtained from two methods were examined by AFB examination with staining Ziehl Neelsen (ZN) and cultured of M. tuberculosis on solid media Ogawa on all patients. Positivity, sensitivity and increased sensitivity of AFB and culture of M. tuberculosis in the non bronchoscopic and bronchoscopic groups were compared. there were differences in the positivity level of AFB with ZN staining between non-bronchoscopic and bronchoscopic groups which were 7/40 (17.5%) vs 20/40 (50.0%) (p<0.001). The differences between the cultures of non-bronchoscopic and bronchoscopic groups were 16/40 (40.0%) vs 23/40 (57.5%) (p=0.039). Bronchoscopic sputum increased the positivity level of the ZN AFB examination by 32.5% (from 17.5% to 50.0%) as well as on culture examination by 17.5% (from 40.0% to 57.5%). Bronchoalveolar lavage can improve the positivity level of smears and cultures in patients

[Evaluation of mycobacterial microscopy and culture results of Sureyyapasa Chest Diseases and Chest Surgery Training and Research Hospital: A 3-year analysis].

PubMed

Akduman Alaşehir, Elçin; Balıkçı, Ahmet; Partal, Mualla; Çatmabacak, Gülay; Yaman, Görkem

2016-09-01

Effective diagnosis of tuberculosis is of great importance for transmission control and treatment success. The purpose of this study is to evaluate microscopic examination results of Ehrlich-Ziehl Neelsen (EZN) and Auramine-Rhodamine staining methods and automated BACTEC MGIT 960™ system and Löwenstein-Jensen (L-J) culture results of various clinical samples in the light of recent data from the world and Turkey. Specimens that were sent from various clinics to Sureyyapasa Chest Diseases and Chest Surgery Training and Research Hospital Microbiology Laboratory from January 2012 to December 2015 were evaluated retrospectively. From a total of 62456 samples; 60923 (97.5%) were pulmonary and 1533 (2.5%) were non-pulmonary samples, especially pleura. 2853 (4.6%) Acid-resistant bacilli (ARB) positivity was detected and mycobacterial culture positivity was in total 12.2%. 7076 (93%) and 535 (7%) mycobacteria other than tuberculosis (MOTT) strains were isolated. In 356 specimens the cultures were negative in despite the positive ARB results. Considering mycobacterial culture as the gold standard; the sensitivity, specificity, positive and negative predictive values of ARB microscopy were 32.8%, 99.4%, 87.5% and 91.4%, respectively. The contamination rates in total were within acceptable limits being 2.7% for L-J and 3.8% for MGIT. Analysis of our data indicated that the sensitivity of microscopy is low and it should be evaluated together with the mycobacterial culture to rule out tuberculosis infection. With the use of fluorescent staining and also L-J and MGIT broth together for routine culture since 2013; ARB false negativity rate was observed to fall to 51.7% from 74.1% compared to the years. The follow-up of data such as the sensitivity of microscopy, culture positivity, false-positivity and false-negativity rates and contamination values is of great importance in terms of assessing compliance with laboratory quality standards and contributing to the surveillance

Molecular epidemiology of Cryptosporidium in HIV/AIDS patients in Malaysia.

PubMed

Asma, I; Sim, B L H; Brent, R D; Johari, S; Yvonne Lim, A L

2015-06-01

Cryptosporidiosis is a particular concern in immunocompromised individuals where symptoms may be severe. The aim of this study was to examine the epidemiological and molecular characteristics of Cryptosporidium infections in HIV/AIDS patients in Malaysia in order to identify risk factors and facilitate control measures. A modified Ziehl-Neelsen acid fast staining method was used to test for the presence of Cryptosporidium oocysts in the stools of 346 HIV/AIDS patients in Malaysia. Standard coproscopical methods were used to identify infections with other protozoan or helminths parasites. To identify the species of Cryptosporidium, DNA was extracted and nested-PCR was used to amplify a portion of the SSU rRNA gene. A total of 43 (12.4%) HIV-infected patients were found to be infected with Cryptosporidium spp. Of the 43 Cryptosporidium-positive HIV patients, 10 (23.3%) also harboured other protozoa, and 15 (34.9%) had both protozoa and helminths. The highest rates of cryptosporidiosis were found in adult males of Malay background, intravenous drug users, and those with low CD4 T cell counts (i.e., < 200 cells/mm3). Most were asymptomatic and had concurrent opportunistic infections mainly with Mycobacterium tuberculosis. DNA sequence analysis of 32 Cryptosporidium isolates identified C. parvum (84.3%), C. hominis (6.3%), C. meleagridis (6.3%), and C. felis (3.1%). The results of the present study revealed a high prevalence of Cryptosporidium infection in hospitalized HIV/AIDS patients. The results also confirmed the potential significance of zoonotic transmission of C. parvum in HIV infected patients, as it was the predominant species found in this study. However, these patients were found to be susceptible to a wide range of Cryptosporidium species. Epidemiological and molecular characterization of Cryptosporidium isolates provides clinicians and researchers with further information regarding the origin of the infection, and may enhance treatment and control

Prevalence of Giardia duodenalis and Cryptosporidium species infections among children and cattle in North Shewa Zone, Ethiopia.

PubMed

Wegayehu, Teklu; Adamu, Haileeyesus; Petros, Beyene

2013-09-08

Giardia and Cryptosporidium are the most common causes of protozoan diarrhea that lead to significant morbidity and mortality worldwide. The purpose of this study was to determine the prevalence of Giardia duodenalis and Cryptosporidium species infections among children and cattle, and to assess the potential risk of zoonotic transmission. This cross-sectional study was conducted between January and April 2009 in Girar Jarso and Dera Districts of North Shewa Zone, Oromia Region, Ethiopia. A total of 768 stool specimens were collected and examined for intestinal parasites using direct wet mount with saline and formalin ether concentration methods. The modified Ziehl-Neelsen staining method was used for the detection of Cryptosporidium species. Statistical analysis was performed using SPSS software version 15. Out of 384 children examined, 53 (13.8%) and 28 (7.3%) were positive for Giardia and Cryptosporidium infections, respectively. Similarly, of the total 384 cattle examined, 9 (2.3%) were positive for Giardia duodenalis and 30 (7.8%) were positive for Cryptosporidium infection. The prevalence of giardiasis was significantly higher among children who had close contact with cattle 33 (18.7%) compared to children who had no contact with cattle 20 (9.6%) (P < 0.05). Higher number of Cryptosporidium infection was also recorded in children who had close contact with cattle 15 (8.5%). Difference in prevalence of giardiasis and cryptosporidiosis among children was not statistically significant between males and females. On the other hand, difference in the prevalence of giardiasis among children was statistically significant between age groups. Higher prevalence of Giardia duodenalis infection detected among children was significantly associated with contact with cattle and manure that the children had. Further analysis using molecular techniques is needed to explain the existence of zoonotic transmission in the study area.

Low sensitivity of the ImmunocardSTAT® Crypto/Giardia Rapid Assay test for the detection of Giardia and Cryptosporidium in fecal samples from children living in Libreville, Central Africa.

PubMed

Bouyou-Akotet, M K; Owono-Medang, M; Moussavou-Boussougou, M N; Mamfoumbi, M Mabika; Mintsa-Nguema, R; Mawili-Mboumba, D P; Kombila, M

2016-12-01

Giardiasis and cryptosporidiosis are now recognized as neglected tropical parasitic diseases. The risk of their dissemination  in developing countries, such as Gabon, is increasing, due to urban crowding and poor sanitation. Accurate, simple and rapid diagnosis tools are thus necessary for the estimation of their real burden. The aim of this study was to evaluate the performances of the ImmunocardSTAT ® Crypto/Giardia Rapid Assay test for the detection of Cryptosporidium ( C. ) spp. and Giardia ( G. ) duodenalis in children living in Libreville, Gabon. Stool samples of 173 healthy children were screened by routine microscopic using the merthiolate iodine formol concentration technique for Giardia , the modified Ziehl Neelsen (ZN) staining for Cryptosporidium and the ImmunocardSTAT ® Crypto/Giardia RDT for the detection of Giardia and Cryptosporidium parasite forms and antigens respectively. G. duodenalis was detected with microscopy and the ImmunocardSTAT ® Crypto/Giardia in 27 (15.6 %) and 22 (13.3 %) fecal samples respectively. C. spp. oocysts were found in 18 (10.4 %) ones, whereas only one sample was positive with the immunochromatographic assay. When microscopic examination was considered as the reference method, sensitivity and specificity of the ImmunocardSTAT ® Crypto/Giardia Rapid Assay were found to be 63.0 %, 96.6 and 5.5 %, 99.3 % for G. duodenalis and C. spp. respectively. The prevalence of G. duodenalis and C. spp. carriage is high in children from Libreville. A low sensitivity of the ImmunocardSTAT ® Crypto/Giardia for the detection of both parasites is observed. It is thus inappropriate as a diagnostic tool for detecting asymptomatic carriers.

Diagnosis of local hepatic tuberculosis through next-generation sequencing: Smarter, faster and better.

PubMed

Ai, Jing-Wen; Li, Yang; Cheng, Qi; Cui, Peng; Wu, Hong-Long; Xu, Bin; Zhang, Wen-Hong

2018-06-01

A 45-year-old man who complained of continuous fever and multiple hepatic masses was admitted to our hospital. Repeated MRI manifestations were similar while each radiological report suggested contradictory diagnosis pointing to infections or malignances respectively. Pathologic examination of the liver tissue showed no direct evidence of either infections or tumor. We performed next-generation sequencing on the liver tissue and peripheral blood to further investigate the possible etiology. High throughput sequencing was performed on the liver lesion tissues using BGISEQ-100 platform, and data was mapped to the Microbial Genome Databases after filtering low quality data and human reads. We identified a total of 299 sequencing reads of Mycobacterium tuberculosis (M. tuberculosis) complex sequences from the liver tissue, including 8, 229 of 4,424,435 of the M. tuberculosis nucleotide sequences, and Mycobacterium africanum, Mycobacterium bovis, and Mycobacterium canettii were also detected due to the 99.9% identical rate among these strains. No specific Mycobacterial tuberculosis nucleotide sequence was detected in the sample of peripheral blood. Patient's symptom quickly recovered after anti-tuberculosis treatment and repeated Ziehl-Neelsen staining of the liver tissue finally identified small numbers of positive bacillus. The diagnosis of this patient was difficult to establish before the next-generation sequencing because of contradictive radiological results and negative pathological findings. More sensitive diagnostic methods are urgently needed. This is the first case reporting hepatic tuberculosis confirmed by the next-generation sequencing, and marks the promising potential of the application of the next-generation sequencing in the diagnosis of hepatic lesions with unknown etiology. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Utility of MPT64 antigen test for differentiating mycobacteria: Can correlation with liquid culture smear morphology add further value?

PubMed

Nerurkar, Vidya; Kattungal, Sushma; Bhatia, Simi

2016-01-01

Clinical presentation of Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) infections may or may not be the same, but the treatment is always different. Hence accurate differentiation between MTBC and NTM is of utmost importance. To assess in parallel, the utility of MPT64 antigen immunochromatography assay (MPT64 ICT) and bacillary morphology on liquid culture smear, for rapid differentiation between MTBC and NTM in clinical isolates. Private sector reference laboratory, prospective. Thousand and ninety-three mycobacterial isolates, recovered using Mycobacteria Growth Indicator Tube 960 liquid culture system (BD, USA), were subjected to MPT64 ICT (Standard Diagnostics Inc., Korea), para amino nitrobenzoicacid (PNB), niacin, and nitrate reduction tests. Smears prepared from culture vials were subjected to Ziehl-Neelsen staining and observed microscopically for typical patterns (chords, single cells, etc.,). PNB, nitrate and niacin tests served as the reference method for MTBC identification. Thousand and fourteen and 79 isolates were identified as MTBC and NTM, respectively. MPT64 ICT correctly identified 955/1014 MTBC and all NTM isolates, yielding sensitivity and specificity of 94.2% and 100%, respectively. 936/1014 (92.3%) MTBC isolates revealed characteristic serpentine chording on culture smear including 56/59 MPT64 ICT negative isolates. Sensitivity and specificity of liquid culture smear were 98.1% and 82.3%, respectively. Correlation of MPT64 ICT results with liquid culture smear was useful, especially in MPT64 ICT negative isolates, where the latter could help to determine need and/or type of additional confirmatory testing. Liquid culture smear, however, lacked specificity and cannot be used as a stand alone test.

[Mycobacterial species repartition: experience of the Antituberculosis Center in Pointe Noire (Republic of Congo)].

PubMed

Ontsira Ngoyi, E N; Obengui; Taty Taty, R; Koumba, E L; Ngala, P; Ossibi Ibara, R B

2014-12-01

The aim of the present work was to describe mycobacteria species isolated in the antituberculosis center of Pointe-Noire city in Congo Brazzaville. It was a descriptive transversal study, conducted between September 2008 and April 2009 (7 months). A simple random sample was established from patients who came to the antituberculosis center of Pointe-Noire City (reference center on diagnosis and treatment of tuberculosis). To those patients consulting with symptoms leading to suspect pulmonary tuberculosis, a sputum sampling in three sessions was conducted. Staining techniques to Ziehl-Neelsen and auramine were performed in Pointe-Noire. Culture, molecular hybridization and antibiotic susceptibility testing to first-line antituberculosis drugs (isoniazid, rifampicin, ethambutol, pyrazinamide or streptomycine) using diffusion method on agar were performed in Cerba Pasteur laboratory in France. In 77 patients, 24 sputum (31.20%) were positive to the microscopic examination and 45 (58.44%) to the culture and identification by molecular hybridization. Mycobacteria species complex isolated were M. tuberculosis with 31 cases (68.9%) and M. africanum with 3 cases (6.67%). Non-tuberculous mycobacteria (NMT) were isolated in association or not with M. tuberculosis in 9 cases (20%) and the most common species were M. intracellulare. In M. tuberculosis species, 7 strains (41.20%) were tested sensitive to the first-line antituberculosis drugs, 8 cases (47%) monoresistance and 2 cases multidrug resistance at both isoniazide and rifampicine (12%) (MDR). This study showed the importance of Mycobacteria species complex and non-mycobacteria species in pulmonary tuberculosis. The data on resistance can help medical physicians in the treatment of pulmonary tuberculosis. Another study with a large population is required to confirm these data.

Operational Implementation of LED Fluorescence Microscopy in Screening Tuberculosis Suspects in an Urban HIV Clinic in Uganda

PubMed Central

Albert, Heidi; Nakiyingi, Lydia; Sempa, Joseph; Mbabazi, Olive; Mukkada, Sheena; Nyesiga, Barnabas; Perkins, Mark D.; Manabe, Yukari C.

2013-01-01

Background Light emitting diode (LED) fluorescence microscopy (FM) is an affordable, technology targeted for use in resource-limited settings and recommended for widespread roll-out by the World Health Organization (WHO). We sought to compare the operational performance of three LED FM methods compared to light microscopy in a cohort of HIV-positive tuberculosis (TB) suspects at an urban clinic in a high TB burden country. Methods Two spot specimens collected from TB suspects were included in the study. Smears were stained using auramine O method and read after blinding by three LED-based FM methods by trained laboratory technicians in the Infectious Diseases Institutelaboratory. Leftover portions of the refrigerated sputum specimens were transported to the FIND Tuberculosis Research Laboratory for Ziehl Neelsen (ZN) smear preparation and reading by experienced technologist as well as liquid and solid culture. Results 174 of 627 (27.8%) specimens collected yielded one or more positive mycobacterial cultures. 94.3% (164/174) were M. tuberculosis complex. LED FM was between 7.3–11.0% more sensitive compared to ZN microscopy. Of the 592 specimens examined by all microscopy methods, there was no significant difference in sensitivity between the three LED FM methods. The specificity of the LED FM methods was between 6.1% and 7.7% lower than ZN microscopy (P<0.001), although exclusion of the single poor reader resulted in over 98% specificity for all FM methods. Conclusions Laboratory technicians in routine settings can be trained to use FM which is more sensitive than ZN microscopy. Despite rigorous proficiency testing, there were operator-dependent accuracy issues which highlight the critical need for intensive quality assurance procedures during LED FM implementation. The low sensitivity of FM for HIV-positive individuals particularly those with low CD4 T cell counts, will limit the number of additional patients found by LED FM in countries with high rates of HIV co

Cryptosporidiosis - an occupational risk and a disregarded disease in Estonia.

PubMed

Lassen, Brian; Ståhl, Marie; Enemark, Heidi L

2014-06-05

Cases of cryptosporidiosis have not been officially reported in Estonia after the year 2000, and the disease appears to be either under-diagnosed or under-reported. Based on a human case of cryptosporidiosis contracted during faecal sampling in dairy farms, cattle considered to be sources of infection were analysed for Cryptosporidium spp. by a modified Ziehl Neelsen technique and molecular typing. C. parvum subtype IIaA16G1R1 was detected from the human case and from calves from one of nine farms enrolled in the study providing strong circumstantial evidence of zoonotic transmission from calves to humans. Cryptosporidiosis presents an occupational risk to people with cattle contact, and may also be a risk to the human population in general. Thus increased public and medical awareness is warranted.

Diagnosing periprosthetic infection: false-positive intraoperative Gram stains.

PubMed

Oethinger, Margret; Warner, Debra K; Schindler, Susan A; Kobayashi, Hideo; Bauer, Thomas W

2011-04-01

Intraoperative Gram stains have a reported low sensitivity but high specificity when used to help diagnose periprosthetic infections. In early 2008, we recognized an unexpectedly high frequency of apparent false-positive Gram stains from revision arthroplasties. The purpose of this report is to describe the cause of these false-positive test results. We calculated the sensitivity and specificity of all intraoperative Gram stains submitted from revision arthroplasty cases during a 3-month interval using microbiologic cultures of the same samples as the gold standard. Methods of specimen harvesting, handling, transport, distribution, specimen processing including tissue grinding/macerating, Gram staining, and interpretation were studied. After a test modification, results of specimens were prospectively collected for a second 3-month interval, and the sensitivity and specificity of intraoperative Gram stains were calculated. The retrospective review of 269 Gram stains submitted from revision arthroplasties indicated historic sensitivity and specificity values of 23% and 92%, respectively. Systematic analysis of all steps of the procedure identified Gram-stained but nonviable bacteria in commercial broth reagents used as diluents for maceration of periprosthetic membranes before Gram staining and culture. Polymerase chain reaction and sequencing showed mixed bacterial DNA. Evaluation of 390 specimens after initiating standardized Millipore filtering of diluent fluid revealed a reduced number of positive Gram stains, yielding 9% sensitivity and 99% specificity. Clusters of false-positive Gram stains have been reported in other clinical conditions. They are apparently rare related to diagnosing periprosthetic infections but have severe consequences if used to guide treatment. Even occasional false-positive Gram stains should prompt review of laboratory methods. Our observations implicate dead bacteria in microbiologic reagents as potential sources of false-positive Gram

[Clinical usefulness of urine-formed elements' information obtained from bacteria detection by flow cytometry method that uses nucleic acid staining].

PubMed

Nakagawa, Hiroko; Yuno, Tomoji; Itho, Kiichi

2009-03-01

Recently, specific detection method for Bacteria, by flow cytometry method using nucleic acid staining, was developed as a function of automated urine formed elements analyzer for routine urine testing. Here, we performed a basic study on this bacteria analysis method. In addition, we also have a comparison among urine sediment analysis, urine Gram staining and urine quantitative cultivation, the conventional methods performed up to now. As a result, the bacteria analysis with flow cytometry method that uses nucleic acid staining was excellent in reproducibility, and higher sensitivity compared with microscopic urinary sediment analysis. Based on the ROC curve analysis, which settled urine culture method as standard, cut-off level of 120/microL was defined and its sensitivity = 85.7%, specificity = 88.2%. In the analysis of scattergram, accompanied with urine culture method, among 90% of rod positive samples, 80% of dots were appeared in the area of 30 degrees from axis X. In addition, one case even indicated that analysis of bacteria by flow cytometry and scattergram of time series analysis might be helpful to trace the progress of causative bacteria therefore the information supposed to be clinically significant. Reporting bacteria information with nucleic acid staining flow cytometry method is expected to contribute to a rapid diagnostics and treatment of urinary tract infections. Besides, the contribution to screening examination of microbiology and clinical chemistry, will deliver a more efficient solution to urine analysis.

Chromosome-specific staining to detect genetic rearrangements

DOEpatents

Gray, Joe W.; Pinkel, Daniel; Tkachuk, Douglas; Westbrook, Carol

2013-04-09

Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyzes. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML) and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.

Periodic acid‑Schiff staining method for function detection of liver cells is affected by 2% horse serum in induction medium.

PubMed

Hui, Hui; Ma, Wenjun; Cui, Jiejie; Gong, Mengjia; Wang, Yi; Zhang, Yuanyuan; He, Tongchuan; Bi, Yang; He, Yun

2017-12-01

Developing a thorough understanding of experimental methods of hepatic differentiation in hepatic progenitor cells (HPCs) should expand the knowledge of hepatocyte induction in vitro and may help to develop cell transplantation therapies for the clinical usage of HPCs in liver diseases. A previous induction method effectively induced differentiation and metabolic abilities in HPCs. Periodic acid‑Schiff (PAS) staining is used to identify glycogen synthesis and hepatocyte function; however, this method failed to detect induced hepatocytes. The present study aimed to investigate the possible factors affecting the previous confusing results of PAS staining. Removal of single induction factors, including dexamethasone, hepatic growth factor and fibroblast growth factor 4 from the induction media did not restore PAS staining, whereas replacement of 2% horse serum (HS) with 10% fetal bovine serum (FBS) significantly increased the number of PAS positive cells. Following 12 days of basal induction, replacing the induction medium with media containing 10% FBS for 12‑72 h significantly improved PAS staining, but did not influence indocyanine green uptake. Furthermore, incubation in induction medium with 10% FBS following 12 days of normal induction did not affect the expression of hepatic markers and mature function of HPCs. Therefore, the present study suggested that 2% HS in the induction medium did not affect the hepatic function of induced cells, but did affect glycogen storage, whereas replacement of medium with 10% FBS in advance of PAS staining may restore the failure of PAS staining in low serum concentrations of induced hepatocytes.

Protocol for vital dye staining of corneal endothelial cells.

PubMed

Park, Sunju; Fong, Alan G; Cho, Hyung; Zhang, Cheng; Gritz, David C; Mian, Gibran; Herzlich, Alexandra A; Gore, Patrick; Morganti, Ashley; Chuck, Roy S

2012-12-01

To describe a step-by-step methodology to establish a reproducible staining protocol for the evaluation of human corneal endothelial cells. Four procedures were performed to determine the best protocol. (1) To determine the optimal trypan blue staining method, goat corneas were stained with 4 dilutions of trypan blue (0.4%, 0.2%, 0.1%, and 0.05%) and 1% alizarin red. (2) To determine the optimal alizarin red staining method, goat corneas were stained with 2 dilutions of alizarin red (1% and 0.5%) and 0.2% trypan blue. (3) To ensure that trypan blue truly stains damaged cells, goat corneas were exposed to either 3% hydrogen peroxide or to balanced salt solution, and then stained with 0.2% trypan blue and 0.5% alizarin red. (4) Finally, fresh human corneal buttons were examined; 1 group was stained with 0.2% trypan blue and another group with 0.4% trypan blue. For the 4 procedures performed, the results are as follows: (1) trypan blue staining was not observed in any of the normal corneal samples; (2) 0.5% alizarin red demonstrated sharper cell borders than 1% alizarin red; (3) positive trypan blue staining was observed in the hydrogen peroxide exposed tissue in damaged areas; (4) 0.4% trypan blue showed more distinct positive staining than 0.2% trypan blue. We were able to determine the optimal vital dye staining conditions for human corneal endothelial cells using 0.4% trypan blue and 0.5% alizarin red.

Screening of protozoan and microsporidian parasites in feces of great cormorant (Phalacrocorax carbo).

PubMed

Rzymski, Piotr; Słodkowicz-Kowalska, Anna; Klimaszyk, Piotr; Solarczyk, Piotr; Poniedziałek, Barbara

2017-04-01

The global population of great cormorants (Phalacrocorax carbo L.) is on the rise. These birds, characterized by rapid metabolism, can deposit large quantities of feces, and because they breed on the land but forage on water, both terrestrial and aquatic environments can be simultaneously affected by their activities. The contribution of great cormorants in the dispersal of bacterial and viral pathogens has been immensely studied; whereas, the occurrence of eukaryotic parasites such as protozoans and microsporidians in these birds is little known. The present study investigated the presence of dispersive stages of potentially zoonotic protozoans belonging to the genera Blastocystis, Giardia and Cryptosporidium, and Microsporidia spores in feces collected from birds inhabiting the breeding colony established at one lake island in Poland, Europe. The feces were examined by coprological techniques (staining with iron hematoxylin, Ziehl-Neelsen, and modified Weber's chromotrope 2R-based trichrome), and with immunofluorescence antibody MERIFLUOR Cryptosporidium/Giardia assay. As found, the Cryptosporidium oocysts were identified rarely in 8% of samples (2/25; 3-5 × 10 3 /g) and no cysts of Giardia and Blastocystis were detected. Microsporidian spores were detected in 4% of samples (1/25) but at very high frequency (4.3 × 10 4 /g). No dispersive stages of parasites were identified in water samples collected from the littoral area near the colony. Despite the profuse defecation of cormorants, their role in the dispersion of the investigated parasites may not be as high as hypothesized.

Primary tuberculosis of glans penis after intravesical Bacillus Calmette Guerin immunotherapy.

PubMed

Sharma, V K; Sethy, P K; Dogra, P N; Singh, Urvashi; Das, P

2011-01-01

A 55-year-old male with carcinoma in situ of urinary bladder was treated with weekly intravesical injections of Bacillus Calmette Guerin (BCG) vaccine. Three days after the sixth injection, he developed low grade fever and multiple grouped punched out, 2-3 mm ulcers around meatus and corona glandis. In addition, multiple, firm, indurated, nontender papules and few deeper nodules were present on the proximal part of glans penis, along with bilateral enlarged, matted and nontender inguinal lymph nodes. There was no history suggestive of sexually transmitted diseases and high risk behavior. Chest X-ray was within normal limits, and Mantoux, Venereal Disease Research Laboratory (VDRL) and HIV antibody tests were negative. The biopsy from the penile ulcer revealed epithelioid cell granuloma with Langhans giant cells. Fine needle aspiration cytology from the lymph node also revealed epithelioid cell granuloma and acid fast bacilli on Ziehl Neelsen's stain. The tissue biopsy grew Mycobacterium tuberculosis. The BCG immunotherapy was stopped and patient was treated with four drug antitubercular therapy with isoniazid, rifampicin, ethambutol, and pyrazinamide in standard daily doses along with pyridoxine. The edema resolved and the ulcers started healing within 2 weeks, and at 6 weeks after starting antitubercular therapy almost complete healing occurred. To the best of our knowledge, we describe the first case of an Indian patient with BCG induced primary tuberculosis of penis after immunotherapy for carcinoma urinary bladder and review the previously described cases to increase awareness of this condition in dermatologists and venereologists.

Initial Data on the Molecular Epidemiology of Cryptosporidiosis in Lebanon

PubMed Central

Osman, Marwan; El Safadi, Dima; Benamrouz, Sadia; Guyot, Karine; Dei-Cas, Eduardo; Aliouat, El Moukhtar; Creusy, Colette; Mallat, Hassan; Hamze, Monzer; Dabboussi, Fouad; Viscogliosi, Eric; Certad, Gabriela

2015-01-01

Cryptosporidium spp. represent a major public health problem worldwide and infect the gastrointestinal tract of both immunocompetent and immunocompromised persons. The prevalence of these parasites varies by geographic region, and no data are currently available in Lebanon. To promote an understanding of the epidemiology of cryptosporidiosisin this country, the main aim of this study was to determine the prevalence Cryptosporidium in symptomatic hospitalized patients, and to analyze the genetic diversity of the corresponding isolates. Fecal specimens were collected in four hospitals in North Lebanon from 163 patients (77 males and 86 females, ranging in age from 1 to 88 years, with a mean age of 22 years) presenting gastrointestinal disorders during the period July to December 2013. The overall prevalence of Cryptosporidium spp. infection obtained by modified Ziehl-Neelsen staining and/or nested PCR was 11%, and children <5 years old showed a higher rate of Cryptosporidium spp. The PCR products of the 15 positive samples were successfully sequenced. Among them, 10 isolates (66.7%) were identified as C. hominis, while the remaining 5 (33.3%) were identified as C. parvum. After analysis of the gp60 locus, C. hominis IdA19, a rare subtype, was found to be predominant. Two C. parvum subtypes were found: IIaA15G1R1 and IIaA15G2R1. The molecular characterization of Cryptosporidium isolates is an important step in improving our understanding of the epidemiology and transmission of the infection. PMID:25950832

Western Blot of Stained Proteins from Dried Polyacrylamide Gels

NASA Technical Reports Server (NTRS)

Gruber, Claudia; Stan-Lotter, Helga

1996-01-01

Western blotting of proteins is customarily performed following their separation on polyacrylamide gels, either prior to staining (1) or, as recently reported, following staining (2). We describe here Western blotting with stained gels, which had been dried and some of which had been stored for years. This procedure permits immunological analysis of proteins, to which antisera may have become available only later, or where the application of newly developed sensitive detection methods is desired. Once rehydration of the gels is achieved, proteins can be-transferred to blotting membranes by any appropriate protocol. Proteins stained with Coomassie Blue have to be detected with a non-chromogenic method, such as the film-based enhanced chemiluminescence (ECL)2) procedure (3). Silver stained proteins, which transfer in the colorless form, may be visualized by any detection method, although, because of the usually very low amounts of proteins, detection by ECL is preferable. Blotting of stained proteins from rehydrated gels is as rapid and as quantitative as from freshly prepared gels, in contrast to blotting from wet stained gels, which requires extensive washing and results in low transfer efficiency (2). Together with a photographic record of the gel pattern, unambiguous identification of immunoreactive proteins from complex mixtures is possible. Some further applications of this work are discussed.

A novel histochemical method of simultaneous detection by a single- or double-immunofluorescence and Bielschowsky's silver staining in teased rat sciatic nerves.

PubMed

Segura-Anaya, Edith; Flores-Miranda, Rommel; Martínez-Gómez, Alejandro; Dent, Myrna A R

2018-07-01

The Golgi silver method has been widely used in neuroscience for the study of normal and pathological morphology of neurons. The method has been steadily improved and Bielschowsky's silver staining method (BSSM) is widely used in various pathological conditions, like Alzheimer's disease. In this work, teased sciatic nerves were silver impregnated using BSSM. We also developed simultaneous staining by silver impregnation and single- or double-immunofluorescence of the same section in teased nerve preparations. We immunostained against non-myelinating Schwann cells and different myelinating Schwann cell domains. BSSM teased nerves show a strong staining of axons (black) and a gold-brown staining of myelinating and non-myelinating Schwann cells. We were also able to stain by immunofluorescence these BSSM teased nerves with specific molecular markers against non-myelinating Schwann cells, also against non-compact myelin such as the Schmidt-Lanterman incisures or paranodal regions and compact myelin, but not axons. In peripheral nerves, several silver impregnation methods have been used to stain nerves in paraffin sections, but not in teased nerves to enable the assessment of isolated nerve fibers. In conclusion, BSSM gives accurate information of nerve morphology and combining the procedure with immunofluorescence it would be very useful to study the molecular nerve domain organization of the nerve fibers, and to study the molecular pathology of axon degeneration, or myelin disorders, or of any peripheral neuropathy, also to study demyelination diseases in the central nervous system. Copyright © 2018. Published by Elsevier B.V.

Histological Stains: A Literature Review and Case Study

PubMed Central

Alturkistani, Hani A; Tashkandi, Faris M; Mohammedsaleh, Zuhair M

2016-01-01

The history of histology indicates that there have been significant changes in the techniques used for histological staining through chemical, molecular biology assays and immunological techniques, collectively referred to as histochemistry. Early histologists used the readily available chemicals to prepare tissues for microscopic studies; these laboratory chemicals were potassium dichromate, alcohol and the mercuric chloride to harden cellular tissues. Staining techniques used were carmine, silver nitrate, Giemsa, Trichrome Stains, Gram Stain and Hematoxylin among others. The purpose of this research was to assess past and current literature reviews, as well as case studies, with the aim of informing ways in which histological stains have been improved in the modern age. Results from the literature review has indicated that there has been an improvement in histopathology and histotechnology in stains used. There has been a rising need for efficient, accurate and less complex staining procedures. Many stain procedures are still in use today, and many others have been replaced with new immunostaining, molecular, non-culture and other advanced staining techniques. Some staining methods have been abandoned because the chemicals required have been medically proven to be toxic. The case studies indicated that in modern histology a combination of different stain techniques are used to enhance the effectiveness of the staining process. Currently, improved histological stains, have been modified and combined with other stains to improve their effectiveness. PMID:26493433

Histological Stains: A Literature Review and Case Study.

PubMed

Alturkistani, Hani A; Tashkandi, Faris M; Mohammedsaleh, Zuhair M

2015-06-25

The history of histology indicates that there have been significant changes in the techniques used for histological staining through chemical, molecular biology assays and immunological techniques, collectively referred to as histochemistry. Early histologists used the readily available chemicals to prepare tissues for microscopic studies; these laboratory chemicals were potassium dichromate, alcohol and the mercuric chloride to harden cellular tissues. Staining techniques used were carmine, silver nitrate, Giemsa, Trichrome Stains, Gram Stain and Hematoxylin among others. The purpose of this research was to assess past and current literature reviews, as well as case studies, with the aim of informing ways in which histological stains have been improved in the modern age. Results from the literature review has indicated that there has been an improvement in histopathology and histotechnology in stains used. There has been a rising need for efficient, accurate and less complex staining procedures. Many stain procedures are still in use today, and many others have been replaced with new immunostaining, molecular, non-culture and other advanced staining techniques. Some staining methods have been abandoned because the chemicals required have been medically proven to be toxic. The case studies indicated that in modern histology a combination of different stain techniques are used to enhance the effectiveness of the staining process. Currently, improved histological stains, have been modified and combined with other stains to improve their effectiveness.

A new technique for Gram staining paraffin-embedded tissue.

PubMed Central

Engbaek, K; Johansen, K S; Jensen, M E

1979-01-01

Five techniques for Gram staining bacteria in paraffin sections were compared on serial sections of pulmonary tissues from eight bacteriological necropsies. Brown and Hopp's method was the most satisfactory for distinguishing Gram-positive and Gram-negative bacteria. However, this method cannot be recommended as the preparations were frequently overstained, and the Gram-negative bacteria were stained indistinctly. A modification of Brown and Hopps' method was developed which stains larger numbers of Gram-negative bacteria and differentiates well between different cell types and connective tissue, and there is no risk of overstaining. PMID:86548

Gram staining apparatus for space station applications

NASA Technical Reports Server (NTRS)

Molina, T. C.; Brown, H. D.; Irbe, R. M.; Pierson, D. L.

1990-01-01

A self-contained, portable Gram staining apparatus (GSA) has been developed for use in the microgravity environment on board the Space Station Freedom. Accuracy and reproducibility of this apparatus compared with the conventional Gram staining method were evaluated by using gram-negative and gram-positive controls and different species of bacteria grown in pure cultures. A subsequent study was designed to assess the performance of the GSA with actual specimens. A set of 60 human and environmental specimens was evaluated with the GSA and the conventional Gram staining procedure. Data obtained from these studies indicated that the GSA will provide the Gram staining capability needed for the microgravity environment of space.

DNA comet Giemsa staining for conventional bright-field microscopy.

PubMed

Osipov, Andreyan; Arkhangelskaya, Ekaterina; Vinokurov, Alexei; Smetaninа, Nadezhda; Zhavoronkov, Alex; Klokov, Dmitry

2014-04-10

This study was undertaken to evaluate the compatibility of Giemsa staining protocol with the comet assay. We showed, for the first time, that DNA comets can be visualized and analyzed using Giemsa staining. We generated DNA damage dose response curves for human peripheral blood lymphocytes exposed to X-ray radiation using the comet assay with either SybrGreen I or Giemsa stain. The dose response curves were fitted by linear regressions (R2>0.977). The SybrGreen I results showed only ~1.2-fold higher slope coefficient (method sensitivity) compared to the Giemsa results. The unexpectedly high sensitivity of Giemsa staining for the comet assay is due to the Romanowsky-Giemsa effect, the stain photo-stability and the higher resolution of bright-field imaging compared to fluorescence imaging. Our results demonstrate that Giemsa staining can effectively be used for measuring DNA damage by the comet assay. The low cost and availability of Giemsa stain makes this method affordable for any low budget research and will facilitate new applications of the comet assay in biology and medicine.

Fluorogenic Ag+–Tetrazolate Aggregation Enables Efficient Fluorescent Biological Silver Staining

PubMed Central

Xie, Sheng; Wong, Alex Y. H.; Kwok, Ryan T. K.; Li, Ying; Su, Huifang; Lam, Jacky W. Y.

2018-01-01

Abstract Silver staining, which exploits the special bioaffinity and the chromogenic reduction of silver ions, is an indispensable visualization method in biology. It is a most popular method for in‐gel protein detection. However, it is limited by run‐to‐run variability, background staining, inability for protein quantification, and limited compatibility with mass spectroscopic (MS) analysis; limitations that are largely attributed to the tricky chromogenic visualization. Herein, we reported a novel water‐soluble fluorogenic Ag+ probe, the sensing mechanism of which is based on an aggregation‐induced emission (AIE) process driven by tetrazolate‐Ag+ interactions. The fluorogenic sensing can substitute the chromogenic reaction, leading to a new fluorescence silver staining method. This new staining method offers sensitive detection of total proteins in polyacrylamide gels with a broad linear dynamic range and robust operations that rival the silver nitrate stain and the best fluorescent stains. PMID:29575702

Protein Stains to Detect Antigen on Membranes.

PubMed

Dsouza, Anil; Scofield, R Hal

2015-01-01

Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after western blotting. "Detection" precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. "Detection" is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical.

Protein stains to detect antigen on membranes.

PubMed

D'souza, Anil; Scofield, R Hal

2009-01-01

Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after electrophoresis. "Detection" precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. Detection is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical.

Counterion dye staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic gel digestion of stained protein for mass spectrometry.

PubMed

Cong, Wei-Tao; Wang, Xu; Hwang, Sun-Young; Jin, Li-Tai; Choi, Jung-Kap

2012-01-01

A fast and matrix-assisted laser desorption/ionization mass spectrometry compatible protein staining method in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. It is based on the counterion dye staining method that employs oppositely charged two dyes, zincon and ethyl violet, to form an ion-pair complex. The protocol, including fixing, staining, and quick washing steps, can be completed in 1-1.5 h, depending upon gel thickness. It has the sensitivity comparable to the colloidal Coomassie Brilliant Blue G stain using phosphoric acid as a component of staining solution (4-8 ng). The counterion dye stain does not induce protein modifications that complicate interpretation of peptide mapping data from mass spectrometry. Considering the speed, sensitivity, and compatibility with mass spectrometry, the counterion dye stain may be more practical than any other dye-based protein stains for routine proteomic researches.

Optimal staining methods for delineation of cortical areas and neuron counts in human brains.

PubMed

Uylings, H B; Zilles, K; Rajkowska, G

1999-04-01

For cytoarchitectonic delineation of cortical areas in human brain, the Gallyas staining for somata with its sharp contrast between cell bodies and neuropil is preferable to the classical Nissl staining, the more so when an image analysis system is used. This Gallyas staining, however, does not appear to be appropriate for counting neuron numbers in pertinent brain areas, due to the lack of distinct cytological features between small neurons and glial cells. For cell counting Nissl is preferable. In an optimal design for cell counting at least both the Gallyas and the Nissl staining must be applied, the former staining for cytoarchitectural delineaton of cortical areas and the latter for counting the number of neurons in the pertinent cortical areas. Copyright 1999 Academic Press.

Development of a stain shade guide to aid the measurement of extrinsic dental stain.

PubMed

Gadhia, K; Shah, R; Swaminathan, D; Wetton, S; Moran, J

2006-05-01

Accurate and reproducible assessment of extrinsic staining is pivotal to determining efficacy of some tooth whitening oral hygiene products. The aim of this study was: (1) to produce a stain shade guide to aid the in vitro and in vivo stain assessment (2) to assess intra- and inter-examiner reproducibility of stain assessment using the stain shade guide. Using chlorhexidine and tea, perspex and acrylic teeth specimens were stained. The amount of staining on the perspex was measured with a spectrophotometer and the values obtained were assigned to the stained acrylic teeth, which were made into a stain guide. Using clinical photographs and a group of 10 volunteers, stain area and intensity were assessed using the stain guide and the recognized Lobene stain index by two examiners. The degree of intra- and inter-examiner reproducibility for these measurements were assessed using Cohen's kappa statistics. For both the clinical examination and use of photographs, intra-examiner reproducibility for stain intensity was improved when using the stain guide compared with the Lobene Index. Similarly, when assessing inter-examiner reproducibility, stain intensity kappa values were greater using the stain guide (kappa = 0.82) compared with the Lobene Index (kappa = 0.57). The findings of this study would suggest that the use of the stain guide could be of importance in the assessment of extrinsic dental stain.

An indirect method for quantitation of cellular zinc content of Timm-stained cerebellar samples by energy dispersive X-ray microanalysis.

PubMed

Farkas, I; Szerdahelyi, P; Kása, P

1988-01-01

The absolute concentration of zinc in the Purkinje cells of the rat cerebellum was determined by means of energy dispersive X-ray microanalysis (EDAX). Gelatine blocks with known zinc concentrations were stained by Timm's sulphide-silver method, and their silver concentrations were measured by EDAX. A linear correlation was found between the zinc and silver concentrations and this linear function was used as a quantitative calibration for evaluation of sulphide-silver staining, after perfusion with sodium-sulphide solution, fixation with glutaraldehyde, cryostat sectioning and staining of cerebellar samples in Timm's reagent.

Gram staining apparatus for space station applications.

PubMed Central

Molina, T C; Brown, H D; Irbe, R M; Pierson, D L

1990-01-01

A self-contained, portable Gram staining apparatus (GSA) has been developed for use in the microgravity environment on board the Space Station Freedom. Accuracy and reproducibility of this apparatus compared with the conventional Gram staining method were evaluated by using gram-negative and gram-positive controls and different species of bacteria grown in pure cultures. A subsequent study was designed to assess the performance of the GSA with actual specimens. A set of 60 human and environmental specimens was evaluated with the GSA and the conventional Gram staining procedure. Data obtained from these studies indicated that the GSA will provide the Gram staining capability needed for the microgravity environment of space. Images PMID:1690529

The minimal carcinoma triple stain is superior to commercially available multiplex immunohistochemical stains: breast triple stain and LC/DC breast cocktail.

PubMed

Ginter, Paula S; Varma, Sonal; Liu, Yi-Fang; Shin, Sandra J

2015-12-01

The Minimal Carcinoma (MC) Triple Stain is a tri-chromogen multiplex immunostain (CK7, p63, and E-cadherin) helpful in classifying morphologically ambiguous and/or small carcinomas as either ductal or lobular and/or in situ or invasive. We compared the utility of this stain with two commercially available duplex/multiplex immunostains: Breast Triple Stain (BTS) (Clarient, Aliso Viejo, CA; CK5, p63, and CK8/18) and LC/DC Breast Cocktail (LCDC) (Biocare, Concord, CA; E-cadherin and p120). Ninety-seven mammary carcinomas stained with the MC Triple Stain, BTS, and LCDC were compared. The MC Triple Stain, LCDC, and BTS were diagnostic in 90 (93%) of 97, 82 (85%) of 97, and 85 (88%) of 97 of cases, respectively. All stains showed decreased diagnostic utility due to variability in tissue integrity, quality of the staining, and/or ease of interpretation. In cases where all immunostains were interpretable, the MC Triple Stain yielded the most information. When technically sufficient, all three immunostains demonstrated relative strengths and weaknesses in their ability to provide diagnostic information with the highest consistency and ease of use. Many cases stained with LCDC were technically insufficient due to a suboptimal staining protocol provided by the company. Overall, the MC Triple Stain outperformed BTS and LCDC by more consistently providing more diagnostic information. The MC Triple Stain is a viable alternative to other multiplex immunostains in evaluating small foci of carcinoma, particularly when both the histologic type and extent of disease (in situ vs invasive) require clarification. Copyright© by the American Society for Clinical Pathology.

Using microabrasive material to remove fluorosis stains.

PubMed

Allen, Kenneth; Agosta, Claudine; Estafan, Denise

2004-03-01

Increased public access to fluoride has decreased the prevalence of caries and increased the prevalence of fluorosis staining. This article provides a case report involving a conservative method of removing fluorosis stain, as well as describes an in vitro test of the method. A healthy man sought treatment at New York University College of Dentistry for removal of severe, dark brown fluorosis staining on his anterior teeth. To remove the stain, the treating clinician used a microabrasive material, which leaves enamel intact, instead of a tooth-whitening agent, which requires removal of all affected enamel. To demonstrate that enamel structure is not disturbed by the microabrasive material, the authors performed a study using scanning electron microscopy, or SEM. They viewed enamel structure under SEM at x1,000 magnification. They viewed untreated microabraded enamel and compared it with enamel that had been treated for 20 seconds with 37 percent phosphoric acid. An etch pattern was not discernible on the tooth treated with the microabrasive material. The enamel prisms remained intact and the cores were not exposed. Microabrasion removes intrinsic fluorosis stain effectively while protecting enamel. In this case, an enamel shade of brown not in the range of any tooth color shade guide was reduced.

Double-Staining Method for Differentiation of Morphological Changes and Membrane Integrity of Campylobacter coli Cells

PubMed Central

Alonso, Jose L.; Mascellaro, Salvatore; Moreno, Yolanda; Ferrús, María A.; Hernández, Javier

2002-01-01

We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells. PMID:12324366

[Tuberculosis of the penis: report of a case and review of the literature].

PubMed

Tanikawa, K; Matsushita, K; Ohkoshi, M

1985-06-01

Tuberculosis of the penis is a rare disease. We report a case of tuberculosis of the penis. A 51-year-old man noticed a painless induration with a central ulceration on the glans penis. He had a history of tuberculosis of cervical lymph-nodes, right epididymis and leg skin. Examination of the other parts showed no evidence of tuberculosis. Tuberculin test was strongly positive. The skin lesion of the glans was excised. The pathology was epithelioid cell granuloma with Langhans' giant cell, indicative of tuberculosis. But acid-fast bacilli were not detected in the Ziehl-Neelsen preparation of the tissue. The patient was treated with isoniazid, rifampicin and cycloserine. After the treatment for approximately 5 months, recurrence was not observed and enlargement of cervical lymphadenopathy improved. We reviewed 39 cases of tuberculosis of the penis reported in Japan during the past 14 years.

Nuclear staining with alum hematoxylin.

PubMed

Llewellyn, B D

2009-08-01

The hematoxylin and eosin stain is the most common method used in anatomic pathology, yet it is a method about which technologists ask numerous questions. Hematoxylin is a natural dye obtained from a tree originally found in Central America, and is easily converted into the dye hematein. This dye forms coordination compounds with mordant metals, such as aluminum, and the resulting lake attaches to cell nuclei. Regressive formulations contain a higher concentration of dye than progressive formulations and may also contain a lower concentration of mordant. The presence of an acid increases the life of the solution and in progressive solutions may also affect selectivity of staining. An appendix lists more than 60 hemalum formulations and the ratio of dye to mordant for each.

Preparation of colloidal gold for staining proteins electrotransferred onto nitrocellulose membranes.

PubMed

Yamaguchi, K; Asakawa, H

1988-07-01

This paper describes a simple method of preparing colloidal gold for staining protein blots. Colloidal gold was prepared from 0.005 or 0.01% HAuCl4 by the addition of formalin as a reductant and potassium hydroxide. Staining of small cell carcinoma tissue extract blotted onto nitrocellulose membranes with this colloidal gold solution resulted in the appearance of a large number of clear wine-red bands. The sensitivity of gold staining was 60 times higher than that of Coomassie brilliant blue staining and almost comparable to that of silver staining of proteins in polyacrylamide gel. The sensitivity of this method was also satisfactory in comparison with that of enzyme immunoblotting. The colloidal gold prepared by this method is usable for routine work.

Automated single-slide staining device

NASA Technical Reports Server (NTRS)

Wilkins, J. R.; Mills, S. M. (Inventor)

1977-01-01

A simple apparatus and method is disclosed for making individual single Gram stains on bacteria inoculated slides to assist in classifying bacteria in the laboratory as Gram-positive or Gram-negative. The apparatus involves positioning a single inoculated slide in a stationary position and thereafter automatically and sequentially flooding the slide with increments of a primary stain, a mordant, a decolorizer, a counterstain and a wash solution in a sequential manner without the individual lab technician touching the slide and with minimum danger of contamination thereof from other slides.

Pleural fluid Gram stain

MedlinePlus

Gram stain of pleural fluid ... mixing it with a violet stain (called a Gram stain). A laboratory specialist uses a microscope to ... reveals an abnormal collection of pleural fluid. The Gram stain can help identify the bacteria that might ...

A simple and rapid microwave-assisted hematoxylin and eosin staining method using 1,1,1 trichloroethane as a dewaxing and a clearing agent.

PubMed

Temel, S G; Noyan, S; Cavusoglu, I; Kahveci, Z

2005-01-01

The use and practicability of microwave-assisted staining procedures in routine histopathology has been well established for more than 17 years. In the study reported here, we aimed to examine an alternative approach that would shorten the duration of dewaxing and clearing steps of hematoxylin and eosin (H & E) staining of paraffin sections by using a microwave oven. Although xylene is one of the most popular dewaxing and clearing agents, its flammability restricts its use in a microwave oven; thus we preferred 1,1,1 trichloroethane, which is not flammable, as the dewaxing and clearing agent in the present study. In Group I and Group II (control groups), intestine was processed with xylene and 1,1,1 trichloroethane, respectively. The sections were then stained with H & E according to the conventional staining protocol at room temperature and subdivided into two groups according to the duration of dewaxing and clearing in xylene. In Groups III and IV (experimental groups) similar tissues were processed with xylene and 1,1,1 trichloroethane, respectively; however, sections from these groups were divided into four subgroups to study the period required for dewaxing and clearing in 1,1,1 trichloroethane, then stained with H & E in the microwave oven at 360 W for 30 sec. Our conventional H & E staining procedure, which includes dewaxing, staining and clearing of sections, requires approximately 90 min, while our method using 1,1,1 trichloroethane and microwave heating required only 2 min. Our alternative method for H & E staining not only reduced the procedure time significantly, but also yielded staining quality equal or superior to those stained the conventional way. Our results suggest that 1,1,1 trichloroethane can be used effectively and safely as a dewaxing and clearing agent for H & E staining in a microwave oven.

A procedure for Alcian blue staining of mucins on polyvinylidene difluoride membranes.

PubMed

Dong, Weijie; Matsuno, Yu-ki; Kameyama, Akihiko

2012-10-16

The isolation and characterization of mucins are critically important for obtaining insight into the molecular pathology of various diseases, including cancers and cystic fibrosis. Recently, we developed a novel membrane electrophoretic method, supported molecular matrix electrophoresis (SMME), which separates mucins on a polyvinylidene difluoride (PVDF) membrane impregnated with a hydrophilic polymer. Alcian blue staining is widely used to visualize mucopolysaccharides and acidic mucins on both blotted membranes and SMME membranes; however, this method cannot be used to stain mucins with a low acidic glycan content. Meanwhile, periodic acid-Schiff staining can selectively visualize glycoproteins, including mucins, but is incompatible with glycan analysis, which is indispensable for mucin characterizations. Here we describe a novel staining method, designated succinylation-Alcian blue staining, for visualizing mucins on a PVDF membrane. This method can visualize mucins regardless of the acidic residue content and shows a sensitivity 2-fold higher than that of Pro-Q Emerald 488, a fluorescent periodate Schiff-base stain. Furthermore, we demonstrate the compatibility of this novel staining procedure with glycan analysis using porcine gastric mucin as a model mucin.

A Novel Contrast Stain for the Rapid Diagnosis of Pityriasis Versicolor: A Comparison of Chicago Sky Blue 6B Stain, Potassium Hydroxide Mount and Culture

PubMed Central

Lodha, Nikita; Poojary, Shital Amin

2015-01-01

Background: The mycological study of pityriasis versicolor is usually done by potassium hydroxide (KOH) mount and culture. However, KOH mount lacks a color contrast and requires a trained eye to interpret, while culture is difficult to perform, time consuming and has low sensitivity. Chicago Sky Blue 6B (CSB) is a new contrast stain that highlights the fungal hyphae and spores, blue against a purplish background. Aims and Objectives: This study was done to compare the utility of a novel contrast stain (CSB stain) with KOH mount and culture. Materials and Methods: Skin scrapings from the lesions of 100 clinically diagnosed cases of P. versicolor were subjected to (1) KOH mount and CSB stain for direct microscopic examination and (2) culture using Sabouraud's dextrose agar. The statistical analysis of CSB stain and culture was done using KOH mount as the reference method, as it is the most commonly performed and practical diagnostic test available for P. versicolor. An interrater reliability analysis using the Cohen's Kappa statistic was performed to determine consistency (agreement) among the different modalities. Observations and Results: Direct microscopy with CSB stain, KOH mount and mycological culture showed positive results in 98 (98%), 92 (92%) and 56 (56%) patients, respectively. Using KOH mount as the reference method, CSB stain had a sensitivity of 100% which was significantly higher than culture (60.9%). Statistically significant fair agreement was found between CSB stain and KOH mount (94% with κ=0.38, P < 0.001). Negligible agreement was found between CSB stain and culture (66%, κ=0.199, P = 0.001) as well as between KOH mount and culture (64%, κ=0.051, P = 0.107). Conclusion: CSB staining of skin scrapings is the most sensitive method for the diagnosis of pityriasis versicolor. Due to the distinct contrast provided by CSB, it is easy to perform, rapid and qualitatively superior to KOH mount. PMID:26288400

Unsupervised color normalisation for H and E stained histopathology image analysis

NASA Astrophysics Data System (ADS)

Celis, Raúl; Romero, Eduardo

2015-12-01

In histology, each dye component attempts to specifically characterise different microscopic structures. In the case of the Hematoxylin-Eosin (H&E) stain, universally used for routine examination, quantitative analysis may often require the inspection of different morphological signatures related mainly to nuclei patterns, but also to stroma distribution. Nevertheless, computer systems for automatic diagnosis are often fraught by color variations ranging from the capturing device to the laboratory specific staining protocol and stains. This paper presents a novel colour normalisation method for H&E stained histopathology images. This method is based upon the opponent process theory and blindly estimates the best color basis for the Hematoxylin and Eosin stains without relying on prior knowledge. Stain Normalisation and Color Separation are transversal to any Framework of Histopathology Image Analysis.

Urine-Based Nested PCR for the Diagnosis of Mycobacterium tuberculosis: A Comparative Study Between HIV-Positive and HIV-Negative Patients.

PubMed

Jamshidi Makiani, Mahin; Davoodian, Parivash; Baghershiroodi, Mahnaz; Nejatizadeh, Abdol Azim; Fakkhar, Farideh; Zangeneh, Mehrangiz; Jahangiri, Nadia

2016-08-01

While tuberculosis (TB) can be diagnosed by microscopy and culture, the sensitivity of Ziehl-Neelsen staining is variable and culture results require 4 - 8 weeks to be determined. Polymerase chain reaction (PCR) and its modifications, including nested PCR, might be promising methods for the rapid diagnosis of TB. This study aimed to evaluate the performance of nested PCR on urine samples of human immunodeficiency virus (HIV)-positive and -negative patients with different manifestations of clinical TB. In a prospective study, three early-morning urine samples from 100 patients with pulmonary TB (PTB) or extrapulmonary TB (EPTB) were evaluated using a molecular target with insertion element IS6110, specific to the Mycobacterium tuberculosis genome, and nested PCR was performed. The results were analyzed with SPSS version 22. A total of 100 patients, including 74 (74%) with PTB and 26 (26%) with EPTB, were enrolled. Positive smears were seen in 38 patients (38%). Lymph nodes were the most commonly involved organ in 14 of the 26 (53.8%) EPTB patients (13.5%). Seven (23.1%) of the EPTB patients were HIV-positive. Urine PCR was positive in only 28 patients (28%). Seven HIV-positive patients with PTB showed positive urine PCR results. Moreover, PCR results were positive in only one of the seven HIV-positive subjects with EPTB. Positive PCR results were found in 20 of the 73 HIV-negative patients (27.4%) and in 8 of the 27 HIV-positive patients (29.6%). Therefore, there was no significant difference between the HIV-negative and HIV-positive patients for urine PCR (sensitivity 29.6%, specificity 72.6%; positive and negative predictive values 28% and 72%, respectively; P = 0.138). Nested PCR showed the same sensitivity in HIV-positive and HIV-negative patients. It can be applied as a rapid technique for the diagnosis of TB.

Comparative performance of Thin Layer Agar and Löwenstein-Jensen culture for diagnosis of tuberculosis.

PubMed

Battaglioli, T; Rintiswati, N; Martin, A; Palupi, K R; Bernaerts, G; Dwihardiani, B; Ahmad, R A; Matthys, F; Mahendradhata, Y; Van der Stuyft, P

2013-11-01

Sputum smear microscopy for the diagnosis of tuberculosis (TB) is cheap and simple but its sensitivity is low. Culture on Löwenstein-Jensen (LJ) is more sensitive but it takes a long time to yield results. Thin-Layer Agar (TLA) culture was suggested as an equally sensitive and faster alternative. We evaluated the performance of TLA for diagnosing TB in Jogjakarta, Indonesia. People with suspected TB presenting from July 2010 to July 2011 to two chest clinics of the National TB Control Programme network of Jogjakarta were eligible for inclusion. A sputum sample was sent to the Gadjah Mada University microbiology laboratory for concentration, smearing, Ziehl-Neelsen staining and culture on LJ and TLA. Sensitivity of cultures was evaluated against a composite reference standard (any positive culture). Time to detection of Mycobacteria was recorded. Out of 1414 samples, 164 (12%) were smear positive, 99 (7%) were scanty and 1151 (81%) were negative. On TLA and LJ respectively, 168 (12%) and 149 (11%) samples were positive, 72 (5%) and 32 (2%) were contaminated (κ = 0.64; 95% CI 0.59-0.69, p <0.01). Using the reference standard, 196 (14%) TB cases were identified. The sensitivity of TLA was 0.86 (95% CI 0.80-0.90), significantly higher (p 0.03) than for LJ (0.76; 95% CI 0.69-0.81). The median time to detection in days was significantly shorter (p <0.01) for TLA (12; 95% CI 11-13) than for LJ (44; 95% CI 43-45). TLA is a rapid and sensitive method for the diagnosis of TB. Implementation studies to evaluate the cost-effectiveness and impact of its introduction into programmatic settings are urgently needed. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

Comparative evaluation of PCR amplification of RLEP, 16S rRNA, rpoT and Sod A gene targets for detection of M. leprae DNA from clinical and environmental samples.

PubMed

Turankar, Ravindra P; Pandey, Shradha; Lavania, Mallika; Singh, Itu; Nigam, Astha; Darlong, Joydeepa; Darlong, Fam; Sengupta, Utpal

2015-03-01

PCR assay is a highly sensitive, specific and reliable diagnostic tool for the identification of pathogens in many infectious diseases. Genome sequencing Mycobacterium leprae revealed several gene targets that could be used for the detection of DNA from clinical and environmental samples. The PCR sensitivity of particular gene targets for specific clinical and environmental isolates has not yet been established. The present study was conducted to compare the sensitivity of RLEP, rpoT, Sod A and 16S rRNA gene targets in the detection of M. leprae in slit skin smear (SSS), blood, soil samples of leprosy patients and their surroundings. Leprosy patients were classified into Paucibacillary (PB) and Multibacillary (MB) types. Ziehl-Neelsen (ZN) staining method for all the SSS samples and Bacteriological Index (BI) was calculated for all patients. Standard laboratory protocol was used for DNA extraction from SSS, blood and soil samples. PCR technique was performed for the detection of M. leprae DNA from all the above-mentioned samples. RLEP gene target was able to detect the presence of M. leprae in 83% of SSS, 100% of blood samples and in 36% of soil samples and was noted to be the best out of all other gene targets (rpoT, Sod A and 16S rRNA). It was noted that the RLEP gene target was able to detect the highest number (53%) of BI-negative leprosy patients amongst all the gene targets used in this study. Amongst all the gene targets used in this study, PCR positivity using RLEP gene target was the highest in all the clinical and environmental samples. Further, the RLEP gene target was able to detect 53% of blood samples as positive in BI-negative leprosy cases indicating its future standardization and use for diagnostic purposes. Copyright © 2015 Asian African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

Acetylcholinesterase and Nissl staining in the same histological section.

PubMed

Shipley, M T; Ennis, M; Behbehani, M M

1989-12-18

Acetylcholinesterase (AChE) enzyme histochemistry and Nissl staining are commonly utilized in neural architectonic studies. However, the opaque reaction deposit produced by the most commonly used AChE histochemical methods is not compatible with satisfactory Nissl staining. As a result, precise correlation of AChE and Nissl staining necessitates time-consuming comparisons of adjacent sections which may have differential shrinkage. Here, we have modified the Koelle-Friedenwald histochemical reaction for AChE by omitting the final intensification steps. The modified reaction yields a non-opaque reaction product that is selectively visualized by darkfield illumination. This non-intensified darkfield AChE (NIDA) reaction allows clear visualization of Nissl staining in the same histological section. This combined AChE-Nissl method greatly facilitates detailed correlation of enzyme and cytoarchitectonic organization.

A Novel Contrast Stain for the Rapid Diagnosis of Pityriasis Versicolor: A Comparison of Chicago Sky Blue 6B Stain, Potassium Hydroxide Mount and Culture.

PubMed

Lodha, Nikita; Poojary, Shital Amin

2015-01-01

The mycological study of pityriasis versicolor is usually done by potassium hydroxide (KOH) mount and culture. However, KOH mount lacks a color contrast and requires a trained eye to interpret, while culture is difficult to perform, time consuming and has low sensitivity. Chicago Sky Blue 6B (CSB) is a new contrast stain that highlights the fungal hyphae and spores, blue against a purplish background. This study was done to compare the utility of a novel contrast stain (CSB stain) with KOH mount and culture. Skin scrapings from the lesions of 100 clinically diagnosed cases of P. versicolor were subjected to (1) KOH mount and CSB stain for direct microscopic examination and (2) culture using Sabouraud's dextrose agar. The statistical analysis of CSB stain and culture was done using KOH mount as the reference method, as it is the most commonly performed and practical diagnostic test available for P. versicolor. An interrater reliability analysis using the Cohen's Kappa statistic was performed to determine consistency (agreement) among the different modalities. Direct microscopy with CSB stain, KOH mount and mycological culture showed positive results in 98 (98%), 92 (92%) and 56 (56%) patients, respectively. Using KOH mount as the reference method, CSB stain had a sensitivity of 100% which was significantly higher than culture (60.9%). Statistically significant fair agreement was found between CSB stain and KOH mount (94% with κ=0.38, P < 0.001). Negligible agreement was found between CSB stain and culture (66%, κ=0.199, P = 0.001) as well as between KOH mount and culture (64%, κ=0.051, P = 0.107). CSB staining of skin scrapings is the most sensitive method for the diagnosis of pityriasis versicolor. Due to the distinct contrast provided by CSB, it is easy to perform, rapid and qualitatively superior to KOH mount.

Mass spectrometry-compatible silver staining of histones resolved on acetic acid-urea-Triton PAGE.

PubMed

Pramod, Khare Satyajeet; Bharat, Khade; Sanjay, Gupta

2009-05-01

Acetic acid-Urea-Triton (AUT) PAGE is commonly used method to separate histone variants and their post-translationally modified forms. Coomassie staining is the preferred method for protein visualization; however, its sensitivity is less than that of silver staining. Though silver staining of histones in AUT-PAGE has been reported, the method is time-consuming, dependent on prior staining by Amido black and has not been reported suitable for mass spectrometry. Here, we propose 'SDS-Silver' method for rapid, sensitive and mass spectrometry-compatible staining of histones resolved on AUT-PAGE.

Quantification and Statistical Analysis Methods for Vessel Wall Components from Stained Images with Masson's Trichrome

PubMed Central

Hernández-Morera, Pablo; Castaño-González, Irene; Travieso-González, Carlos M.; Mompeó-Corredera, Blanca; Ortega-Santana, Francisco

2016-01-01

Purpose To develop a digital image processing method to quantify structural components (smooth muscle fibers and extracellular matrix) in the vessel wall stained with Masson’s trichrome, and a statistical method suitable for small sample sizes to analyze the results previously obtained. Methods The quantification method comprises two stages. The pre-processing stage improves tissue image appearance and the vessel wall area is delimited. In the feature extraction stage, the vessel wall components are segmented by grouping pixels with a similar color. The area of each component is calculated by normalizing the number of pixels of each group by the vessel wall area. Statistical analyses are implemented by permutation tests, based on resampling without replacement from the set of the observed data to obtain a sampling distribution of an estimator. The implementation can be parallelized on a multicore machine to reduce execution time. Results The methods have been tested on 48 vessel wall samples of the internal saphenous vein stained with Masson’s trichrome. The results show that the segmented areas are consistent with the perception of a team of doctors and demonstrate good correlation between the expert judgments and the measured parameters for evaluating vessel wall changes. Conclusion The proposed methodology offers a powerful tool to quantify some components of the vessel wall. It is more objective, sensitive and accurate than the biochemical and qualitative methods traditionally used. The permutation tests are suitable statistical techniques to analyze the numerical measurements obtained when the underlying assumptions of the other statistical techniques are not met. PMID:26761643

Can Feulgen Stain be a Reliable Biomarker over PAP Stain for Estimation of Micronuclei Score?

PubMed Central

Prasad, Umesh Chandra; Chandolia, Betina; Manjunath, S M; Basu, Shiva; Verma, Silvie

2016-01-01

Introduction Malignant transformation of the Potentially Malignant Lesions (PML) in the oral cavity is associated with elevated mortality rate because of its aggressive and exceedingly invasive nature. Meticulous diagnosis and prompt therapy of PML may help prevent malignant conversion in oral lesions. Carcinogenic insult to oral cells results in chromosomal damage and formation of Micronuclei (Mn), before the development of clinical symptoms. Aim To determine the genotoxic effect of smoking and chewing tobacco on target tissue using Mn assay and to evaluate the prevalence of other nuclear anomalies associated with it and to determine the reliability of feulgen stain for Mn assay over Papaincolau (PAP) stain. Materials and Methods PAP and feulgen staining was done to study Mn in individuals who were having tobacco habits (smoking and chewing) without lesion (n=30), individuals who were having tobacco habit (smoking and chewing) with PML (n=30) and apparently healthy subjects (n=30). Data was analysed for statistical significance using SPSS 17.0 by Kruskal - Wallis Test and Bonferronii test. Results Tobacco habits in the form of smoking and chewing have mutagenic effects on human chromosomes which is indicated by increased frequency of Mn in oral exfoliative cells. The mean Mn frequency using feulgen stain was found to be 12.27 with lesion, 10.23 with without lesion and 3.87 in controls. Whereas, metanucleated analysis revealed no significant correlation with the formation of Mn. Non-specific DNA stain (PAP) showed high numbers of Mn cells in all the groups compared to feulgen. Statistically significant difference (p<0.0001) was observed when both the stains were compared for Mn numbers. Conclusion These findings indicate that the individuals having tobacco habits (smoking and chewing) with lesion have high number of Mn cells, thus supporting the assay to be used as a reliable biomarker to assess the genotoxic effect of tobacco in the oral mucosa. The reason for

A brief update on physical and optical disector applications and sectioning-staining methods in neuroscience.

PubMed

Yurt, Kıymet Kübra; Kivrak, Elfide Gizem; Altun, Gamze; Mohamed, Hamza; Ali, Fathelrahman; Gasmalla, Hosam Eldeen; Kaplan, Suleyman

2018-02-26

A quantitative description of a three-dimensional (3D) object based on two-dimensional images can be made using stereological methods These methods involve unbiased approaches and provide reliable results with quantitative data. The quantitative morphology of the nervous system has been thoroughly researched in this context. In particular, various novel methods such as design-based stereological approaches have been applied in neuoromorphological studies. The main foundations of these methods are systematic random sampling and a 3D approach to structures such as tissues and organs. One key point in these methods is that selected samples should represent the entire structure. Quantification of neurons, i.e. particles, is important for revealing degrees of neurodegeneration and regeneration in an organ or system. One of the most crucial morphometric parameters in biological studies is thus the "number". The disector counting method introduced by Sterio in 1984 is an efficient and reliable solution for particle number estimation. In order to obtain precise results by means of stereological analysis, counting items should be seen clearly in the tissue. If an item in the tissue cannot be seen, these cannot be analyzed even using unbiased stereological techniques. Staining and sectioning processes therefore play a critical role in stereological analysis. The purpose of this review is to evaluate current neuroscientific studies using optical and physical disector counting methods and to discuss their definitions and methodological characteristics. Although the efficiency of the optical disector method in light microscopic studies has been revealed in recent years, the physical disector method is more easily performed in electron microscopic studies. Also, we offered to readers summaries of some common basic staining and sectioning methods, which can be used for stereological techniques in this review. Copyright © 2018 Elsevier B.V. All rights reserved.

An easy and inexpensive method for quantitative analysis of endothelial damage by using vital dye staining and Adobe Photoshop software.

PubMed

Saad, Hisham A; Terry, Mark A; Shamie, Neda; Chen, Edwin S; Friend, Daniel F; Holiman, Jeffrey D; Stoeger, Christopher

2008-08-01

We developed a simple, practical, and inexpensive technique to analyze areas of endothelial cell loss and/or damage over the entire corneal area after vital dye staining by using a readily available, off-the-shelf, consumer software program, Adobe Photoshop. The purpose of this article is to convey a method of quantifying areas of cell loss and/or damage. Descemet-stripping automated endothelial keratoplasty corneal transplant surgery was performed by using 5 precut corneas on a human cadaver eye. Corneas were removed and stained with trypan blue and alizarin red S and subsequently photographed. Quantitative assessment of endothelial damage was performed by using Adobe Photoshop 7.0 software. The average difference for cell area damage for analyses performed by 1 observer twice was 1.41%. For analyses performed by 2 observers, the average difference was 1.71%. Three masked observers were 100% successful in matching the randomized stained corneas to their randomized processed Adobe images. Vital dye staining of corneal endothelial cells can be combined with Adobe Photoshop software to yield a quantitative assessment of areas of acute endothelial cell loss and/or damage. This described technique holds promise for a more consistent and accurate method to evaluate the surgical trauma to the endothelial cell layer in laboratory models. This method of quantitative analysis can probably be generalized to any area of research that involves areas that are differentiated by color or contrast.

Application of immunogold-silver staining and immunoenzymatic methods in multiple labelling of human pancreatic Langerhans islet cells.

PubMed

Krenács, T; Lászik, Z; Dobó, E

1989-01-01

The use of immunogold-silver staining (IGSS) combined with immunoperoxidase and/or immunoalkaline phosphatase methods for the simultaneous demonstration of pancreatic islet cell hormones on routinely fixed paraffin-embedded human tissue sections was examined. If IGSS was applied first, the black colour of silver-enhanced colloidal gold on doubly immunostained sections contrasted with the colours of most of the chromogens used generally in the 2 immunoenzymatic methods. If IGSS was followed by immunoalkaline phosphatase and immunoperoxidase techniques in optional sequence, 3 different hormone-containing cell types could be stained simultaneously without non-specific cross-reactions. IGSS and immunoalkaline phosphatase methods, together with 2 kinds of non-cross-reacting immunoperoxidase systems, permitted the detection of 4 distinct antigens on the same tissue section. Multiple immunohistochemical labelling of the endocrine pancreas provides an opportunity for the correct and rapid analysis of the topographic and morphometric relationships between different hormone-producing cell populations under both normal and pathological conditions. IGSS is of great potential for the simultaneous immunolabelling of antigens situated within separate cells.

Luna stain, an improved selective stain for detection of microsporidian spores in histologic sections.

PubMed

Peterson, Tracy S; Spitsbergen, Jan M; Feist, Stephen W; Kent, Michael L

2011-06-16

Microsporidia in histologic sections are most often diagnosed by observing spores in host tissues. Spores are easy to identify if they occur in large aggregates or xenomas when sections are stained with hematoxylin and eosin (H&E). However, individual spores are not frequently detected in host tissues with conventional H&E staining, particularly if spores are scattered within the tissues, areas of inflammation, or small spores in nuclei (i.e. Nucleospora salmonis). Hence, a variety of selective stains that enhance visualization of spores is recommended. We discovered that the Luna stain, used to highlight eosinophils, red blood cells, and chitin in arthropods and other invertebrates, also stains spores of Pseudoloma neurophilia. We compared this stain to the Gram, Fite's acid fast, Giemsa, and H&E stains on 8 aquatic microsporidian organisms that were readily available in our 2 laboratories: Loma salmonae, Glugea anomala, Pseudoloma neurophilia, Pleistophora hyphessobryconis, Pleistophora vermiformis, Glugea sp., Steinhausia mytilovum, and an unidentified microsporidian from UK mitten crabs Eriocheir sinensis. Based on tinctorial properties and background staining, the Luna stain performed better for detection of 6 of the 8 microsporidia. Gram stain was superior for the 2 microsporidia from invertebrates: S. mytilovum and the unidentified microsporidian from E. sinensis.

Definite (microbiologically confirmed) tuberculous meningitis: predictors and prognostic impact.

PubMed

Jha, Sneh Kumar; Garg, Ravindra Kumar; Jain, Amita; Malhotra, Hardeep Singh; Verma, Rajesh; Sharma, Praveen Kumar

2015-12-01

Microbiological confirmation cannot be obtained in approximately two-third patients with tuberculous meningitis. In this study, we sought to identify epidemiological, clinical, cerebrospinal fluid, and imaging parameters that could indicate the possibility of microbiological confirmation among patients with suspected tuberculous meningitis. In this prospective observational study, patients with tuberculous meningitis were evaluated for clinical, laboratory (cerebrospinal fluid microscopy, culture, and polymerase chain reaction), and neuroimaging parameters. All patients received anti-tuberculosis drugs and corticosteroids. The patients were followed for a period of 6 months. Among 118 cases of tuberculous meningitis, there were 43 (36 %) definite (microbiologically confirmed) cases, 59 (50 %) probable and 16 (14 %) possible cases. Among 43 definite cases, tuberculosis polymerase chain reaction (PCR) was positive in 42 (35 %) patients, culture was positive in 1 case and microscopy, after Ziehl-Neelsen staining, was positive in 3 cases. Three factors, modified Barthel index score at admission ≤12 (p = 0.008), cerebrospinal fluid total cell count >100/mm(3) (p = 0.016), and basal exudates on imaging (p = 0.015), were significantly associated with definite tuberculous meningitis. Among 20 patients who died within 6 months, 13 belonged to definite tuberculous meningitis group (p < 0.001). Stage III tuberculous meningitis (p = 0.004), baseline-modified Barthel index score ≤12 (p = 0.013), and positive tuberculosis PCR (p = 0.007) were independently associated with poor outcome on multivariate analysis. Severe disability, cerebrospinal fluid cells >100 mm(3), and basal exudates are significantly related to the presence of microbiologically confirmed definite tuberculous meningitis. Microbiologically confirmed tuberculous meningitis is associated with poorer outcome.

[Primary mucosal tuberculosis of head and neck region: a clinicopathologic analysis of 47 cases].

PubMed

Wang, Shu-yi; Zhu, Jia-xing

2013-10-01

To study the clinicopathologic features, histologic diagnosis and differential diagnosis of primary mucosal tuberculosis (TB) in the head and neck region. Forty-seven cases of primary mucosal TB of the head and neck region were studied by hematoxylin-eosin and Ziehl-Neelsen stains. The clinical and pathologic features were analyzed with review of the literature. The patients included 26 male and 21 female, with mean age 47.1 years (range 14-84 years). There were three sinonasal TB, 19 nasopharyngeal TB, two oropharyngeal TB, 18 laryngeal TB, four middle ear TB, one salivary gland TB and one laryngeal TB complicating laryngeal cancer. The initial symptoms were nasal obstruction, mucopurulent rhinorrhea, epistaxis, snoring, hoarseness, dysphagia, odynophagia, serous otitis, hearing loss, tinnitus, and otalgia. Physical examination result was variable, from an apparently normal mucosa, to an evident mass, or a mucosa with an adenotic or swollen appearance, ulcers, leukoplakic areas, and various combinations thereof. CT and MRI findings included diffuse thickening, a soft-tissue mass, calcification within the mass and bone destruction resembling malignancy. Histologic examination showed granulomas with a central necrotic focus surrounded by epithelioid histiocytes and multinucleated Langhan's giant cells. Acid-fast bacilli were difficult to demonstrate but found in 13/45 cases. Follow-up data were available in 42 patients. Primary TB arising in the head and neck mucosa is rare. It may mimic or co-exist with other conditions. The characteristic histopathology is a granuloma with central caseous necrosis and Langhans'giant cells. Identification of acid-fast bacilli and bacteriologic culture confirm the diagnosis of mycobacterial disease.

Occurrence of Cryptosporidium and Giardia in recycled waters used for irrigation and first description of Cryptosporidium parvum and C. muris in Greece.

PubMed

Spanakos, Gregory; Biba, Anastasia; Mavridou, Athena; Karanis, Panagiotis

2015-05-01

Here, we present the first time findings regarding the occurrence of Cryptosporidium and Giardia in sewage waters and the first molecular characterization of Cryptosporidium species in Greece. Biological treatment plants from three regions in Greece have been investigated. The detection of Cryptosporidium oocysts was by modified Ziehl-Neelsen acid fast (MZN-AF) and by immunofluorescence microscopy (IFT) for Cryptosporidium and Giardia (oo)cysts, whereas nested PCR based on the SSU rDNA assay was used for molecular detection of Cryptosporidium followed by sequencing for the genetic characterization of the species. In total, 73 samples (37 raw sewage samples and 38 of treated water samples) were collected and analyzed. Of the 73 water samples, 4 samples were Cryptosporidium-positive by IFT and staining, 12 samples were Cryptosporidium-positive by nested PCR; 9 samples were Giardia-positive by IFT. We showed that Cryptosporidium cysts are found both in the input and the discharge of the biological treatment plants. Molecular characterization of Cryptosporidium based on the small subunit ribosomal DNA gene resulted in the determination of Cryptosporidium parvum and Cryptosporidium muris Greek isolates. This is the first report of Cryptosporidium and Giardia occurrence in wastewaters and the first molecular identification of Cryptosporidium species in Greek environments. As the treated water is used for irrigation, or it is discharged into the sea, our findings indicate that biological treatment facilities constitute a possible risk for public health because the related species are prevalent in humans; the results invite for further epidemiological investigations to evaluate the real public health risk in Greece.

[BCG infection of the glans penis after intravesical BCG therapy].

PubMed

Michelet, N; Spenatto, N; Viraben, R; Cuny, J-F; Mazet, J; Trechot, P; Barbaud, A; Schmutz, J-L

2008-01-01

BCG therapy is an effective adjuvant treatment for superficial bladder tumors. Therapy involves intravesical instillation of live attenuated Calmette-Guérin bacilli. BCG infection of the glans is a rare local complication associated with this treatment, two cases of which are reported below. Case 1: A 77-year-old man presented relapsing urothelial bladder carcinoma treated by endoscopic resection and BCG therapy. One week after the seventh instillation, severe balanitis developed. Three months later, examination revealed massive painful perimeatal ulceration with yellowish papules in the peripheral regions. Histology revealed epithelioid giant-cell granulomas. Ziehl-Neelsen staining was positive. Slow cure of the lesions was achieved within 12months using double antitubercular antibiotic therapy. Case 2: In a 61-year-old man receiving BCG therapy for relapsing bladder carcinoma in situ, the sixth instillation was considered traumatic since it was highly painful. One week later, papular nodules appeared on the glans with a sclerosing lesion of the balanopreputial sac, dark purple perimeatal papules and a mass beneath the mucosa of the glans. Antibiotic treatment comprising ofloxacin followed by rifampicin for two months proved ineffective. Histology revealed granulomatous dermal lesions with eosinophilic necrosis. Triple antitubercular antibiotic therapy was initiated. The first reported case of BCG infection of the glans in patients undergoing intravesical BCG therapy was published in 1992. Since then, there have been nine other reports. There is no stereotypical clinical presentation. In most cases, an infiltrated erythematosus plaque is seen together with yellowish papules in certain patients. Diagnosis is based upon history and histological examination.

Genotypes and subtypes of Cryptosporidium spp. in diarrheic lambs and goat kids in northern Greece.

PubMed

Papanikolopoulou, Vasiliki; Baroudi, Djamel; Guo, Yaqiong; Wang, Yuanfei; Papadopoulos, Elias; Lafi, Shwakat Q; Abd El-Tawab, Mohamed M; Diakou, Anastasia; Giadinis, Nektarios D; Feng, Yaoyu; Xiao, Lihua

2018-08-01

Inconsistent data exist on the distribution of zoonotic Cryptosporidium species and subtypes in sheep and goats in European countries, and few such data are available from Greece. In this study, 280 fecal specimens were collected from 132 diarrheic lambs and 148 diarrheic goat kids aged 4 to 15 days on 15 farms in northern Greece, and examined for Cryptosporidium spp. using microscopy of Ziehl-Neelsen-stained fecal smears. Cryptosporidium spp. in 80 microscopy-positive fecal specimens (39 from lambs and 41 from goat kids) were genotyped by PCR-RFLP analysis of the small subunit rRNA gene and subtyped by sequence analysis the 60 kDa glycoprotein gene. Among the 33 specimens successfully genotyped, C. parvum was found in 32 and C. xiaoi in one. Seven subtypes belonging to two subtype families (IIa and IId) were identified among the 29 C. parvum specimens successfully subtyped, including IIaA14G2R1 (1/29), IIaA15G2R1 (6/29), IIaA20G1R1 (7/29), IIdA14G2 (1/29), IIdA15G1 (9/29), IIdA16G1 (3/29), and IIdA23G1 (2/29). Lambs were more commonly infected with C. parvum IIa subtypes, whereas goat kids were more with IId subtypes. The results illustrate that C. parvum is prevalent in diarrheic lambs and goat kids in northern Greece and these animals could potentially play a role in epidemiology of human cryptosporidiosis. Copyright © 2018 Elsevier B.V. All rights reserved.

ADENOCARCINOMA AND TUBERCULOSIS OF THE SIGMOID COLON AND FALLOPIAN TUBE--A RARE ASSOCIATION. A CASE REPORT AND REVIEW OF THE LITERATURE.

PubMed

Ionescu, Lidia; Dănilă, R; Ciobanu, Delia; Ciortescu, Irina; Livadariu, Roxana; Timofte, D

2016-01-01

Association of adenocarcinoma and tuberculosis (TB) of the sigmoid colon is a rare clinical condition even in an endemic country as Romania, with challenging diagnosis and treatment. Case report. We present the case of a 57-year-old female patient who was admitted on emergency basis for a diagnosis of obstructive sigmoid adenocarcinoma. The patient was operated on and it an obstructive sigmoid tumor with serosal invasion, adherent (invading) to the body of uterus and left adnexa and urinary bladder serosa, no liver or peritoneal metastases. A sigmoidectomy was performed "en bloc" with subtotal hysterectomy, left adnexectomy and extramucosal cistectomy. The histopathological exam showed a moderately differentiated, ulcerated adenocarcinoma, widely infiltrating the colon wall invading the myometrium. Ziehl Neelsen (ZN) stain identified the presence of metachromatic bacillary structures in the colonic wall, lymph nodes and adnexal areas. Postoperative course was uneventful and the patient was discharged 10 days postoperatively in good clinical condition. After one year when the patient completed the full course of anti-tubercular drugs, a thorough work-up was performed. Colonoscopy, CT of the thorax, abdomen, pelvis showed no signs of recurrence while tumoral marker CEA (1.62 ng/ml - n<3.4) and QFT (Quantiferon-TB Gold) test were within normal range. Discussion and conclusion. Although digestive tuberculosis is included in differential diagnosis for those patients presenting abdominal pain or obstructive digestive symptoms in endemic regions, in this case the absence of TB infection criteria and positive endoscopic biopsy for colonic adenocarcinoma did not allow a complete pre- or perioperative diagnosis.

Prevalence of zoonotic intestinal parasites in domestic and stray dogs in a rural area of Iran.

PubMed

Beiromvand, Molouk; Akhlaghi, Lame; Fattahi Massom, Seyed Hossein; Meamar, Ahmad Reza; Motevalian, Abbas; Oormazdi, Hormozd; Razmjou, Elham

2013-04-01

Certain zoonotic parasites are enteropathogens in dogs that cause serious human disease such as cystic echinococcosis, human alveolar echinococcosis, visceral larva migrans, and ocular larva migrans. This study investigated the prevalence of intestinal parasites in dogs in the Chenaran County, Razavi Khorasan Province, Iran. Sampling was carried out randomly in 17 villages from November 2009 to January 2010. Seventy-seven fecal samples from 28 domestic and 49 stray dogs were examined using sieving/flotation and modified Ziehl-Neelsen staining. Intestinal parasites were found in 51 of the 77 (66%) dogs most common being Toxascaris leonina (29%, 22/77), Toxocara spp. (25%, 19/77), Eimeria spp. (19%, 15/77), Taenia/Echinococcus spp. (18%, 14/77), Sarcocystis spp. (17%, 13/77), and Dicrocoelium dendriticum (14%, 11/77). Lower infection rates of parasites were observed for Trichuris vulpis (6%, 5/77), Cryptosporidium spp. (5%, 4/77), and Physaloptera spp. (3%, 2/77). Prevalence of infection by Dipylidium caninum, Capillaria spp., Cystoisospora spp., and hookworms was similar (1%, 1/77). This study is the first report of the prevalence of intestinal parasites of domestic and stray dogs in Chenaran County, Northeast Iran. The higher prevalence of zoonotic intestinal parasites such as Toxascaris leonina, Toxocara spp. and Taenia/Echinococcus spp. compared to other parasites indicates the need for control programs to minimize the risk of transmission of zoonotic disease, particularly cystic echinococcosis, alveolar echinococcosis, visceral larva migrans, and ocular larva migrans to people living in these areas. Copyright © 2012 Elsevier B.V. All rights reserved.

A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual erythrocytes. II. Further improvements of the staining procedure and some observations with glucose-6-phosphate dehydrogenase deficiency.

PubMed

Van Noorden, C J; Vogels, I M

1985-05-01

A cytochemical method for staining glucose-6-phosphate dehydrogenase (G6PD) activity in individual erythrocytes as reported previously has been optimized further by the incorporation of a number of technical improvements. Analysis of the enzyme content in erythrocytes of normal individuals as well as patients suffering from G6PD deficiency in the homozygous and heterozygous forms allows these three categories to be easily distinguished. Considerable formazan production occurs in most erythrocytes of a healthy person and only a small percentage of the cells appeared to be negative. Two cell populations of almost equal size could be discerned in heterozygotes for G6PD deficiency, one completely negative, the other with a variable amount of formazan per cell. Homozygous deficiency leads to a population of negative cells with a few positive ones after staining. It is concluded that a reliable method has been found for analysis of G6PD deficiency in erythrocytes at the single cell level.

Detection Of Concrete Deterioration By Staining

DOEpatents

Guthrie, Jr., George D.; Carey, J. William

1999-09-21

A method using concentrated aqueous solutions of sodium cobaltinitrite and a rhodamine dye is described which can be used to identify concrete that contains gels formed by the alkali-silica reaction (ASR), and to identify degraded concrete which results in a porous or semi-permeable paste due to carbonation or leaching. These solutions present little health or environmental risk, are readily applied, and rapidly discriminate between two chemically distinct gels; K-rich, Na--K--Ca--Si gels are identified by yellow staining, and alkali-poor, Ca--Si gels are identified by pink staining.

Multispectral image enhancement for H&E stained pathological tissue specimens

NASA Astrophysics Data System (ADS)

Bautista, Pinky A.; Abe, Tokiya; Yamaguchi, Masahiro; Ohyama, Nagaaki; Yagi, Yukako

2008-03-01

The presence of a liver disease such as cirrhosis can be determined by examining the proliferation of collagen fiber from a tissue slide stained with special stain such as the Masson's trichrome(MT) stain. Collagen fiber and smooth muscle, which are both stained the same in an H&E stained slide, are stained blue and pink respectively in an MT-stained slide. In this paper we show that with multispectral imaging the difference between collagen fiber and smooth muscle can be visualized even from an H&E stained image. In the method M KL bases are derived using the spectral data of those H&E stained tissue components which can be easily differentiated from each other, i.e. nucleus, cytoplasm, red blood cells, etc. and based on the spectral residual error of fiber weighting factors are determined to enhance spectral features at certain wavelengths. Results of our experiment demonstrate the capability of multispectral imaging and its advantage compared to the conventional RGB imaging systems to delineate tissue structures with subtle colorimetric difference.

Utility of Gram staining for diagnosis of Malassezia folliculitis.

PubMed

Tu, Wei-Ting; Chin, Szu-Ying; Chou, Chia-Lun; Hsu, Che-Yuan; Chen, Yu-Tsung; Liu, Donald; Lee, Woan-Ruoh; Shih, Yi-Hsien

2018-02-01

Malassezia folliculitis (MalF) mimics acne vulgaris and bacterial folliculitis in clinical presentations. The role of Gram staining in rapid diagnosis of MalF has not been well studied. In our study, 32 patients were included to investigate the utility of Gram staining for MalF diagnosis. The final diagnoses of MalF were determined according to clinical presentation, pathological result and treatment response to antifungal agents. Our results show that the sensitivity and specificity of Gram staining are 84.6% and 100%, respectively. In conclusion, Gram staining is a rapid, non-invasive, sensitive and specific method for MalF diagnosis. © 2017 Japanese Dermatological Association.

The Bacterial Endospore Stain on Schaeffer Fulton using Variation of Methylene Blue Solution

NASA Astrophysics Data System (ADS)

Oktari, A.; Supriatin, Y.; Kamal, M.; Syafrullah, H.

2017-02-01

Endospores staining is the type of staining to recognize the presence spore in bacterial vegetative cells. The bacterial endospores need a staining which can penetrate wall thickness of spore bacteria. A method of endospores staining is Schaeffer Fulton method that used Malachite Green. It is an alkaline substance staining that can staining the spore bacteria. In this research, it have found the alternative staining that can replace Malachite Green solution in spore bacterial stain. The alternative staining used is Methylene Blue solution (0,5 %, 0,7%, and 1% concentration) with pH variation (10, 11, and 12), and varyous heating time (3, 4, and 5 minutes). The all treatments staining have been effect on bacterial spores staining results. The warming time greatly affect the dye to penetrate the walls of bacterial spores, this can be seen in the results with various concentration at pH 10, indicates that the not long warm-up time 3 and 4 minutes, bacterial spores are not stained, while in the longer heating time is 5 minutes bacterial spores stained. This is caused because the longer heating time can make the pores of spore wall is open so that can facilitate the dye to get into the bacterial spores.

Staining Method for Protein Analysis by Capillary Gel Electrophoresis

PubMed Central

Wu, Shuqing; Lu, Joann J; Wang, Shili; Peck, Kristy L.; Li, Guigen; Liu, Shaorong

2009-01-01

A novel staining method and the associated fluorescent dye were developed for protein analysis by capillary SDS-PAGE. The method strategy is to synthesize a pseudo-SDS dye and use it to replace some of the SDS in SDS–protein complexes so that the protein can be fluorescently detected. The pseudo-SDS dye consists of a long, straight alkyl chain connected to a negative charged fluorescent head and binds to proteins just as SDS. The number of dye molecules incorporated with a protein depends on the dye concentration relative to SDS in the sample solution, since SDS and dye bind to proteins competitively. In this work, we synthesized a series of pseudo-SDS dyes, and tested their performances for capillary SDS-PAGE. FT-16 (a fluorescein molecule linked with a hexadodecyl group) seemed to be the best among all the dyes tested. Although the numbers of dye molecules bound to proteins (and the fluorescence signals from these protein complexes) were maximized in the absence of SDS, high-quality separations were obtained when co-complexes of SDS–protein–dye were formed. The migration time correlates well with protein size even after some of the SDS in the SDS–protein complexes was replaced by the pseudo-SDS dye. Under optimized experimental conditions and using a laser-induced fluorescence detector, limits of detection of as low as 0.13 ng/mL (bovine serum albumin) and dynamic ranges over 5 orders of magnitude in which fluorescence response is proportional to the square root of analyte concentration were obtained. The method and dye were also tested for separations of real-world samples from E. coli. PMID:17874848

Post-staining electroblotting for efficient and reliable peptide blotting.

PubMed

Lee, Der-Yen; Chang, Geen-Dong

2015-01-01

Post-staining electroblotting has been previously described to transfer Coomassie blue-stained proteins from polyacrylamide gel onto polyvinylidene difluoride (PVDF) membranes. Actually, stained peptides can also be efficiently and reliably transferred. Because of selective staining procedures for peptides and increased retention of stained peptides on the membrane, even peptides with molecular masses less than 2 kDa such as bacitracin and granuliberin R are transferred with satisfactory results. For comparison, post-staining electroblotting is about 16-fold more sensitive than the conventional electroblotting for visualization of insulin on the membrane. Therefore, the peptide blots become practicable and more accessible to further applications, e.g., blot overlay detection or immunoblotting analysis. In addition, the efficiency of peptide transfer is favorable for N-terminal sequence analysis. With this method, peptide blotting can be normalized for further analysis such as blot overlay assay, immunoblotting, and N-terminal sequencing for identification of peptide in crude or partially purified samples.

Clinical utility of an automated instrument for gram staining single slides.

PubMed

Baron, Ellen Jo; Mix, Samantha; Moradi, Wais

2010-06-01

Gram stains of 87 different clinical samples were prepared by the laboratory's conventional methods (automated or manual) and by a new single-slide-type automated staining instrument, GG&B AGS-1000. Gram stains from either heat- or methanol-fixed slides stained with the new instrument were easy to interpret, and results were essentially the same as those from the methanol-fixed slides prepared as a part of the routine workflow. This instrument is well suited to a rapid-response laboratory where Gram stain requests are commonly received on a stat basis.

Utility of Modified Ultrafast Papanicolaou Stain in Cytological Diagnosis

PubMed Central

Arakeri, Surekha Ulhas

2017-01-01

Introduction Need for minimal turnaround time for assessing Fine Needle Aspiration Cytology (FNAC) has encouraged innovations in staining techniques that require lesser staining time with unequivocal cell morphology. The standard protocol for conventional Papanicolaou (PAP) stain requires about 40 minutes. To overcome this, Ultrafast Papanicolaou (UFP) stain was introduced which reduces staining time to 90 seconds and also enhances the quality. However, reagents required for this were not easily available hence, Modified Ultrafast Papanicolaou (MUFP) stain was introduced subsequently. Aim To assess the efficacy of MUFP staining by comparing the quality of MUFP stain with conventional PAP stain. Materials and Methods FNAC procedure was performed by using 10 ml disposable syringe and 22-23 G needle. Total 131 FNAC cases were studied which were lymph node (30), thyroid (38), breast (22), skin and soft tissue (24), salivary gland (11) and visceral organs (6). Two smears were prepared and stained by MUFP and conventional PAP stain. Scores were given on four parameters: background of smears, overall staining pattern, cell morphology and nuclear staining. Quality Index (QI) was calculated from ratio of total score achieved to maximum score possible. Statistical analysis using chi square test was applied to each of the four parameters before obtaining the QI in both stains. Students t-test was applied to evaluate the efficacy of MUFP in comparison with conventional PAP stain. Results The QI of MUFP for thyroid, breast, lymph node, skin and soft tissue, salivary gland and visceral organs was 0.89, 0.85, 0.89, 0.83, 0.92, and 0.78 respectively. Compared to conventional PAP stain QI of MUFP smears was better in all except visceral organ cases and was statistically significant. MUFP showed clear red blood cell background, transparent cytoplasm and crisp nuclear features. Conclusion MUFP is fast, reliable and can be done with locally available reagents with unequivocal

Whole Blood Cell Staining Device

NASA Technical Reports Server (NTRS)

Sams, Clarence F.; Clift, Vaughan L.; McDonald, Kelly E.

2000-01-01

An apparatus and method for staining particular cell markers is disclosed. The apparatus includes a flexible tube that is reversibly pinched into compartments with one or more clamps. Each compartment of the tube contains a separate reagent and is in selective fluid communication with adjoining compartments.

The Luna stain, an improved selective stain for detection of microsporidian spores in histologic sections

PubMed Central

Peterson, Tracy S.; Spitsbergen, Jan M.; Feist, Stephen W.; Kent, Michael L.

2014-01-01

Microsporidia in histologic sections are most often diagnosed by observing spores in host tissues. Spores are easy to identify if they occur in large aggregates or xenomas when sections are stained with hematoxylin and eosin (H&E). However, individual spores are not frequently detected in host tissues with conventional H&E staining, particularly if spores are scattered within the tissues, areas of inflammation or small spores in nuclei (i.e., Nucleospora salmonis). Hence, a variety of selective stains that enhance visualization of spores are recommended. We discovered that the Luna stain, used to highlight eosinophils, red blood cells and chitin in arthropods and other invertebrates, also stains spores of Pseudoloma neurophilia. We compared this stain to the Gram, Fite’s acid fast, Giemsa, and H&E stains on eight aquatic microsporidian organisms that were readily available in our two laboratories: Loma salmonae, Glugea anomala, Pseudoloma neurophilia, Pleistophora hyphessobryconis, Pleistophora vermiformis, Glugea sp., Steinhausia mytilovum and an unidentified microsporidian from E. sinensis, UK. Based on tinctorial properties and background staining, the Luna stain performed better for detection of 6 of the 8 microsporidia. Gram stain was superior for the two microsporidia from invertebrates, Steinhausia mytilovum and the unidentified microsporidian from E. sinensis. PMID:21848126

Circular Mixture Modeling of Color Distribution for Blind Stain Separation in Pathology Images.

PubMed

Li, Xingyu; Plataniotis, Konstantinos N

2017-01-01

In digital pathology, to address color variation and histological component colocalization in pathology images, stain decomposition is usually performed preceding spectral normalization and tissue component segmentation. This paper examines the problem of stain decomposition, which is a naturally nonnegative matrix factorization (NMF) problem in algebra, and introduces a systematical and analytical solution consisting of a circular color analysis module and an NMF-based computation module. Unlike the paradigm of existing stain decomposition algorithms where stain proportions are computed from estimated stain spectra using a matrix inverse operation directly, the introduced solution estimates stain spectra and stain depths via probabilistic reasoning individually. Since the proposed method pays extra attentions to achromatic pixels in color analysis and stain co-occurrence in pixel clustering, it achieves consistent and reliable stain decomposition with minimum decomposition residue. Particularly, aware of the periodic and angular nature of hue, we propose the use of a circular von Mises mixture model to analyze the hue distribution, and provide a complete color-based pixel soft-clustering solution to address color mixing introduced by stain overlap. This innovation combined with saturation-weighted computation makes our study effective for weak stains and broad-spectrum stains. Extensive experimentation on multiple public pathology datasets suggests that our approach outperforms state-of-the-art blind stain separation methods in terms of decomposition effectiveness.

Laser therapy in plastic surgery: decolorization in port wine stains

NASA Astrophysics Data System (ADS)

Peszynski-Drews, Cezary; Wolf, Leszek

1996-03-01

For the first time laserotherapy is described as a method of port wine stain decolorization in plastic surgery. The authors present their 20-year experience in the treatment of port wine stains with the argon laser and dye laser.

[Improvement of Phi bodies stain and its clinical significance].

PubMed

Gong, Xu-Bo; Lu, Xing-Guo; Yan, Li-Juan; Xiao, Xi-Bin; Wu, Dong; Xu, Gen-Bo; Zhang, Xiao-Hong; Zhao, Xiao-Ying

2009-02-01

The aim of this study was to improve the dyeing method of hydroperoxidase (HPO), to analyze the morphologic features of Phi bodies and to evaluate the clinical application of this method. 128 bone marrow or peripheral blood smears from patients with myeloid and lymphoid malignancies were stained by improved HPO staining. The Phi bodies were observed with detection rate of Phi bodies in different leukemias. 69 acute myeloid leukemia (AML) specimens were chosen randomly, the positive rate and the number of Phi bodies between the improved HPO and POX stain based on the same substrate of 3, 3'diaminobenzidine were compared. The results showed that the shape of bundle-like Phi bodies was variable, long or short. while the nubbly Phi bodies often presented oval and smooth. Club-like Phi bodies were found in M(3). The detection rates of bundle-like Phi bodies in AML M(1)-M(5) were 42.9% (6/14), 83.3% (15/18), 92.0% (23/25), 52.3% (11/21), 33.3% (5/15) respectively, and those of nubbly Phi bodies were 28.6% (4/14), 66.7% (12/18), 11.1% (3/25), 33.3% (7/21), 20.0% (3/15) respectively. The detection rate of bundle-like Phi bodies in M(3) was significantly higher than that in (M(1) + M(2)) or (M(4) + M(5)) groups. The detection rate of nubbly Phi bodies in (M(1) + M(2)) group was higher than that in M(3) group. In conclusion, after improvement of staining method, the HPO stain becomes simple, the detection rate of Phi bodies is higher than that by the previous method, the positive granules are more obvious, and the results become stable. This improved method plays an important role in differentiating AML from ALL, subtyping AML, and evaluating the therapeutic results.

Comparison of staining of mitotic figures by haematoxylin and eosin-and crystal violet stains, in oral epithelial dysplasia and squamous cell carcinoma.

PubMed

Ankle, Madhuri R; Kale, Alka D; Charantimath, Seema

2007-01-01

Mitosis of cells gives rise to tissue integrity. Defects during mitosis bring about abnormalities. Excessive proliferation of cells due to increased mitosis is one such outcome, which is the hallmark in precancer and cancer. The localization of proliferating cells or their precursors may not be obvious and easy. Establishing an easy way to distinguish these mitotic cells will help in grading and understanding their biological potential. Although immunohistochemistry is an advanced method in use, the cost and time factor makes it less feasible for many laboratories. Selective histochemical stains like toluidine blue, giemsa and crystal violet have been used in tissues including the developing brain, neural tissue and skin. 1) To compare the staining of mitotic cells in haematoxylin and eosin with that in crystal violet. 2) To compare the number of mitotic figures present in normal oral mucosa, epithelial dysplasia and oral squamous cell carcinoma in crystal violet-stained sections with that in H and E-stained sections. Ten tissues of normal oral mucosa and 15 tissues each of oral epithelial dysplasia seen in tobacco-associated leukoplakia and squamous cell carcinoma were studied to evaluate the selectivity of 1% crystal violet for mitotic figures. The staining was compared with standard H and E staining. Statistical analysis was done using Mann-Whitney U test. A statistically significant increase in the mean mitotic count was observed in crystal violet-stained sections of epithelial dysplasia as compared to the H and E-stained sections (p=0.0327). A similar increase in the mitotic counts was noted in crystal violet-stained sections of oral squamous cell carcinoma as compared to the H and E-stained sections.(p=0.0443). No significant difference was found in the mitotic counts determined in dysplasia or carcinoma by either the crystal violet (p=0.4429) or the H and E-staining techniques (p=0.2717). One per cent crystal violet provides a definite advantage over the H

Comparison of various staining methods for the detection of Cryptosporidium in cell-free culture.

PubMed

Boxell, Annika; Hijjawi, Nawal; Monis, Paul; Ryan, Una

2008-09-01

The complete development of Cryptosporidium in host cell-free medium first described in 2004, represented a significant advance that can facilitate many aspects of Cryptosporidium research. A current limitation of host cell-free cultivation is the difficulty involved in visualising the life-cycle stages as they are very small in size, morphologically difficult to identify and dispersed throughout the media. This is in contrast to conventional cell culture methods for Cryptosporidium, where it is possible to focus on the host cells and view the foci of infection on the host cells. In the present study, we compared three specific and three non-specific techniques for visualising Cryptosporidium parvum life-cycle stages in cell-free culture; antibody staining using anti-sporozoite and anti-oocyst wall antibodies (Sporo-Glo and Crypto Cel), fluorescent in-situ hybridization (FISH) using a Cryptosporidium specific rRNA oligonucleotide probe and the non-specific dyes; Texas Red, carboxyfluorescein diacetate succinimidyl ester (CFSE) and 4,6' diamino-2-phenylindole dihydrochloride (DAPI). Results revealed that a combination of Sporo-Glo and Crypto Cel staining resulted in easy and reliable identification of all life-cycle stages.

Structure-Preserving Color Normalization and Sparse Stain Separation for Histological Images.

PubMed

Vahadane, Abhishek; Peng, Tingying; Sethi, Amit; Albarqouni, Shadi; Wang, Lichao; Baust, Maximilian; Steiger, Katja; Schlitter, Anna Melissa; Esposito, Irene; Navab, Nassir

2016-08-01

Staining and scanning of tissue samples for microscopic examination is fraught with undesirable color variations arising from differences in raw materials and manufacturing techniques of stain vendors, staining protocols of labs, and color responses of digital scanners. When comparing tissue samples, color normalization and stain separation of the tissue images can be helpful for both pathologists and software. Techniques that are used for natural images fail to utilize structural properties of stained tissue samples and produce undesirable color distortions. The stain concentration cannot be negative. Tissue samples are stained with only a few stains and most tissue regions are characterized by at most one effective stain. We model these physical phenomena that define the tissue structure by first decomposing images in an unsupervised manner into stain density maps that are sparse and non-negative. For a given image, we combine its stain density maps with stain color basis of a pathologist-preferred target image, thus altering only its color while preserving its structure described by the maps. Stain density correlation with ground truth and preference by pathologists were higher for images normalized using our method when compared to other alternatives. We also propose a computationally faster extension of this technique for large whole-slide images that selects an appropriate patch sample instead of using the entire image to compute the stain color basis.

Gram staining for the treatment of peritonsillar abscess.

PubMed

Takenaka, Yukinori; Takeda, Kazuya; Yoshii, Tadashi; Hashimoto, Michiko; Inohara, Hidenori

2012-01-01

Objective. To examine whether Gram staining can influence the choice of antibiotic for the treatment of peritonsillar abscess. Methods. Between 2005 and 2009, a total of 57 cases of peritonsillar abscess were analyzed with regard to cultured bacteria and Gram staining. Results. Only aerobes were cultured in 16% of cases, and only anaerobes were cultured in 51% of cases. Mixed growth of aerobes and anaerobes was observed in 21% of cases. The cultured bacteria were mainly aerobic Streptococcus, anaerobic Gram-positive cocci, and anaerobic Gram-negative rods. Phagocytosis of bacteria on Gram staining was observed in 9 cases. The bacteria cultured from these cases were aerobic Streptococcus, anaerobic Gram-positive cocci, and anaerobic Gram-negative rods. The sensitivity of Gram staining for the Gram-positive cocci and Gram-negative rods was 90% and 64%, respectively. The specificity of Gram staining for the Gram-positive cocci and Gram-negative rods was 62% and 76%, respectively. Most of the Gram-positive cocci were sensitive to penicillin, but some of anaerobic Gram-negative rods were resistant to penicillin. Conclusion. When Gram staining shows only Gram-positive cocci, penicillin is the treatment of choice. In other cases, antibiotics effective for the penicillin-resistant organisms should be used.

Sudan stain of fecal fat: new insight into an old test.

PubMed

Khouri, M R; Huang, G; Shiau, Y F

1989-02-01

The 72-h fecal fat determination is used as the gold standard to document the presence of steatorrhea. Although the Sudan stain for fecal fat is advocated as a sensitive screening test, a quantitative correlation between the 72-h fecal fat quantitation and the fecal Sudan stain is lacking. This study was designed to examine the staining properties of different classes of purified lipids in an experimentally defined artificial matrix, and to elucidate the reasons for the lack of quantitative correlation between these two tests. Our results indicate that the "neutral fat" stain without acidification or heating identifies triglyceride; and at an appropriate pH, the "neutral stain" also identifies fatty acid. The "split fat" stain with acidification and heating identifies both triglyceride and fatty acid. After acidification, fatty acid soaps are converted to the nonionized fatty acid. Thus, fatty acid soaps can be identified indirectly as fat droplets that are stained by the split fat stain. Although cholesterol is stained with Sudan stain after heating, upon cooling, cholesterol forms crystals of anhydrous cholesterol, making its staining pattern distinct. Neither the neutral fat nor the split fat stain can detect phospholipid or cholesteryl ester. The 72-h fecal fat determination is a measure of the total fatty acid content after a specimen is saponified. The resulting fatty acids are derived from a variety of endogenous and exogenous sources, including free fatty acids, soaps of fatty acids, triglycerides, cholesterol esters, and phospholipids. Therefore, the 72-h fecal fat quantitation does not differentiate between the primary sources of the measured fatty acid. It is concluded that the 72-h fecal fat determination is not specific for documenting triglyceride (fat) malabsorption. Until new methods are developed that specifically measure fecal triglyceride and fatty acid, the Sudan stain of fecal fat appears to be a more specific method for detecting the presence

A flow-cytometric gram-staining technique for milk-associated bacteria.

PubMed

Holm, Claus; Jespersen, Lene

2003-05-01

A Gram-staining technique combining staining with two fluorescent stains, Oregon Green-conjugated wheat germ agglutinin (WGA) and hexidium iodide (HI) followed by flow-cytometric detection is described. WGA stains gram-positive bacteria while HI binds to the DNA of all bacteria after permeabilization by EDTA and incubation at 50 degrees C for 15 min. For WGA to bind to gram-positive bacteria, a 3 M potassium chloride solution was found to give the highest fluorescence intensity. A total of 12 strains representing some of the predominant bacterial species in bulk tank milk and mixtures of these were stained and analyzed by flow cytometry. Overall, the staining method showed a clear differentiation between gram-positive and gram-negative bacterial populations. For stationary-stage cultures of seven gram-positive bacteria and five gram-negative bacteria, an average of 99% of the cells were correctly interpreted. The method was only slightly influenced by the growth phase of the bacteria or conditions such as freezing at -18 degrees C for 24 h. For any of these conditions, an average of at least 95% of the cells were correctly interpreted. When stationary-stage cultures were stored at 5 degrees C for 14 days, an average of 86% of the cells were correctly interpreted. The Gram-staining technique was applied to the flow cytometry analysis of bulk tank milk inoculated with Staphylococcus aureus and Escherichia coli. These results demonstrate that the technique is suitable for analyzing milk samples without precultivation.

Xpert MTB/RIF assay for diagnosis of pulmonary tuberculosis in sputum specimens in remote health care facility.

PubMed

Geleta, Dereje Assefa; Megerssa, Yoseph Cherinet; Gudeta, Adugna Negussie; Akalu, Gizachew Taddesse; Debele, Melaku Tesfaye; Tulu, Kassu Desta

2015-10-19

Xpert MTB/RIF assay is considered as a great advance over conventional smear and culture in the diagnosis of TB and MDR-TB by simultaneously detecting M.tuberculosis and rifampicin resistance bacilli. However, very little information regarding the performance characteristics of Xpert MTB/RIF assay is available in Ethiopia. Therefore, the purpose of this study was to evaluate the performance of Xpert MTB/RIF assay compared to conventional sputum smear and culture methods for the diagnosis of pulmonary tuberculosis in remote health care facility. A paired expectorated sputum samples were obtained from 227 consecutively recruited patients with signs and symptoms suggestive of tuberculosis at Karamara hospital during December 2013 to May 2014. One of the sputum specimen was tested directly by Ziehl-Neelsen staining and Xpert MTB/RIF assay without NALC-NaOH decontamination. The other of pair of sputa specimen was cultured for isolation of TB bacilli by conventional methods. Diagnostic performance of Xpert MTB/RIF assay and AFB smear microscopy were calculated against culture as the gold standard. Overall 25.5% (58/227) samples were positive for Mycobacterium tuberculosis complex (MTBC) by MGIT and/or LJ media of which 36.2% (21/58) and 65.5% (35/58) were positive by AFB smear microscopy and Xpert MTB/RIF respectively. The sensitivity, specificity, as well as the positive and negative predictive value of Xpert MTB/RIF assay were 65.5% (95% CI: 53.3-77.7%), 96.3% (95% CI: 93.4-99.2%), 86.4% (95% CI: 76.2-96.5%), and 88.6% (95% CI: 83.9-93.3%) respectively. Eighteen of 58 (31%) cases that were smear microscopy negative, were positive by Xpert MTB/RIF assay. Although Xpert MTB/RIF assay demonstrated high sensitivity in detecting MTBC in sputum specimens compared with conventional AFB smear microscopy, it demonstrated suboptimal sensitivity in smear negative patients compared to conventional culture.

Development of a quantitative validation method for forensic investigation of human spermatozoa using a commercial fluorescence staining kit (SPERM HY-LITER™ Express).

PubMed

Takamura, Ayari; Watanabe, Ken; Akutsu, Tomoko

2016-11-01

In investigations of sexual assaults, as well as in identifying a suspect, the detection of human sperm is important. Recently, a kit for fluorescent staining of human spermatozoa, SPERM HY-LITER™, has become available. This kit allows for microscopic observation of the heads of human sperm using an antibody tagged with a fluorescent dye. This kit is specific to human sperm and provides easy detection by luminescence. However, criteria need to be established to objectively evaluate the fluorescent signals and to evaluate the staining efficiency of this kit. These criteria will be indispensable for investigation of forensic samples. In the present study, the SPERM HY-LITER™ Express kit, which is an improved version of SPERM HY-LITER™, was evaluated using an image analysis procedure using Laplacian and Gaussian methods. This method could be used to automatically select important regions of fluorescence produced by sperm. The fluorescence staining performance was evaluated and compared under various experimental conditions, such as for aged traces and in combination with other chemical staining methods. The morphological characteristics of human sperm were incorporated into the criteria for objective identification of sperm, based on quantified features of the fluorescent spots. Using the criteria, non-specific or insignificant fluorescent spots were excluded, and the specificity of the kit for human sperm was confirmed. The image analysis method and criteria established in this study are universal and could be applied under any experimental conditions. These criteria will increase the reliability of operator judgment in the analysis of human sperm samples in forensics.

Staining of Tissue Sections for Electron Microscopy with Heavy Metals

PubMed Central

Watson, Michael L.

1958-01-01

Descriptions of three heavy metal stains and methods of application to tissue sections for electron microscopy are presented. Lead hydroxide stains rather selectively two types of particles in liver: those associated with the endoplasmic reticulum and containing ribonucleic acid and other somewhat larger particles. Barium hydroxide emphasizes certain bodies within vesicles of the Golgi region of hepatic cells. Alkalized lead acetate is useful as a general stain, as are also lead and barium hydroxides. PMID:13610936

Gram staining with an automatic machine.

PubMed

Felek, S; Arslan, A

1999-01-01

This study was undertaken to develop a new Gram-staining machine controlled by a micro-controller and to investigate the quality of slides that were stained in the machine. The machine was designed and produced by the authors. It uses standard 220 V AC. Staining, washing, and drying periods are controlled by a timer built in the micro-controller. A software was made that contains a certain algorithm and time intervals for the staining mode. One-hundred and forty smears were prepared from Escherichia coli, Staphylococcus aureus, Neisseria sp., blood culture, trypticase soy broth, direct pus and sputum smears for comparison studies. Half of the slides in each group were stained with the machine, the other half by hand and then examined by four different microbiologists. Machine-stained slides had a higher clarity and less debris than the hand-stained slides (p < 0.05). In hand-stained slides, some Gram-positive organisms showed poor Gram-positive staining features (p < 0.05). In conclusion, we suggest that Gram staining with the automatic machine increases the staining quality and helps to decrease the work load in a busy diagnostic laboratory.

Clinical Utility of an Automated Instrument for Gram Staining Single Slides ▿

PubMed Central

Baron, Ellen Jo; Mix, Samantha; Moradi, Wais

2010-01-01

Gram stains of 87 different clinical samples were prepared by the laboratory's conventional methods (automated or manual) and by a new single-slide-type automated staining instrument, GG&B AGS-1000. Gram stains from either heat- or methanol-fixed slides stained with the new instrument were easy to interpret, and results were essentially the same as those from the methanol-fixed slides prepared as a part of the routine workflow. This instrument is well suited to a rapid-response laboratory where Gram stain requests are commonly received on a stat basis. PMID:20410348

An improved silver staining procedure for schizodeme analysis in polyacrylamide gradient gels.

PubMed

Gonçalves, A M; Nehme, N S; Morel, C M

1990-01-01

A simple protocol is described for the silver staining of polyacrylamide gradient gels used for the separation of restriction fragments of kinetoplast DNA [schizodeme analysis of trypanosomatids (Morel et al., 1980)]. The method overcomes the problems of non-uniform staining and strong background color which are frequently encountered when conventional protocols for silver staining of linear gels are applied to gradient gels. The method described has proven to be of general applicability for DNA, RNA and protein separations in gradient gels.

A Flow-Cytometric Gram-Staining Technique for Milk-Associated Bacteria

PubMed Central

Holm, Claus; Jespersen, Lene

2003-01-01

A Gram-staining technique combining staining with two fluorescent stains, Oregon Green-conjugated wheat germ agglutinin (WGA) and hexidium iodide (HI) followed by flow-cytometric detection is described. WGA stains gram-positive bacteria while HI binds to the DNA of all bacteria after permeabilization by EDTA and incubation at 50°C for 15 min. For WGA to bind to gram-positive bacteria, a 3 M potassium chloride solution was found to give the highest fluorescence intensity. A total of 12 strains representing some of the predominant bacterial species in bulk tank milk and mixtures of these were stained and analyzed by flow cytometry. Overall, the staining method showed a clear differentiation between gram-positive and gram-negative bacterial populations. For stationary-stage cultures of seven gram-positive bacteria and five gram-negative bacteria, an average of 99% of the cells were correctly interpreted. The method was only slightly influenced by the growth phase of the bacteria or conditions such as freezing at −18°C for 24 h. For any of these conditions, an average of at least 95% of the cells were correctly interpreted. When stationary-stage cultures were stored at 5°C for 14 days, an average of 86% of the cells were correctly interpreted. The Gram-staining technique was applied to the flow cytometry analysis of bulk tank milk inoculated with Staphylococcus aureus and Escherichia coli. These results demonstrate that the technique is suitable for analyzing milk samples without precultivation. PMID:12732558

Putting vital stains in context.

PubMed

Efron, Nathan

2013-07-01

While vital staining remains a cornerstone in the diagnosis of ocular disease and contact lens complications, there are many misconceptions regarding the properties of commonly used dyes by eye-care practitioners and what is and what is not corneal staining after instillation of sodium fluorescein. Similarly, the proper use and diagnostic utility of rose Bengal and lissamine green B, the other two ophthalmic dyes commonly used for assessing ocular complications, have similarly remained unclear. Due to the limitations of vital stains for definitive diagnosis, concomitant signs and symptoms in addition to a complete patient history are required. Over the past decade, there have been many reports of a type of corneal staining--often referred to as solution-induced corneal staining (SICS)--that is observed with the use of multipurpose solutions in combination with soft lenses, more specifically silicone hydrogel lenses. Some authors believe that SICS is a sign of lens/solution incompatibility; however, new research shows that SICS may be neither a measure of lens/solution biocompatibility nor 'true' corneal staining, as that observed in pathological situations. A large component of SICS may be a benign phenomenon, known as preservative-associated transient hyperfluorescence (PATH). There is a lack of correlated signs and/or symptoms with SICS/PATH. Several properties of SICS/PATH, such as appearance and duration, differentiate it from pathological corneal staining. This paper reviews the properties of vital stains, their use and limitations in assessment of the ocular surface, the aetiology of corneal staining, characteristics of SICS/PATH that differentiate it from pathological corneal staining and what the SICS/PATH phenomenon means for contact lens-wearing patients. © 2012 The Author. Clinical and Experimental Optometry © 2012 Optometrists Association Australia.

'Ease of interpretation' of cytological smears stained with modified ultrafast papanicolaou stain: Interobserver agreement and reproducibility.

PubMed

Uthamalingam, Preithy; Sathish Kumar, Thabasum; Venus, Albina; Sekar, Preethi; Muthusamy, Rajeshwari K; Mehta, Sangita

2018-04-01

Since its inception in 1995, the Ultrafast Papanicoloau (UFPAP) cytological stain has undergone a number of modifications to suit the local availability of reagents and cost in different set ups. However, the reported results have been uniformly encouraging. We designed a study to investigate the inter-observer agreement in 'perceived ease of interpretation' of cytological smears stained with Modified Ultrafast Papanicoloau stain (MUFPAP). After a small pilot study, we prospectively stained air-dried fine needle aspirate smears (FNACs) and Body Fluid smears with the standardized MUFPAP stain. The MUFPAP stained slides were evaluated in tandem with other routine cytological stains as well as independently by two pathologists. Two rater kappa was used to determine the agreement. The study included 93 fluids and 34 FNACs. A vast majority of the cases stained with MUFPAP were rated 'better' than the routine stains in terms of 'overall ease of interpretation' with considerable agreement. The agreement tended to be better for FNACs than fluid specimens. Cases with malignant pathology demonstrated a perfect agreement (kappa = 1) between the raters in terms of 'overall ease of interpretation' (91.7% cases were rated 'very good' by each pathologist) when compared to cases with benign pathology (kappa = 0.52). Nuclear characteristics were appreciated with a better agreement than other parameters. Modified UFPAP stain appears to be quick, reliable, cost-effective alternative in cytology, especially for detecting malignant cells in smears with low cellularity. Its specific advantage is robust nuclear staining against a clear background. © 2018 Wiley Periodicals, Inc.

[Automated analysis of bacterial preparations manufactured on automatic heat fixation and staining equipment].

PubMed

2012-01-01

Heat fixation of preparations was made in the fixation bath designed by EMKO (Russia). Programmable "Emkosteiner" (EMKO, Russia) was used for trial staining. Reagents set Micko-GRAM-NITsF was applied for Gram's method of staining. It was demostrated that automatic smear fixation equipment and programmable staining ensure high-quality imaging (1% chromaticity variation) good enough for standardization of Gram's staining of microbial preparations.

Comparison of the haematoxylin basic fuchsin picric acid method and the fluorescence of haematoxylin and eosin stained sections for the identification of early myocardial infarction.

PubMed Central

Al-Rufaie, H K; Florio, R A; Olsen, E G

1983-01-01

A retrospective study has been carried out on the necropsy material from 30 patients who have died after a clinically diagnosed myocardial infarction. This study has been undertaken to compare the reliability of the fluorescence of infarcted myocardium when stained by haematoxylin and eosin and an adjacent section stained by the haematoxylin basic fuchsin picric acid (HBFP) method to detect early ischaemia. The results showed that the fluorescence technique is reliable, reproducible and coincides with the findings obtained by HBFP stain. Images PMID:6189866

Staining of retinal neurons in the isolated eyecup by extracellular horseradish peroxidase injection.

PubMed

Amthor, F R; Jackson, C A

1986-01-01

A reliable method has been developed for the staining of mammalian retinal neurons in the isolated eyecup. Small injections of horseradish peroxidase are made at a number of places on the retinal surface while the hemisected eyecup preparation floats in Eagle's medium. After 30 min, the retinas are removed, fixed, and reacted with diaminobenzidine. Golgi-like staining of fine dendrites, spines and axons of ganglion cells is achieved. The method also stains amacrine, horizontal and bipolar cells.

Fluorescent staining for leukocyte chemotaxis. Eosinophil-specific fluorescence with aniline blue.

PubMed

McCrone, E L; Lucey, D R; Weller, P F

1988-11-10

To overcome problems associated with the quantitation of human eosinophil chemotaxis in micropore filters, we have developed a fluorescent method of specifically staining eosinophils in chemotactic filters. A neutral solution of aniline blue yielded bright green fluorescent staining of the cytoplasmic granules of eosinophils. Other leukocytes and contaminating neutrophils potentially present with eosinophils did not fluoresce with aniline blue. The fluorescent staining eosinophils within filters provided bright, non-fading images that facilitated visual microscopic counting and were of sufficiently high contrast, unlike those with conventional eosinophil stains, to allow image analyzer based enumeration of eosinophil chemotactic responses at levels through the filters. Although not cell type-specific, congo red and ethidium bromide also provided high contrast, fluorescent images of all leukocyte types within chemotactic filters. Fluorescent staining with aniline blue constitutes a rapid, stable and eosinophil-specific stain that facilitates the visual or image analyzer-based quantitation of eosinophil chemotaxis.

Splenitis in 33 Dogs.

PubMed

Ferri, F; Zini, E; Auriemma, E; Castagnaro, M; Coppola, L M; Peano, A; Martella, V; Decaro, N; Kuhnert, P; Ferro, S

2017-01-01

Splenitis is uncommonly reported in dogs. Herein, the authors describe its prevalence, clinical findings and outcomes, histologic patterns, and causes. Splenic samples of dogs diagnosed with splenitis between 2005 and 2013 were collected and stained with hematoxylin and eosin, Gram, green-Gram, Giemsa, periodic acid-Schiff, and Ziehl-Neelsen. Samples were processed for polymerase chain reaction (PCR) to detect bacteria, fungi, and protozoa ( Leishmania infantum, Hepatozoon canis). Thirty-three of 660 splenic samples (5%) had splenitis. Clinical findings and outcomes were available in 19 dogs (58%); 49% had weakness, 33% had fever, and 84% survived. The most frequent inflammatory patterns included purulent splenitis (27%), pyogranulomatous splenitis (24%), and neutrophilic perisplenitis (15%). One dog had a putative diagnosis of primary splenitis; in 8 dogs, microorganisms were identified histologically or by PCR in the spleen without obvious comorbidities. Twenty-four dogs (73%) had concurrent diseases; a permissive role in the development of splenitis was suspected in 21 of these cases. Histologic examination identified the cause of splenitis in 10 dogs. Bacteria were identified by PCR in 23 cases, but the bacteria were confirmed histologically in only 6 of these. Leishmania was detected with PCR in 6 dogs. Leishmania was identified in 1 dog and H. canis in another histologically, but both were PCR negative. Fungi were identified in 8 spleens by PCR and in 1 by histology. This study suggests that splenitis is uncommon in dogs and is frequently associated with systemic diseases. Prognosis is favorable in most cases. Identification of bacteria, fungi, and protozoa in the spleens of affected dogs with PCR should be interpreted cautiously, because the findings are not confirmed histologically in many cases.

Rapid Diagnosis of Tuberculosis by Real-Time High-Resolution Imaging of Mycobacterium tuberculosis Colonies.

PubMed

Ghodbane, Ramzi; Asmar, Shady; Betzner, Marlena; Linet, Marie; Pierquin, Joseph; Raoult, Didier; Drancourt, Michel

2015-08-01

Culture remains the cornerstone of diagnosis for pulmonary tuberculosis, but the fastidiousness of Mycobacterium tuberculosis may delay culture-based diagnosis for weeks. We evaluated the performance of real-time high-resolution imaging for the rapid detection of M. tuberculosis colonies growing on a solid medium. A total of 50 clinical specimens, including 42 sputum specimens, 4 stool specimens, 2 bronchoalveolar lavage fluid specimens, and 2 bronchial aspirate fluid specimens were prospectively inoculated into (i) a commercially available Middlebrook broth and evaluated for mycobacterial growth indirectly detected by measuring oxygen consumption (standard protocol) and (ii) a home-made solid medium incubated in an incubator featuring real-time high-resolution imaging of colonies (real-time protocol). Isolates were identified by Ziehl-Neelsen staining and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Use of the standard protocol yielded 14/50 (28%) M. tuberculosis isolates, which is not significantly different from the 13/50 (26%) M. tuberculosis isolates found using the real-time protocol (P = 1.00 by Fisher's exact test), and the contamination rate of 1/50 (2%) was not significantly different from the contamination rate of 2/50 (4%) using the real-time protocol (P = 1.00). The real-time imaging protocol showed a 4.4-fold reduction in time to detection, 82 ± 54 h versus 360 ± 142 h (P < 0.05). These preliminary data give the proof of concept that real-time high-resolution imaging of M. tuberculosis colonies is a new technology that shortens the time to growth detection and the laboratory diagnosis of pulmonary tuberculosis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Experimental mycobacteriosis in striped bass Morone saxatilis

USGS Publications Warehouse

Gauthier, David T.; Rhodes, M.W.; Vogelbein, W.K.; Kator, H.; Ottinger, C.A.

2003-01-01

Striped bass Morone saxatilis were infected intraperitoneally with approximately 105 Mycobacterium marinum, M. shottsii sp. nov., or M. gordonae. Infected fish were maintained in a flow-through freshwater system at 18 to 21??C, and were examined histologically and bacteriologically at 2, 4, 6, 8, 17, 26, 36 and 45 wk post-infection (p.i.). M. marinum caused acute peritonitis, followed by extensive granuloma development in the mesenteries, spleen and anterior kidney. Granulomas in these tissues underwent a temporal progression of distinct morphological stages, culminating in well-circumscribed lesions surrounded by normal or healing tissue. Mycobacteria were cultured in high numbers from splenic tissue at all times p.i. Standard Ziehl-Neelsen staining, however, did not demonstrate acid-fast rods in most early inflammatory foci and granulomas. Large numbers of acid-fast rods were present in granulomas beginning at 8 wk p.i. Between 26 and 45 wk p.i., reactivation of disease was observed in some fish, with disintegration of granulomas, renewed inflammation, and elevated splenic bacterial densities approaching 109 colony-forming units g-1. Infection with M. shottsii or M. gordonae did not produce severe pathology. Mild peritonitis was followed by granuloma formation in the mesenteries, but, with 1 exception, granulomas were not observed in the spleen or anterior kidney. M. shottsii and M. gordonae both established persistent infections in the spleen, but were present at densities at least 2 orders of magnitude less than M. marinum at all time points observed. Granulomas in the mesenteries of M. shottsii- and M. gordonae-infected fish resolved over time, and no reactivation of disease was observed.

Opportunistic and other intestinal parasitic infections in AIDS patients, HIV seropositive healthy carriers and HIV seronegative individuals in southwest Ethiopia.

PubMed

Mariam, Zelalem T; Abebe, Gemeda; Mulu, Andargachew

2008-12-01

Human Immunodeficiency Virus (HIV) infection leads to acquired immunodeficiency syndrome (AIDS) and major causes of morbidity and mortality of such patients are opportunistic infections caused by viral, bacterial, fungal and parasitic pathogens. To determine the magnitude of opportunistic and non-opportunistic intestinal parasitic infections among AIDS patients and HIV positive carrier individuals. Cross-sectional study was conducted among AIDS patients, HIV positive healthy carriers and HIV negative individuals in Jimma University Hospital, Mother Theresa Missionary Charity Centre, Medan Acts Projects and Mekdim HIV positive persons and AIDS orphans' national association from January to May, 2004. Convenient sampling technique was employed to identify the study subjects and hence a total of 160 subjects were included. A pre-tested structured questionnaire was used to collect socio-demographic data of the patients. Stool samples were examined by direct saline, iodine wet mount, formol-ether sedimentation concentration, oocyst concentration and modified Ziehl-Neelsen staining technique. Out of 160 persons enrolled in this study 100 (62.5%) (i.e. 65 male and 35 female) were infected with one or more intestinal parasites. The highest rate 36 (69.2%) of intestinal parasites were observed among HIV/AIDS patients, followed by HIV positive healthy carriers 35 (61.4%) of and HIV negative individuals (29 (56.9%). Isospora belli 2 (3.9%), Cryptosporidum parvum 8 (15.4%), Strongyloides stercoralis 6 (11.5%) and Blastocystis 2 (3.9%) were found only in HIV/AIDS groups I. belli, C. parvum, S. stercoralis and Blastocystis are the major opportunistic intestinal parasites observed in HIV/AIDS patients. Therefore, early detection and treatment of these parasites are important to improve the quality of life of HIV/AIDS patients with diarrhoea.

Common bacterial isolates, clinical outcome and TB meningitis in children admitted at Morogoro Regional Referral Hospital, Tanzania.

PubMed

Chambuso, Ramadhani Salum; Mkhoi, Mkhoi Lord; Kaambo, Evelyn

2017-01-01

Bacterial meningitis is still one of the major causes of deaths, disabilities, and mental retardation in children in Morogoro region. To study the current meningitis burden, we evaluated the common bacterial isolates and clinical outcome of the disease in the region. We conducted a hospital-based prospective study on 1352 children aged between 7 days and 12 years admitted in pediatric wards at Morogoro Regional Referral Hospital for 7 months. Cerebrospinal fluid (CSF) for laboratory microbiological examination was collected by lumbar puncture in 72 children with signs and symptoms of meningitis. Latex agglutination test was used to confirm the bacterial colonies in the culture. Chi-square test was used for relative risk with 95% confidence intervals; statistical analysis and tests were considered statistically significant when P < 0.05. Among 72 CSF samples, 23 (31.9%) were positive for Streptococcus pneumoniae, 6 (8.3%) for Haemophilus influenzae, 5 (6.9%) for Group B Streptococcus, 3 (4.2%) for Escherichia coli, and 1 (1.4%) was positive for Mycobacterium tuberculosis. Furthermore, 34 CSF samples showed no bacteria growth in the culture media. In addition, 39 children (54.2%) did not respond to the treatment, whereas 79.5% (n = 39) of them died, while 20.5% (n = 39) of them were referred to a tertiary hospital. Nevertheless, the incidence of meningitis infection was 5.3% (n = 1352) among the admitted children. S. pneumoniae was the major laboratory-confirmed bacterial isolate associated with meningitis in children. We report for the first time the presence of tuberculous meningitis in Morogoro region. Ziehl-Neelsen staining for acid-fast bacilli should be mandatory for any case clinically suspected for meningitis.

Digital simulation of staining in histopathology multispectral images: enhancement and linear transformation of spectral transmittance.

PubMed

Bautista, Pinky A; Yagi, Yukako

2012-05-01

Hematoxylin and eosin (H&E) stain is currently the most popular for routine histopathology staining. Special and/or immuno-histochemical (IHC) staining is often requested to further corroborate the initial diagnosis on H&E stained tissue sections. Digital simulation of staining (or digital staining) can be a very valuable tool to produce the desired stained images from the H&E stained tissue sections instantaneously. We present an approach to digital staining of histopathology multispectral images by combining the effects of spectral enhancement and spectral transformation. Spectral enhancement is accomplished by shifting the N-band original spectrum of the multispectral pixel with the weighted difference between the pixel's original and estimated spectrum; the spectrum is estimated using M < N principal component (PC) vectors. The pixel's enhanced spectrum is transformed to the spectral configuration associated to its reaction to a specific stain by utilizing an N × N transformation matrix, which is derived through application of least mean squares method to the enhanced and target spectral transmittance samples of the different tissue components found in the image. Results of our experiments on the digital conversion of an H&E stained multispectral image to its Masson's trichrome stained equivalent show the viability of the method.

Evaluation of Papanicolaou stain for studying micronuclei in buccal cells under field conditions.

PubMed

Ayyad, Sohair B A; Israel, Ebenezer; El-Setouhy, Maged; Nasr, Ghada Radwan; Mohamed, Mostafa K; Loffredo, Christopher A

2006-01-01

To compare Papanicolaou (Pap) and May-Grünwald Giemsa (MGG) stain as 2 techniques for staining for buccal mucosal cells to detect micronuclei (MN) infield studies. Eighty cytologic smears (2 per individual) were taken from the buccal mucosa of 40 cigarette smokers recruited at a rural village in Egypt. Forty smears were stained with Pap stain and 40 with MGG stain. All were assessed for cellularity and scored for MN. Pap stain was faster and easier to process and transport in the field study than was MGG stain. Regarding MGG smears, bacteria and cell debris masked the MN as compared to Pap smears, in which the fixative destroyed the bacteria and made the cell boundaries clearly demarcated. Using Pap stain, MN were seen easily in transparent cytoplasm. Pap stain is the preferred method infield studies for scoring and detecting MN in cells of buccal mucosa.

Application of immunohistochemical staining to detect antigen destruction as a measure of tissue damage.

PubMed

Onul, Abdullah; Colvard, Michael D; Paradise, William A; Elseth, Kim M; Vesper, Benjamin J; Gouvas, Eftychia; Deliu, Zane; Garcia, Kelly D; Pestle, William J; Radosevich, James A

2012-09-01

Electrocautery and directed energy devices (DEDs) such as lasers, which are used in surgery, result in tissue damage that cannot be readily detected by traditional histological methods, such as hematoxylin and eosin staining. Alternative staining methods, including 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to stain live tissue, have been reported. Despite providing superior detection of damaged tissue relative to the hematoxylin and eosin (H&E) method, the MTT method possesses a number of drawbacks, most notably that it must be carried out on live tissue samples. Herein, we report the development of a novel staining method, "antigen destruction immunohistochemistry" (ADI), which can be carried out on paraffin-embedded tissue. The ADI method takes advantage of epitope loss to define the area of tissue damage and provides many of the benefits of live tissue MTT staining without the drawbacks inherent to that method. In addition, the authors provide data to support the use of antibodies directed at a number of gene products for use in animal tissue for which there are no species-specific antibodies commercially available, as well as an example of a species-specific direct antibody. Data are provided that support the use of this method in many tissue models, as well as evidence that ADI is comparable to the live tissue MTT method.

Application of Immunohistochemical Staining to Detect Antigen Destruction as a Measure of Tissue Damage

PubMed Central

Onul, Abdullah; Colvard, Michael D.; Paradise, William A.; Elseth, Kim M.; Vesper, Benjamin J.; Gouvas, Eftychia; Deliu, Zane; Garcia, Kelly D.; Pestle, William J.

2012-01-01

Electrocautery and directed energy devices (DEDs) such as lasers, which are used in surgery, result in tissue damage that cannot be readily detected by traditional histological methods, such as hematoxylin and eosin staining. Alternative staining methods, including 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to stain live tissue, have been reported. Despite providing superior detection of damaged tissue relative to the hematoxylin and eosin (H&E) method, the MTT method possesses a number of drawbacks, most notably that it must be carried out on live tissue samples. Herein, we report the development of a novel staining method, “antigen destruction immunohistochemistry” (ADI), which can be carried out on paraffin-embedded tissue. The ADI method takes advantage of epitope loss to define the area of tissue damage and provides many of the benefits of live tissue MTT staining without the drawbacks inherent to that method. In addition, the authors provide data to support the use of antibodies directed at a number of gene products for use in animal tissue for which there are no species-specific antibodies commercially available, as well as an example of a species-specific direct antibody. Data are provided that support the use of this method in many tissue models, as well as evidence that ADI is comparable to the live tissue MTT method. PMID:22723525

Improvement of malaria diagnostic system based on acridine orange staining.

PubMed

Kimura, Masatsugu; Teramoto, Isao; Chan, Chim W; Idris, Zulkarnain Md; Kongere, James; Kagaya, Wataru; Kawamoto, Fumihiko; Asada, Ryoko; Isozumi, Rie; Kaneko, Akira

2018-02-07

Rapid diagnosis of malaria using acridine orange (AO) staining and a light microscope with a halogen lamp and interference filter was deployed in some malaria-endemic countries. However, it has not been widely adopted because: (1) the lamp was weak as an excitation light and the set-up did not work well under unstable power supply; and, (2) the staining of samples was frequently inconsistent. The halogen lamp was replaced by a low-cost, blue light-emitting diode (LED) lamp. Using a reformulated AO solution, the staining protocol was revised to make use of a concentration gradient instead of uniform staining. To evaluate this new AO diagnostic system, a pilot field study was conducted in the Lake Victoria basin in Kenya. Without staining failure, malaria infection status of about 100 samples was determined on-site per one microscopist per day, using the improved AO diagnostic system. The improved AO diagnosis had both higher overall sensitivity (46.1 vs 38.9%: p = 0.08) and specificity (99.0 vs 96.3%) than the Giemsa method (N = 1018), using PCR diagnosis as the standard. Consistent AO staining of thin blood films and rapid evaluation of malaria parasitaemia with the revised protocol produced superior results relative to the Giemsa method. This AO diagnostic system can be set up easily at low cost using an ordinary light microscope. It may supplement rapid diagnostic tests currently used in clinical settings in malaria-endemic countries, and may be considered as an inexpensive tool for case surveillance in malaria-eliminating countries.

Platinum blue staining of cells grown in electrospun scaffolds.

PubMed

Yusuf, Mohammed; Millas, Ana Luiza G; Estandarte, Ana Katrina C; Bhella, Gurdeep K; McKean, Robert; Bittencourt, Edison; Robinson, Ian K

2014-01-01

Fibroblast cells grown in electrospun polymer scaffolds were stained with platinum blue, a heavy metal stain, and imaged using scanning electron microscopy. Good contrast on the cells was achieved compared with samples that were gold sputter coated. The cell morphology could be clearly observed, and the cells could be distinguished from the scaffold fibers. Here we optimized the required concentration of platinum blue for imaging cells grown in scaffolds and show that a higher concentration causes platinum aggregation. Overall, platinum blue is a useful stain for imaging cells because of its enhanced contrast using scanning electron microscopy (SEM). In the future it would be useful to investigate cell growth and morphology using three-dimensional imaging methods.

Effect of Photo-Fenton Bleaching on Tetracycline-stained Dentin in vitro.

PubMed

Bennett, Zackary Yale; Walsh, Laurence James

2015-02-01

Tetracycline-stained tooth structure is difficult to bleach using nightguard tray methods. The possible benefits of in-office light-accelerated bleaching systems based on the photo-Fenton reaction are of interest as possible adjunctive treatments. This study was a proof of concept for possible benefits of this approach, using dentine slabs from human tooth roots stained in a reproducible manner with the tetracycline antibiotic demeclocycline hydrochloride. Color changes overtime in tetra-cycline stained roots from single rooted teeth treated using gel (Zoom! WhiteSpeed(®)) alone, blue LED light alone, or gel plus light in combination were tracked using standardized digital photography. Controls received no treatment. Changes in color channel data were tracked overtime, for each treatment group (N = 20 per group). Dentin was lighter after bleaching, with significant improvements in the dentin color for the blue channel (yellow shade) followed by the green channel and luminosity. The greatest changes occurred with gel activated by light (p < 0.0001), which was superior to effects seen with gel alone. Use of the light alone did not significantly alter shade. This proof of concept study demonstrates that bleaching using the photo-Fenton chemistry is capable of lightening tetracycline-stained dentine. Further investigation of the use of this method for treating tetracycline-stained teeth in clinical settings appears warranted. Because tetracycline staining may respond to bleaching treatments based on the photo-Fenton reaction, systems, such as Zoom! WhiteSpeed, may have benefits as adjuncts to home bleaching for patients with tetracycline-staining.

Identification of active fluorescence stained bacteria by Raman spectroscopy

NASA Astrophysics Data System (ADS)

Krause, Mario; Beyer, Beatrice; Pietsch, Christian; Radt, Benno; Harz, Michaela; Rösch, Petra; Popp, Jürgen

2008-04-01

Microorganisms can be found everywhere e.g. in food both as useful ingredients or harmful contaminations causing food spoilage. Therefore, a fast and easy to handle analysis method is needed to detect bacteria in different kinds of samples like meat, juice or air to decide if the sample is contaminated by harmful microorganisms. Conventional identification methods in microbiology require always cultivation and therefore are time consuming. In this contribution we present an analysis approach to identify fluorescence stained bacteria on strain level by means of Raman spectroscopy. The stained bacteria are highlighted and can be localized easier against a complex sample environment e.g. in food. The use of Raman spectroscopy in combination with chemometrical methods allows the identification of single bacteria within minutes.

Differences in staining intensities affect reported occurrences and concentrations of Giardia spp. in surface drinking water sources.

PubMed

Alderisio, K A; Villegas, L F; Ware, M W; McDonald, L A; Xiao, L; Villegas, E N

2017-12-01

USEPA Method 1623, or its equivalent, is currently used to monitor for protozoan contamination of surface drinking water sources worldwide. At least three approved staining kits used for detecting Cryptosporidium and Giardia are commercially available. This study focuses on understanding the differences among staining kits used for Method 1623. Merifluor and EasyStain labelling kits were used to monitor Cryptosporidium oocyst and Giardia cyst densities in New York City's raw surface water sources. In the year following a change to the approved staining kits for use with Method 1623, an anomaly was noted in the occurrence of Giardia cysts in New York City's raw surface water. Specifically, Merifluor-stained samples had higher Giardia cyst densities as compared with those stained with EasyStain. Side by side comparison revealed significantly lower fluorescence intensities of Giardia muris as compared with Giardia duodenalis cysts when labelled with EasyStain. This study showed very poor fluorescence intensity signals by EasyStain on G. muris cysts resulting in lower cyst counts, while Merifluor, with its broader Giardia cyst staining specificity, resulted in higher cyst counts, when using Methods 1623. These results suggest that detected Giardia cyst concentrations are dependent on the staining kits used, which can result in a more or less conservative estimation of occurrences and densities of zoonotic Giardia cysts by detecting a broader range of Giardia species/Assemblages. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

A high precision method for length-based separation of carbon nanotubes using bio-conjugation, SDS-PAGE and silver staining.

PubMed

Borzooeian, Zahra; Taslim, Mohammad E; Ghasemi, Omid; Rezvani, Saina; Borzooeian, Giti; Nourbakhsh, Amirhasan

2018-01-01

Parametric separation of carbon nanotubes, especially based on their length is a challenge for a number of nano-tech researchers. We demonstrate a method to combine bio-conjugation, SDS-PAGE, and silver staining in order to separate carbon nanotubes on the basis of length. Egg-white lysozyme, conjugated covalently onto the single-walled carbon nanotubes surfaces using carbodiimide method. The proposed conjugation of a biomolecule onto the carbon nanotubes surfaces is a novel idea and a significant step forward for creating an indicator for length-based carbon nanotubes separation. The conjugation step was followed by SDS-PAGE and the nanotube fragments were precisely visualized using silver staining. This high precision, inexpensive, rapid and simple separation method obviates the need for centrifugation, additional chemical analyses, and expensive spectroscopic techniques such as Raman spectroscopy to visualize carbon nanotube bands. In this method, we measured the length of nanotubes using different image analysis techniques which is based on a simplified hydrodynamic model. The method has high precision and resolution and is effective in separating the nanotubes by length which would be a valuable quality control tool for the manufacture of carbon nanotubes of specific lengths in bulk quantities. To this end, we were also able to measure the carbon nanotubes of different length, produced from different sonication time intervals.

Cell permeability and nuclear DNA staining by propidium iodide in basidiomycetous yeasts.

PubMed

Zhang, Ning; Fan, Yuxuan; Li, Chen; Wang, Qiming; Leksawasdi, Noppol; Li, Fuli; Wang, Shi'an

2018-05-01

Non-model yeasts within basidiomycetes have considerable importance in agriculture, industry, and environment, but they are not as well studied as ascomycetous yeasts. Serving as a basic technique, nuclear DNA staining is widely used in physiology, ecology, cell biology, and genetics. However, it is unclear whether the classical nuclear DNA staining method for ascomycetous yeasts is applicable to basidiomycetous yeasts. In this study, 5 yeasts ineffectively stained by the classical propidium iodide (PI) staining method were identified from 23 representative basidiomycetous yeasts. Pretreatment of cells using dimethyl sulfoxide (DMSO) or snailase markedly improved cell penetration to PI and thus enabled DNA content determination by flow cytometry on the recalcitrant yeasts. The pretreatments are efficient, simple, and fast, avoiding tedious mutagenesis or genetic engineering used in previous reports. The heterogeneity of cell penetration to PI among basidiomycetous yeasts was attributed to the discrepancy in cell wall polysaccharides instead of capsule or plasma membrane. This study also indicated that care must be taken in attributing PI-negative staining as viable cells when studying non-model microorganisms.

Detection of the multiphoton signals in stained tissue using nonlinear optical microscopy

NASA Astrophysics Data System (ADS)

Zeng, Yaping; Xu, Jian; Kang, Deyong; Lin, Jiangbo; Chen, Jianxin

2016-10-01

Multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging, has become a powerful, important tool for tissue imaging at the molecular level. Recently, MPM is also used to image hematoxylin and eosin (H and E)-stained sections in cancer diagnostics. However, several studies have showed that the MPM images of tissue stained with H and E are significantly different from unstained tissue sections. Our aim was to detect of the multiphoton signals in stained tissue by using MPM. In this paper, MPM was used to image histological sections of esophageal invasive carcinoma tissues stained with H, E, H and E and fresh tissue. To detect of the multiphoton signals in stained tissue, the emission spectroscopic of tissue stained with H, E, H and E were obtained. For comparison, the fresh tissues were also investigated. Our results showed that the tissue stained with H, E, H and E could be detected by their TPEF signals. While the tissue stained with H and fresh tissue could be detected by their TPEF and SHG signals. In this work, we detect of the multiphoton signals in stained tissue. These findings will be useful for choosing suitable staining method so to improve the quality of MPM imaging in the future.

A memory-efficient staining algorithm in 3D seismic modelling and imaging

NASA Astrophysics Data System (ADS)

Jia, Xiaofeng; Yang, Lu

2017-08-01

The staining algorithm has been proven to generate high signal-to-noise ratio (S/N) images in poorly illuminated areas in two-dimensional cases. In the staining algorithm, the stained wavefield relevant to the target area and the regular source wavefield forward propagate synchronously. Cross-correlating these two wavefields with the backward propagated receiver wavefield separately, we obtain two images: the local image of the target area and the conventional reverse time migration (RTM) image. This imaging process costs massive computer memory for wavefield storage, especially in large scale three-dimensional cases. To make the staining algorithm applicable to three-dimensional RTM, we develop a method to implement the staining algorithm in three-dimensional acoustic modelling in a standard staggered grid finite difference (FD) scheme. The implementation is adaptive to the order of spatial accuracy of the FD operator. The method can be applied to elastic, electromagnetic, and other wave equations. Taking the memory requirement into account, we adopt a random boundary condition (RBC) to backward extrapolate the receiver wavefield and reconstruct it by reverse propagation using the final wavefield snapshot only. Meanwhile, we forward simulate the stained wavefield and source wavefield simultaneously using the nearly perfectly matched layer (NPML) boundary condition. Experiments on a complex geologic model indicate that the RBC-NPML collaborative strategy not only minimizes the memory consumption but also guarantees high quality imaging results. We apply the staining algorithm to three-dimensional RTM via the proposed strategy. Numerical results show that our staining algorithm can produce high S/N images in the target areas with other structures effectively muted.

Utility of Gram stain for the microbiological analysis of burn wound surfaces.

PubMed

Elsayed, Sameer; Gregson, Daniel B; Lloyd, Tracie; Crichton, Marilyn; Church, Deirdre L

2003-11-01

Surface swab cultures have attracted attention as a potential alternative to biopsy histology or quantitative culture methods for microbiological burn wound monitoring. To our knowledge, the utility of adding a Gram-stained slide in this context has not been evaluated previously. To determine the degree of correlation of Gram stain with culture for the microbiological analysis of burn wound surfaces. Prospective laboratory analysis. Urban health region/centralized diagnostic microbiology laboratory. Burn patients hospitalized in any Calgary Health Region burn center from November 2000 to September 2001. Gram stain plus culture of burn wound surface swab specimens obtained during routine dressing changes or based on clinical signs of infection. Degree of correlation (complete, high, partial, none), including weighted kappa statistic (kappa(w)), of Gram stain with culture based on quantitative microscopy and degree of culture growth. A total of 375 specimens from 50 burn patients were evaluated. Of these, 239 were negative by culture and Gram stain, 7 were positive by Gram stain only, 89 were positive by culture only, and 40 were positive by both methods. The degree of complete, high, partial, and no correlation of Gram stain with culture was 70.9% (266/375), 1.1% (4/375), 2.4% (9/375), and 25.6% (96/375), respectively. The degree of correlation for all 375 specimens, as expressed by the weighted kappa statistic, was found to be fair (kappa(w) = 0.32).Conclusion.-The Gram stain is not suitable for the microbiological analysis of burn wound surfaces.

Considerations for point-of-care diagnostics: evaluation of acridine orange staining and postprocessing methods for a three-part leukocyte differential test

NASA Astrophysics Data System (ADS)

Powless, Amy J.; Conley, Roxanna J.; Freeman, Karan A.; Muldoon, Timothy J.

2017-03-01

There exists a broad range of techniques that can be used to classify and count white blood cells in a point-of-care (POC) three-part leukocyte differential test. Improvements in lenses, light sources, and cameras for image-based POC systems have renewed interest in acridine orange (AO) as a contrast agent, whereby subpopulations of leukocytes can be differentiated by colorimetric analysis of AO fluorescence emission. We evaluated the effect on test accuracy using different AO staining and postprocessing methods in the context of an image-based POC colorimetric cell classification scheme. Thirty blood specimens were measured for percent cell counts using our POC system and a conventional hematology analyzer for comparison. Controlling the AO concentration used during whole-blood staining, the incubation time with AO, and the colorimetric ratios among the three population of leukocytes yielded a percent deviation of 0.706%, -1.534%, and -0.645% for the lymphocytes, monocytes, and granulocytes, respectively. Overall, we demonstrated that a redshift in AO fluorescence was observed at elevated AO concentrations, which lead to reproducible inaccuracy of cell counts. This study demonstrates there is a need for a strict control of the AO staining and postprocessing methods to improve test accuracy in these POC systems.

A novel, modernized Golgi-Cox stain optimized for CLARITY cleared tissue.

PubMed

Kassem, Mustafa S; Fok, Sandra Y Y; Smith, Kristie L; Kuligowski, Michael; Balleine, Bernard W

2018-01-15

High resolution neuronal information is extraordinarily useful in understanding the brain's functionality. The development of the Golgi-Cox stain allowed observation of the neuron in its entirety with unrivalled detail. Tissue clearing techniques, e.g., CLARITY and CUBIC, provide the potential to observe entire neuronal circuits intact within tissue and without previous restrictions with regard to section thickness. Here we describe an improved Golgi-Cox stain method, optimised for use with CLARITY and CUBIC that can be used in both fresh and fixed tissue. Using this method, we were able to observe neurons in their entirety within a fraction of the time traditionally taken to clear tissue (48h). We were also able to show for the first-time that Golgi stained tissue is fluorescent when visualized using a multi-photon microscope, allowing us to image synaptic spines with a detail previously unachievable. These novel methods provide cheap and easy to use techniques to investigate the morphology of cellular processes in the brain at a new-found depth, speed, utility and detail, without previous restrictions of time, tissue type and section thickness. This is the first application of a Golgi-Cox stain to cleared brain tissue, it is investigated and discussed in detail, describing different methodologies that may be used, a comparison between the different clearing techniques and lastly the novel interaction of these techniques with this ultra-rapid stain. Copyright © 2017 Elsevier B.V. All rights reserved.

A fluorescence in situ staining method for investigating spores and vegetative cells of Clostridia by confocal laser scanning microscopy and structured illuminated microscopy.

PubMed

D'Incecco, P; Ong, L; Gras, S; Pellegrino, L

2018-04-18

Non-pathogenic spore-forming Clostridia are of increasing interest due to their application in biogas production and their capability to spoil different food products. The life cycle for Clostridium includes a spore stage that can assist in survival under environmentally stressful conditions, such as extremes of temperature or pH. Due to their size, spores can be investigated by a range of microscopic techniques, many of which involve sample pre-treatment. We have developed a quick, simple and non-destructive fluorescent staining procedure that allows a clear differentiation between spores and vegetative cells and effectively stains spores, allowing recovery and tracking in subsequent experiments. Hoechst 34580, Propidium iodide and wheat germ agglutinin WGA 488 were used in combination to stain four strains of Clostridia at different life cycle stages. Staining was conducted without drying the sample, preventing changes induced by dehydration and cells observed by confocal laser scanner microscopy or using a super-resolution microscope equipped with a 3D-structured illumination module. Dual staining with Hoechst/Propidium iodide differentiated spores from vegetative cells, provided information on the viability of cells and was successfully applied to follow spore production induced by heating. Super-resolution microscopy of spores probed by Hoechst 34580 also allowed chromatin to be visualised. Direct staining of a cheese specimen using Nile Red and Fast Green allowed in situ observation of spores within the cheese and their position within the cheese matrix. The proposed staining method has broad applicability and can potentially be applied to follow Clostridium spore behaviour in a range of different environments. Copyright © 2018 Elsevier Ltd. All rights reserved.

Molecular identification of leishmania species using samples obtained from negative stained smears.

PubMed

Mohaghegh, Ma; Fata, A; Salehi, Gh; Berenji, F; Bazzaz, M Mousavi; Rafatpanah, H; Parian, M; Movahedi, A

2013-04-01

Cutaneous Leishmaniasis (CL) is a parasitic skin disease. Diagnosis primarily is based on clinical signs and microscopic observation of parasite on direct stained smears or tissue sections. Sensitivity of direct smear is not as high as molecular methods. The aim of this study was to identify and characterize Leishmania species among the negative direct smears obtained from skin ulcers suspected to CL by PCR method. Among 81 patients with suspicious skin lesions to CL referred to the Parasitology lab, negative Giemsa stained smears were collected. DNA extraction performed by scraping stained smears, then PCR was performed. Among the DNA extracted from smears, L. tropica was isolated from 9 (11.1%) of the smears and L.major was not isolated from any samples. Direct microscopy on stained smears for diagnosis of leishmaniasis is not enough accurate. PCR is recommended for clinically suspected lesions with negative result of direct smear.

Detection of Cryptosporidium and Cyclospora Oocysts from Environmental Water for Drinking and Recreational Activities in Sarawak, Malaysia.

PubMed

Bilung, Lesley Maurice; Tahar, Ahmad Syatir; Yunos, Nur Emyliana; Apun, Kasing; Lim, Yvonne Ai-Lian; Nillian, Elexson; Hashim, Hashimatul Fatma

2017-01-01

Cryptosporidiosis and cyclosporiasis are caused by waterborne coccidian protozoan parasites of the genera Cryptosporidium and Cyclospora, respectively. This study was conducted to detect Cryptosporidium and Cyclospora oocysts from environmental water abstracted by drinking water treatment plants and recreational activities in Sarawak, Malaysia. Water samples (12 each) were collected from Sungai Sarawak Kanan in Bau and Sungai Sarawak Kiri in Batu Kitang, respectively. In addition, 6 water samples each were collected from Ranchan Recreational Park and UNIMAS Lake at Universiti Malaysia Sarawak, Kota Samarahan, respectively. Water physicochemical parameters were also recorded. All samples were concentrated by the iron sulfate flocculation method followed by the sucrose floatation technique. Cryptosporidium and Cyclospora were detected by modified Ziehl-Neelsen technique. Correlation of the parasites distribution with water physicochemical parameters was analysed using bivariate Pearson correlation. Based on the 24 total samples of environmental water abstracted by drinking water treatment plants, all the samples (24/24; 100%) were positive with Cryptosporidium , and only 2 samples (2/24; 8.33%) were positive with Cyclospora . Based on the 12 total samples of water for recreational activities, 4 samples (4/12; 33%) were positive with Cryptosporidium , while 2 samples (2/12; 17%) were positive with Cyclospora . Cryptosporidium oocysts were negatively correlated with dissolved oxygen (DO).

Detection of Cryptosporidium and Cyclospora Oocysts from Environmental Water for Drinking and Recreational Activities in Sarawak, Malaysia

PubMed Central

Tahar, Ahmad Syatir; Yunos, Nur Emyliana; Apun, Kasing; Nillian, Elexson; Hashim, Hashimatul Fatma

2017-01-01

Cryptosporidiosis and cyclosporiasis are caused by waterborne coccidian protozoan parasites of the genera Cryptosporidium and Cyclospora, respectively. This study was conducted to detect Cryptosporidium and Cyclospora oocysts from environmental water abstracted by drinking water treatment plants and recreational activities in Sarawak, Malaysia. Water samples (12 each) were collected from Sungai Sarawak Kanan in Bau and Sungai Sarawak Kiri in Batu Kitang, respectively. In addition, 6 water samples each were collected from Ranchan Recreational Park and UNIMAS Lake at Universiti Malaysia Sarawak, Kota Samarahan, respectively. Water physicochemical parameters were also recorded. All samples were concentrated by the iron sulfate flocculation method followed by the sucrose floatation technique. Cryptosporidium and Cyclospora were detected by modified Ziehl-Neelsen technique. Correlation of the parasites distribution with water physicochemical parameters was analysed using bivariate Pearson correlation. Based on the 24 total samples of environmental water abstracted by drinking water treatment plants, all the samples (24/24; 100%) were positive with Cryptosporidium, and only 2 samples (2/24; 8.33%) were positive with Cyclospora. Based on the 12 total samples of water for recreational activities, 4 samples (4/12; 33%) were positive with Cryptosporidium, while 2 samples (2/12; 17%) were positive with Cyclospora. Cryptosporidium oocysts were negatively correlated with dissolved oxygen (DO). PMID:29234679

Immunogold Staining of Ultrathin Thawed Cryosections for Transmission Electron Microscopy (TEM).

PubMed

Skepper, Jeremy N; Powell, Janet M

2008-06-01

INTRODUCTIONA pre-embedding method of immunochemical staining is used if antigens are damaged by resin embedding, or if the best preservation of membranes is required. Applying immunogold reagents to sections of lightly fixed tissue, free of embedding medium, can be a very sensitive method of immunochemical staining. Cells or tissues are fixed as strongly as possible and then treated with a cryoprotectant, which is usually a mixture of sucrose and polyvinylpyrrolidone (PVP). They are frozen onto pins in liquid nitrogen and sectioned at approximately -100°C. The frozen sections are thaw-mounted on to Formvar/nickel film grids and the cryoprotectant is removed by floating the grids on drops of phosphate-buffered saline (PBS). The immunogold staining is performed on the unembedded sections, which are subsequently contrast counterstained and infiltrated with a mixture of methylcellulose and uranyl acetate. In this protocol, samples are sectioned at low temperature, thaw-mounted onto film grids, immunochemically stained, contrast counterstained, and embedded/encapsulated in situ on the grid before viewing by transmission electron microscopy (TEM).

Immunoperoxidase staining for identification of Aspergillus species in routinely processed tissue sections.

PubMed Central

Verweij, P E; Smedts, F; Poot, T; Bult, P; Hoogkamp-Korstanje, J A; Meis, J F

1996-01-01

AIMS: To evaluate the performance of an immunoperoxidase stain using the monoclonal antibody EB-A1 to detect Aspergillus species in formalin fixed, paraffin wax embedded tissue. METHODS: The monoclonal antibody EB-A1 directed against galactomannan was used to detect Aspergillus species in 23 patients with suspected or confirmed invasive aspergillosis. Immunostaining was performed on formalin fixed, paraffin wax embedded tissue using the streptavidin-biotin method and compared with conventional haematoxylin and eosin, periodic acid-Schiff, and Gomori-Grocott stains. Results of immunostaining were semiquantitatively analysed with regard to both intensity of staining and number of positively staining micro-organisms. Tissue sections from 16 patients with confirmed invasive mycoses due to Candida species, Apophysomyces elegans, Rhizopus oryzae, Pseudallescheria boydii and Histoplasma capsulatum were used as controls. RESULTS: In 19 (83%) of 23 cases invasive aspergillosis was confirmed by both histological examination and culture (18 Aspergillus fumigatus and one A flavus). Immunoperoxidase stains were positive in 17 (89%) of 19 cases including one case of disseminated infection due to A flavus. Furthermore, the immunoperoxidase stain was positive in a culture negative tissue section with histological evidence of mycelial development, indicating the presence of Aspergillus species. Some cross-reactivity was observed with the highly related fungus P boydii, although the number of mycelial elements that stained was low. CONCLUSIONS: Immunoperoxidase staining using the monoclonal antibody EB-A1 performs well on routinely processed tissue sections and permits detection and generic identification of Aspergillus species, although it was no better than conventional histopathology in identifying the presence of an infection. An additional advantage is that the immunostain may help to provide an aetiological diagnosis when cultures remain negative. Images PMID:8943743

Reliability of a rapid hematology stain for sputum cytology*

PubMed Central

Gonçalves, Jéssica; Pizzichini, Emilio; Pizzichini, Marcia Margaret Menezes; Steidle, Leila John Marques; Rocha, Cristiane Cinara; Ferreira, Samira Cardoso; Zimmermann, Célia Tânia

2014-01-01

Objective: To determine the reliability of a rapid hematology stain for the cytological analysis of induced sputum samples. Methods: This was a cross-sectional study comparing the standard technique (May-Grünwald-Giemsa stain) with a rapid hematology stain (Diff-Quik). Of the 50 subjects included in the study, 21 had asthma, 19 had COPD, and 10 were healthy (controls). From the induced sputum samples collected, we prepared four slides: two were stained with May-Grünwald-Giemsa, and two were stained with Diff-Quik. The slides were read independently by two trained researchers blinded to the identification of the slides. The reliability for cell counting using the two techniques was evaluated by determining the intraclass correlation coefficients (ICCs) for intraobserver and interobserver agreement. Agreement in the identification of neutrophilic and eosinophilic sputum between the observers and between the stains was evaluated with kappa statistics. Results: In our comparison of the two staining techniques, the ICCs indicated almost perfect interobserver agreement for neutrophil, eosinophil, and macrophage counts (ICC: 0.98-1.00), as well as substantial agreement for lymphocyte counts (ICC: 0.76-0.83). Intraobserver agreement was almost perfect for neutrophil, eosinophil, and macrophage counts (ICC: 0.96-0.99), whereas it was moderate to substantial for lymphocyte counts (ICC = 0.65 and 0.75 for the two observers, respectively). Interobserver agreement for the identification of eosinophilic and neutrophilic sputum using the two techniques ranged from substantial to almost perfect (kappa range: 0.91-1.00). Conclusions: The use of Diff-Quik can be considered a reliable alternative for the processing of sputum samples. PMID:25029648

[Clinical epidemiology of cryptosporidiosis in calves].

PubMed

Weber, S E; Lippuner, C; Corti, S; Deplazes, P; Hässig, M

2016-05-01

The aim of this study was to determine whether there is an association between Cryptosporidium infections in calves and immunological factors, as well as farm-related factors or the application of the anti-cryptosporidiosis drug Halofuginone. From January to June 2010, 63 cow-calf-pairs from 20 different farms near Zürich, Switzerland have been investigated. Each cowcalf- pair was visited three times within the first 6 weeks of life to collect data of the farm and animals, as well as blood, faecal, colostral and milk samples. An ELISA using sporozoite antigen was developed for the specific detection of anti-Cryptosporidium-IgG in blood- and colostral serum. The IgG concentration in the bloodand colostral serum was determined using radial immuno diffusion test (RID). White blood cell isolation and differential blood cell counts and California Mastitis Test were performed. Bacteriological studies on quarter-milk-samples were carried out. Cryptosporidium oocysts were diagnosed with the modified Ziehl-Neelsen staining, other protozoa with the SAFC method and Eimeria oocysts and helminth eggs were diagnosed with the combined sedimentation/floatation test. ELISAs were performed for the detection of rota- and coronavirus, E. coli F5 and Cryptosporidium spp. in bovine feces (bio-X Diagnostics®, Belgium). The highest prevalence of Cryptosporidium oocysts was 54.0% and found 7 to 20 days post natum, whereas 47.1% were suffering from diarrhea. The transfer of total IgG with the colostrum and the humoral immunity of the calf could not prevent any infection with Cryptosporidium, but the severity of the diarrhea symptoms decreased with increasing total IgG concentrations. Calves housed in open sheds showed significantly more often diarrhea, i. e. they shed more Cryptosporidium oocysts during the first 4 days and 7 to 20 days post natum, respectively. Halofuginone (Halocur®) is approved for prophylaxis against cryptosporidiosis, but it showed no effect on the excretion of

Gram's Stain Does Not Cross the Bacterial Cytoplasmic Membrane.

PubMed

Wilhelm, Michael J; Sheffield, Joel B; Sharifian Gh, Mohammad; Wu, Yajing; Spahr, Christian; Gonella, Grazia; Xu, Bolei; Dai, Hai-Lung

2015-07-17

For well over a century, Hans Christian Gram's famous staining protocol has been the standard go-to diagnostic for characterizing unknown bacteria. Despite continuous and ubiquitous use, we now demonstrate that the current understanding of the molecular mechanism for this differential stain is largely incorrect. Using the fully complementary time-resolved methods: second-harmonic light-scattering and bright-field transmission microscopy, we present a real-time and membrane specific quantitative characterization of the bacterial uptake of crystal-violet (CV), the dye used in Gram's protocol. Our observations contradict the currently accepted mechanism which depicts that, for both Gram-negative and Gram-positive bacteria, CV readily traverses the peptidoglycan mesh (PM) and cytoplasmic membrane (CM) before equilibrating within the cytosol. We find that not only is CV unable to traverse the CM but, on the time-scale of the Gram-stain procedure, CV is kinetically trapped within the PM. Our results indicate that CV, rather than dyes which rapidly traverse the PM, is uniquely suited as the Gram stain.

Iron Stain on Wood

Treesearch

Mark Knaebe

2013-01-01

Iron stain, an unsightly blue–black or gray discoloration, can occur on nearly all woods. Oak, redwood, cypress, and cedar are particularly prone to iron stain because these woods contain large amounts of tannin-like extractives. The discoloration is caused by a chemical reaction between extractives in the wood and iron in steel products, such as nails, screws, and...

An Innovative Method for Obtaining Consistent Images and Quantification of Histochemically Stained Specimens

PubMed Central

Sedgewick, Gerald J.; Ericson, Marna

2015-01-01

Obtaining digital images of color brightfield microscopy is an important aspect of biomedical research and the clinical practice of diagnostic pathology. Although the field of digital pathology has had tremendous advances in whole-slide imaging systems, little effort has been directed toward standardizing color brightfield digital imaging to maintain image-to-image consistency and tonal linearity. Using a single camera and microscope to obtain digital images of three stains, we show that microscope and camera systems inherently produce image-to-image variation. Moreover, we demonstrate that post-processing with a widely used raster graphics editor software program does not completely correct for session-to-session inconsistency. We introduce a reliable method for creating consistent images with a hardware/software solution (ChromaCal™; Datacolor Inc., NJ) along with its features for creating color standardization, preserving linear tonal levels, providing automated white balancing and setting automated brightness to consistent levels. The resulting image consistency using this method will also streamline mean density and morphometry measurements, as images are easily segmented and single thresholds can be used. We suggest that this is a superior method for color brightfield imaging, which can be used for quantification and can be readily incorporated into workflows. PMID:25575568

A staining protocol for identifying secondary compounds in Myrtaceae1

PubMed Central

Retamales, Hernan A.; Scharaschkin, Tanya

2014-01-01

• Premise of the study: Here we propose a staining protocol using toluidine blue (TBO) and ruthenium red to reliably identify secondary compounds in the leaves of some species of Myrtaceae. • Methods and Results: Leaves of 10 species representing 10 different genera of Myrtaceae were processed and stained using five different combinations of ruthenium red and TBO. Optimal staining conditions were determined as 1 min of ruthenium red (0.05% aqueous) and 45 s of TBO (0.1% aqueous). Secondary compounds clearly identified under this treatment include mucilage in the mesophyll, polyphenols in the cuticle, lignin in fibers and xylem, tannins and carboxylated polysaccharides in the epidermis, and pectic substances in the primary cell walls. • Conclusions: Potential applications of this protocol include systematic, phytochemical, and ecological investigations in Myrtaceae. It might be applicable to other plant families rich in secondary compounds and could be used as a preliminary screening method for extraction of these elements. PMID:25309840

Porcine Intestinal Mast Cells. Evaluation of Different Fixatives for Histochemical Staining Techniques Considering Tissue Shrinkage

PubMed Central

Rieger, J.; Twardziok, S.; Huenigen, H.; Hirschberg, R.M.; Plendl, J.

2013-01-01

Staining of mast cells (MCs), including porcine ones, is critically dependent upon the fixation and staining technique. In the pig, mucosal and submucosal MCs do not stain or stain only faintly after formalin fixation. Some fixation methods are particularly recommended for MC staining, for example the fixation with Carnoy or lead salts. Zinc salt fixation (ZSF) has been reported to work excellently for the preservation of fixation-sensitive antigens. The aim of this study was to establish a reliable histological method for counting of MCs in the porcine intestinum. For this purpose, different tissue fixation and staining methods that also allow potential subsequent immunohistochemical investigations were evaluated in the porcine mucosa, as well as submucosa of small and large intestine. Tissues were fixed in Carnoy, lead acetate, lead nitrate, Zamboni and ZSF and stained subsequently with either polychromatic methylene blue, alcian blue or toluidine blue. For the first time our study reveals that ZSF, a heavy metal fixative, preserves metachromatic staining of porcine MCs. Zamboni fixation was not suitable for histochemical visualization of MCs in the pig intestine. All other tested fixatives were suitable. Alcian blue and toluidine blue co-stained intestinal goblet cells which made a prima facie identification of MCs difficult. The polychromatic methylene blue proved to be the optimal staining. In order to compare MC counting results of the different fixation methods, tissue shrinkage was taken into account. As even the same fixation caused shrinkagedifferences between tissue from small and large intestine, different factors for each single fixation and intestinal localization had to be calculated. Tissue shrinkage varied between 19% and 57%, the highest tissue shrinkage was found after fixation with ZSF in the large intestine, the lowest one in the small intestine after lead acetate fixation. Our study emphasizes that MC counting results from data using different

Detection of alkali-silica reaction swelling in concrete by staining

DOEpatents

Guthrie, G.D. Jr.; Carey, J.W.

1998-04-14

A method using concentrated aqueous solutions of sodium cobalt nitrite and rhodamine B is described which can be used to identify concrete that contains gels formed by the alkali-silica reaction (ASR). These solutions present little health or environmental risk, are readily applied, and rapidly discriminate between two chemically distinct gels; K-rich, Na-K-Ca-Si gels are identified by yellow staining, and alkali-poor, Ca-Si gels are identified by pink staining.

Detection of alkali-silica reaction swelling in concrete by staining

DOEpatents

Guthrie, Jr., George D.; Carey, J. William

1998-01-01

A method using concentrated aqueous solutions of sodium cobaltinitrite and rhodamine B is described which can be used to identify concrete that contains gels formed by the alkali-silica reaction (ASR). These solutions present little health or environmental risk, are readily applied, and rapidly discriminate between two chemically distinct gels; K-rich, Na--K--Ca--Si gels are identified by yellow staining, and alkali-poor, Ca--Si gels are identified by pink staining.

Ghost mycobacteria on Gram stain.

PubMed Central

Trifiro, S; Bourgault, A M; Lebel, F; René, P

1990-01-01

The Gram stain is a key tool in diagnostic microbiology. Its usefulness with respect to mycobacteria is undefined. The neutrality of mycobacteria other than Mycobacterium tuberculosis on Gram staining of various clinical specimens is described. Images PMID:1688872

Detection of Catheter-Related Bloodstream Infections by the Differential-Time-to-Positivity Method and Gram Stain-Acridine Orange Leukocyte Cytospin Test in Neutropenic Patients after Hematopoietic Stem Cell Transplantation

PubMed Central

Krause, R.; Auner, H. W.; Gorkiewicz, G.; Wölfler, A.; Daxboeck, F.; Linkesch, W.; Krejs, G. J.; Wenisch, C.; Reisinger, E. C.

2004-01-01

For febrile neutropenic patients who received hematopoietic stem cell transplantation, the Gram stain-acridine orange leukocyte cytospin (AOLC) test and the differential-time-to-positivity method (DTP) were performed. As a diagnostic tool for catheter-related bloodstream infections in these patients, the Gram stain-AOLC test has a lower sensitivity than does the DTP method but acceptable positive and negative predictive values. PMID:15472355

Gram Stain

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

Digital enhancement of haematoxylin- and eosin-stained histological images for red-green colour-blind observers.

PubMed

Landini, G; Perryer, G

2009-06-01

Individuals with red-green colour-blindness (CB) commonly experience great difficulty differentiating between certain histological stain pairs, notably haematoxylin-eosin (H&E). The prevalence of red-green CB is high (6-10% of males), including among medical and laboratory personnel, and raises two major concerns: first, accessibility and equity issues during the education and training of individuals with this disability, and second, the likelihood of errors in critical tasks such as interpreting histological images. Here we show two methods to enhance images of H&E-stained samples so the differently stained tissues can be well discriminated by red-green CBs while remaining usable by people with normal vision. Method 1 involves rotating and stretching the range of H&E hues in the image to span the perceptual range of the CB observers. Method 2 digitally unmixes the original dyes using colour deconvolution into two separate images and repositions the information into hues that are more distinctly perceived. The benefits of these methods were tested in 36 volunteers with normal vision and 11 with red-green CB using a variety of H&E stained tissue sections paired with their enhanced versions. CB subjects reported they could better perceive the different stains using the enhanced images for 85% of preparations (method 1: 90%, method 2: 73%), compared to the H&E-stained original images. Many subjects with normal vision also preferred the enhanced images to the original H&E. The results suggest that these colour manipulations confer considerable advantage for those with red-green colour vision deficiency while not disadvantaging people with normal colour vision.

Silver stain for electron microscopy

NASA Technical Reports Server (NTRS)

Corbett, R. L.

1972-01-01

Ammoniacal silver stain used for light microscopy was adapted advantageously for use with very thin biological sections required for electron microscopy. Silver stain can be performed in short time, has more contrast, and is especially useful for low power electron microscopy.

Single step modified ink staining for Tzanck test: quick detection of herpetic giant cells in Tzanck smear.

PubMed

Mizutani, Hitoshi; Akeda, Tomoko; Yamanaka, Kei-Ichi; Isoda, Kenichi; Gabazza, Esteban C

2012-02-01

Tzanck test has been recently re-evaluated as a method for the diagnosis of herpes virus infection. Giemsa staining for the Tzanck test is time-consuming and laborious. There is a need to develop simple and quick staining methods for bedside diagnosis of this disease. We report a single step and quick method for staining herpes giant cells in Tzanck smears using routinely available inks and physiological saline. A keratinocyte cell line (HaCaT) was cultured on a slide glass and stained with various commercially available blue, blue-black and black inks serially diluted with physiological saline. Clinical smear samples from herpes lesions were also stained with these solutions without specific pretreatment. The nuclei of HaCaT were clearly stained showing high contrast with the cytoplasm using 5% Parker-Quink blue-black ink saline solution. Concentration of ink solution higher or lower than 5% resulted in less contrast. Blue or black inks or other manufacturers' inks can also be used, but staining of the cultured keratinocytes was less clear. Smear of clinical samples from herpes lesions were also stained with 5% ink solution. The nuclei of the multinucleated giant cells were clearly stained, and the sample could be immediately used for microscopic examination. One step staining of Tzanck smear using this diluted ink solution is an inexpensive and a convenient bedside diagnostic tool for the dermatologist. © 2011 Japanese Dermatological Association.

Whole blood staining in suspension for nonspecific esterase and alkaline phosphatase analyzed with a Technicon H-1.

PubMed

Ross, D W; Bishop, C; Henderson, A; Kaplow, L

1990-01-01

We adapted previously published methods for nonspecific esterase and alkaline phosphatase staining of white blood cells in suspension for use on a Technicon H-1 hematology analyzer. The objective was to develop a semiautomated method using whole blood that could be employed on a large scale for hematology laboratory applications, including toxicology studies, measurement of neutrophil left shift, and cytochemical classification of myeloid leukemias. The nonspecific esterase method uses the pararosaniline stain, generating the unstable substrate from two stable precursors. Whole blood is added to the substrate plus dye mix. Next, acid lysis and fixation steps destroy red cells and stabilize the monocyte staining. The alkaline phosphatase stain employs a stable naphthyl phosphate substrate and fast blue B coupling dye. The red cells are lysed with a pH 10.3 propanediol buffer, and the white blood cells are then stabilized with formalin fixation. For both methods the staining is performed off-line, and the sample is then diluted with propanediol to match the refractive index of the sheath on the H-1 analyzer, before aspiration into the direct cytometry port. A cytogram of scattered versus absorbed light is obtained. The number of cells staining and the intensity of the stain can be quantified from the cytogram.

Photodynamic therapy for port wine stains

NASA Astrophysics Data System (ADS)

Li, Junheng

1998-08-01

Therapies for port wine stains including conventional laser irradiation usually cause unacceptable scarring or obtain poor effect. Pulsed dye laser has better approach, but only few patients obtain complete fading after multiple laser treatment. Because port wine stain is a congenital vasculopathy consisting of an abnormal network of capillaries in the upper dermis with an overlying normal epidermis and the researchers found that tumor blood vessels were occluded accompanying the necrosis of the tumor after PDT. It is though to be the effect primarily by thrombus formation in vessels and shut down of the blood supply to the tumor as well as direct tumor cells kill. The author and his colleagues started a series of animal and clinical studies since 1991 about photodynamic therapy for port wine stains and they established the method of PDT for PWS. An experimental study showed that Hpd appeared rapidly within the human vascular endothelial cells in culture fluid. Animal study using chicken combs as PWS models treated by PDT revealed the possibility of selective destruction of the malformative vasculature in PWS. The clinical studies of over 1700 cases proved that PWS can be cured without scar formation by PDT because there is no thermal effect involved. No relapse was found within a maximum follow-up of seven years. The differences and mechanism between the treatments of PDT and conventional lasers are discussed.

Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining1

PubMed Central

Behbehani, Gregory K.; Thom, Colin; Zunder, Eli R.; Finck, Rachel; Gaudilliere, Brice; Fragiadakis, Gabriela K.; Fantl, Wendy J.; Nolan, Garry P.

2015-01-01

Fluorescent cellular barcoding and mass-tag cellular barcoding are cytometric methods that enable high sample throughput, minimize inter-sample variation, and reduce reagent consumption. Previously employed barcoding protocols require that barcoding be performed after surface marker staining, complicating combining the technique with measurement of alcohol-sensitive surface epitopes. This report describes a method of barcoding fixed cells after a transient partial permeabilization with 0.02% saponin that results in efficient and consistent barcode staining with fluorescent or mass-tagged reagents while preserving surface marker staining. This approach simplifies barcoding protocols and allows direct comparison of surface marker staining of multiple samples without concern for variations in the antibody cocktail volume, antigen-antibody ratio, or machine sensitivity. Using this protocol, cellular barcoding can be used to reliably detect subtle differences in surface marker expression. PMID:25274027

ENHANCED DAPI STAINING FOR CRYPTOSPORIDIUM IN WATER SAMPLES

EPA Science Inventory

The U.S. Environmental Protection Agency's Method 1623 is used to detect and quantify the presence of {ital Cryptosporidium} spp. oocysts in water. The protocol consists of concentrating a sample, staining this concentrate with a fluorescent antibody, and examining the sample mi...

Crystal violet stain as a selective stain for the assessment of mitotic figures in oral epithelial dysplasia and oral squamous cell carcinoma.

PubMed

Jadhav, Kiran B; Ahmed Mujib, B R; Gupta, Nidhi

2012-01-01

Assessment of mitotic figures (MFs) is routinely practiced as prognostic indicator in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC), but identification of MFs poses a problem in terms of staining characteristics. To evaluate effectiveness of crystal violet stain for staining of MFs and its comparison with hematoxylin and eosin (H and E) stain. Study sample includes archival tissues embedded in paraffin blocks diagnosed as OED (n = 30) and OSCC (n = 30). The control group comprised of tissue specimen from oral mucosa of healthy volunteers (n = 30). Two serial sections of each tissue specimen were stained separately with H and E stain and 1% crystal violet stain. The stained sections were observed under microscope for identification and counting of MFs. Data obtained was statistically analyzed by using the Man-Whitney U test. A significant increase in number of MFs was observed in OED and OSCC in comparison with normal oral mucosa. There was a highly significant increase in number of MFs in crystal violet stained tissue sections when compared with H and E stain. Metaphase is the most commonly observed phase of mitosis in crystal violet stain when compared with H and E stain for all three groups. Crystal violet stain can be considered as selective stain for mitotic figures.

Differential staining of bacteria: acid fast stain.

PubMed

Reynolds, Jackie; Moyes, Rita B; Breakwell, Donald P

2009-11-01

Acid-fastness is an uncommon characteristic shared by the genera Mycobacterium (Section 10A) and Nocardia. Because of this feature, this stain is extremely helpful in identification of these bacteria. Although Gram positive, acid-fast bacteria do not take the crystal violet into the wall well, appearing very light purple rather than the deep purple of normal Gram-positive bacteria. (c) 2009 by John Wiley & Sons, Inc.

Rorschach inkblot port-wine stain.

PubMed

Coots, N V; Elston, D M

1997-01-01

We present an infant born with bilaterally symmetric, anterior and posterior port-wine stains. These lesions presented a striking resemblance to Rorschach inkblots, a phenomenon not previously reported. A discussion of the case as well as a discussion of syndromes associated with port-wine stains is provided.

Compact, Automated Centrifugal Slide-Staining System

NASA Technical Reports Server (NTRS)

Feeback, Daniel L.; Clarke, Mark S. F.

2004-01-01

The Directional Acceleration Vector-Driven Displacement of Fluids (DAVD-DOF) system, under development at the time of reporting the information for this article, would be a relatively compact, automated, centrifugally actuated system for staining blood smears and other microbiological samples on glass microscope slides in either a microgravitational or a normal Earth gravitational environment. The DAVD-DOF concept is a successor to the centrifuge-operated slide stainer (COSS) concept, which was reported in Slide-Staining System for Microgravity or Gravity (MSC-22949), NASA Tech Briefs, Vol. 25, No. 1 (January, 2001), page 64. The COSS includes reservoirs and a staining chamber that contains a microscope slide to which a biological sample is affixed. The staining chamber is sequentially filled with and drained of staining and related liquids from the reservoirs by use of a weighted plunger to force liquid from one reservoir to another at a constant level of hypergravity maintained in a standard swing-bucket centrifuge. In the DAVD-DOF system, a staining chamber containing a sample would also be sequentially filled and emptied, but with important differences. Instead of a simple microscope slide, one would use a special microscope slide on which would be fabricated a network of very small reservoirs and narrow channels connected to a staining chamber (see figure). Unlike in the COSS, displacement of liquid would be effected by use of the weight of the liquid itself, rather than the weight of a plunger.

The role of histopathology in establishing the diagnosis of tuberculous pericardial effusions in the presence of HIV.

PubMed

Reuter, H; Burgess, L J; Schneider, J; Van Vuuren, W; Doubell, A F

2006-02-01

To establish the influence of human immunodeficiency virus (HIV) infection on the histopathological features of patients presenting with tuberculous pericarditis. A prospective study was carried out at Tygerberg Academic Hospital, South Africa; 36 patients with large pericardial effusions had open pericardial biopsies under general anaesthesia and were included in the study. Patients underwent pericardiocentesis, followed by daily intermittent catheter drainage; a comprehensive diagnostic work-up (including histopathology of the pericardial tissue) was also performed. Histological tuberculous pericarditis was diagnosed according to predetermined criteria. Tuberculous pericarditis was identified in 25 patients, five of whom were HIV+. The presence of granulomatous inflammation (with or without necrosis) and/or Ziehl-Neelsen positivity yielded the best test results (sensitivity 64%, specificity 100% and diagnostic efficiency 75%). Co-infection with HIV impacts on the histopathological features of pericardial tuberculosis and leads to a decrease in the sensitivity of the test. In areas which have a high prevalence of tuberculosis, the combination of a sensitive test such as adenosine deaminase, chest X-ray and clinical features has a higher diagnostic efficiency than pericardial biopsy in diagnosing tuberculous pericarditis.

Paratuberculosis in buffaloes in Northeast Brazil.

PubMed

de Farias Brito, Marilene; Dos Santos Belo-Reis, Alessandra; Barbosa, José Diomedes; Ubiali, Daniel Guimarães; de Castro Pires, Ana Paula; de Medeiros, Elizabeth Sampaio; de Melo, Renata Pimentel Bandeira; de Albuquerque, Pedro Paulo Feitosa; Yamasaki, Elise; Mota, Rinaldo Aparecido

2016-10-01

Several farms in the Northeast of Brazil were investigated for Mycobacterium avium subsp. paratuberculosis infection in order to identify the occurrence of paratuberculosis in buffaloes. Samples were obtained from 17 farms, two slaughter houses, and a quarantine area in the Northeast. About 15,000 buffaloes of the Murrah, Mediterranean, and Jafarabadi breed as well as their crossbreeds were evaluated for meat, dairy, and mixed farms with semi-intensive or extensive breeding practices. For diagnostic purposes, postmortem and histopathological examination, including Ziehl-Neelsen test of fecal smears and scraped intestinal mucosa were performed. PCR was applied for fecal samples, mesenteric lymph nodes, and intestines. Six Johne's disease-positive farms, which together with those previously identified, indicate that the disease is spread through the Brazilian Northeast, similar to what occurs in cattle herds in other regions of the country. The increase in prevalence of paratuberculosis is a consequence of introduction of animals from other regions without adequate veterinary assistance and due to the little official attention paid to this initially silent and chronic disease.

Short communication: Passive shedding of Mycobacterium avium ssp. paratuberculosis in commercial dairy goats in Brazil.

PubMed

Schwarz, D G G; Lima, M C; Barros, M; Valente, F L; Scatamburlo, T M; Rosado, N; Oliveira, C T S A M; Oliveira, L L; Moreira, M A S

2017-10-01

Goat farming is a low-cost alternative to dairy production in developing countries. In Brazil, goat production has increased in recent years due in part to the implementation of programs encouraging this activity. Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis, a disease that causes chronic granulomatous enteritis in ruminants, but MAP transmission dynamics are still poorly understood in goats. In a previously published study of our research group, 10 dairy goat farms (467 animals) from Minas Gerais state were analyzed for MAP detection; 2 fecal cultures and 11 milk samples tested positive for MAP by conventional PCR and were confirmed by sequencing. Because no clinical signs were observed over 1 yr of monitoring, we hypothesized that these MAP-positive goats could be passive shedders. Thus, in the present study, 4 positive goats (4/13) from the previous study were purchased and feces and milk samples were collected for evaluation (twice, with an interval of 3 mo between tests) by culture of MAP, IS900 PCR, or both. All analyses were negative for MAP. At the last time point, blood samples were collected for ELISA, the animals were killed, and tissues collected for tissue culture and histopathology. At necropsy, no macroscopic lesions related to paratuberculosis were observed. Similarly, no histological changes were observed and MAP in samples stained by Ziehl-Neelsen was not detected. These animals were characterized as potential passive shedders with upward contamination of the teat canal by MAP. This is the first report of the passive shedding phenomenon in goats in Brazil and it highlights the importance of identifying these animals for control programs and to ensure the quality of dairy products. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

DIAGNOSIS AND IMPLICATIONS OF MYCOBACTERIUM BOVIS INFECTION IN BANDED MONGOOSES (MUNGOS MUNGO) IN THE KRUGER NATIONAL PARK, SOUTH AFRICA.

PubMed

Brüns, Angela C; Tanner, Manfred; Williams, Mark C; Botha, Louise; O'Brien, Amanda; Fosgate, Geoffrey T; van Helden, Paul D; Clarke, John; Michel, Anita L

2017-01-01

Bovine tuberculosis (bTB) was first diagnosed in the Kruger National Park (KNP) in 1990. Research has since focused on the maintenance host, the African buffalo ( Syncerus caffer ) and clinically affected lion ( Panthera leo ). However, little is known about the role of small predators in tuberculosis epidemiology. During 2011-12, we screened banded mongooses ( Mungos mungo ) in the bTB high-prevalence zone of the KNP for Mycobacterium tuberculosis complex members. Fecal swabs, tracheal swabs, and tracheal lavages of 76 banded mongooses caught in cage traps within a 2-km radius of Skukuza Rest Camp were submitted for Mycobacterium culture, isolation, and species identification. Lesions and lymph node samples collected from 12 animals at postmortem examination were submitted for culture and histopathology. In lung and lymph nodes of two banded mongooses, well demarcated, irregularly margined, gray-yellow nodules of up to 5 mm diameter were identified with either central necrosis or calcification, characterized on histopathology as caseating necrosis with epithelioid macrophages or necrogranuloma with calcified centre. No acid fast bacteria were identified with Ziehl-Neelsen stain. We isolated Mycobacterium bovis from lung, lymph node, and liver samples, as well as from tracheal lavages and tracheal swab from the same two banded mongooses. Blood samples were positive by ElephantTB STAT-PAK® Assay for 12 and Enferplex™ TB Assay for five animals. Only the two banded mongooses positive on pathology and M. bovis culture were positive on both serologic assays. We provide evidence of bTB infection in banded mongooses in the KNP, demonstrate their ability to shed M. bovis , and propose a possible antemortem diagnostic algorithm. Our findings open the discussion around possible sources of infection and their significance at the human/wildlife interface in and around Skukuza.

Characterization of mycobacteria in HIV/AIDS patients of Nepal.

PubMed

Dhungana, G P; Ghimire, P; Sharma, S; Rijal, B P

2008-01-01

Besides Mycobacterium tuberculosis, a number of other Mycobacterium species are also occasional human pathogens. Tuberculosis due to Mycobacterium avium complex (MAC) and Mycobacterium kansasii is particularly prevalent in AIDS patients as compared to the normal population. A cross-sectional study was carried out during January 2004 to August 2005 in 100 HIV-infected persons visiting Tribhuvan University, Teaching Hospital, and about a dozen of HIV/AIDS care centers of Kathmandu with the objectives to characterize the different mycobacterial species in HIV/AIDS patients. Three sputum specimens from each person were used to investigate tuberculosis by Ziehl-Neelsen staining, culture and identification tests. Among the 100 HIV-infected cases, 66 (66%) were males and 34 (34%) were females. Sixty percent of the cases were in the age group of 21-30 years. Mycobacteria were detected in 23 (23%) HIV cases of which 15 (65.2%) were in the age group of 21-30 years ; 17(74%) were males and 6 (26 %) were females. Among 23 co-infected cases, 22 were culture positive for mycobacteria. Among these, the predominant one was Mycobacterium avium complex (MAC), 9 (41%), followed by M. tuberculosis, 6 (27%), M .kansasii, 4 (18%), M. fortuitum, 2 (10%) and M. chelonae 1 (4%). Significant relationship was established between smoking/alcoholism and the subsequent development of tuberculosis (chi(2)=7.24, p<0.05 for smoking habit and chi(2)=4.39, p<0.05 for alcoholism). Fourteen (61%) co-infected cases presented with weight loss and cough whereas diarrhea was presented only by those patients with atypical mycobacterial co-infection, which was as high as 5 (56%) in patients with MAC co-infection. This study demonstrated the predominance of atypical mycobacteria, mainly MAC, in HIV/AIDS cases and most of them were from sputum smear-negative cases.

Intestinal parasitic infections among children under five years of age presenting with diarrhoeal diseases to two public health facilities in Hawassa, South Ethiopia.

PubMed

Mulatu, Getamesay; Zeynudin, Ahmed; Zemene, Endalew; Debalke, Serkadis; Beyene, Getenet

2015-11-04

Diarrhoea is the leading cause of morbidity and mortality in children under 5 years of age in developing countries, including Ethiopia. It is caused by a wide range of pathogens, including parasites, bacteria and viruses. The aim of this study was to determine the prevalence of infection with intestinal parasites (IPs) (and types) among children under 5 years of age with diarrhoeal diseases. A cross-sectional study was conducted at Adare Hospital and Millennium Health Centre, both located in Hawassa, South Ethiopia, from June 6 to October 28, 2011. Children under 5 years of age with diarrhoea who visited these health facilities during the study period were included in the study. Data relating to demography and risk factors associated with intestinal parasitic infections (IPIs) were gathered using a structured questionnaire. Single, fresh stool specimens were examined for IPs using the direct wet mount examination, followed by Ziehl-Neelsen staining of formol-ether concentrated samples, as per standard procedures. Data were analysed using SPSS Statistics 20 software. A total of 158 children (51.3 % male and 48.7 % female) participated in the study. Overall, the prevalence of IPs was 26.6 % (42/158). Two species of IPs were detected in six children (3.8 %). Entamoeba histolytica/dispar/moshkovskii was the predominant parasite identified (11.4 %), followed by Giardia duodenalis (7.0 %). The multivariable analysis revealed that the age group ≥24 months was significantly associated (AOR = 0.221, 95 %CI: 0.085-0.576) with prevalence of IPIs. This study found that intestinal parasites are common among children with diarrheal diseases. The most frequently detected species was E. histolytica/dispar/moshkovskii. Health information about how to prevent diarrheal diseases in general and IPIs in particular should be provided to parents of young children.

Prevalence, characteristics and correlates of enteric pathogenic protozoa in drinking water sources in Molyko and Bomaka, Cameroon: a cross-sectional study.

PubMed

Nsoh, Fuh Anold; Wung, Buh Amos; Atashili, Julius; Benjamin, Pokam Thumamo; Marvlyn, Eba; Ivo, Keumami Katte; Nguedia, Assob Jules Clément

2016-11-08

Access to potable water remains a major challenge particularly in resource-limited settings. Although the potential contaminants of water are varied, enteric pathogenic protozoa are known to cause waterborne diseases greatly. This study aimed at investigating the prevalence, characteristics and correlates of enteric pathogenic protozoa in drinking water sources in Buea, Cameroon. A cross-sectional study was conducted using 155 water samples collected from various drinking sources (boreholes, springs, taps and wells). Each sample was subjected to physicochemical examinations (pH, turbidity, odour and sliminess) and parasitological analysis (wet mount, modified Ziehl-Neelsen stain) to determine the presence of enteric pathogenic protozoa. A data collection tool was used to note characteristics of collected samples and the data was analysed using EPI-INFO Version 3.5.3. The overall prevalence of enteric pathogenic protozoa in water sources was 62.6 %. Eight species of enteric protozoa were observed with Cryptosporidium parvum being the most predominant (45.8 %). Spring water was the most contaminated source with enteric protozoa (85.7 %) while pipe borne water had all eight species of protozoa identified. A pH of 6 was the only significant factor associated with the prevalence of these pathogens in water sources. The prevalence of enteric protozoa in water sources in Molyko and Bomaka is high, spring water is the most contaminated water source and Cryptosporidium parvum is the most common protozoa contaminating water. A water pH of 6 is associated to the prevalence of protozoa. Community members need to be educated to treat water before drinking to avoid infection by enteric protozoa in water and further studies with larger samples of water need to be conducted to find other correlates of the presence of protozoa in water.

Al adjuvants can be tracked in viable cells by lumogallion staining.

PubMed

Mile, Irene; Svensson, Andreas; Darabi, Anna; Mold, Matthew; Siesjö, Peter; Eriksson, Håkan

2015-07-01

The mechanism behind the adjuvant effect of aluminum salts is poorly understood notwithstanding that aluminum salts have been used for decades in clinical vaccines. In an aqueous environment and at a nearly neutral pH, the aluminum salts form particulate aggregates, and one plausible explanation of the lack of information regarding the mechanisms could be the absence of an efficient method of tracking phagocytosed aluminum adjuvants and thereby the intracellular location of the adjuvant. In this paper, we want to report upon the use of lumogallion staining enabling the detection of phagocytosed aluminum adjuvants inside viable cells. Including micromolar concentrations of lumogallion in the culture medium resulted in a strong fluorescence signal from cells that had phagocytosed the aluminum adjuvant. The fluorescence appeared as spots in the cytoplasm and by confocal microscopy and co-staining with probes presenting fluorescence in the far-red region of the spectrum, aluminum adjuvants could to a certain extent be identified as localized in acidic vesicles, i.e., lysosomes. Staining and detection of intracellular aluminum adjuvants was achieved not only by diffusion of lumogallion into the cytoplasm, thereby highlighting the presence of the adjuvant, but also by pre-staining the aluminum adjuvant prior to incubation with cells. Pre-staining of aluminum adjuvants resulted in bright fluorescent particulate aggregates that remained fluorescent for weeks and with only a minor reduction of fluorescence upon extensive washing or incubation with cells. Both aluminum oxyhydroxide and aluminum hydroxyphosphate, two of the most commonly used aluminum adjuvants in clinical vaccines, could be pre-stained with lumogallion and were easily tracked intracellularly after incubation with phagocytosing cells. Staining of viable cells using lumogallion will be a useful method in investigations of the mechanisms behind aluminum adjuvants' differentiation of antigen-presenting cells

Rapid alkaline methylene blue supravital staining for assessment of anterior segment infections.

PubMed

Kiuchi, Katsuji

2016-01-01

To present the Löffler's alkaline methylene blue technique of staining eye discharges in eyes with anterior segment infections. The Löffler's alkaline methylene blue staining method is a simple staining technique that can be used to differentiate bacterial, viral, and fungal infections. It is a cationic dye that stains cells blue because the positively charged dye is attracted to negatively charged particles such as polyphosphates, DNAs, and RNAs. Specimens collected from patients by swabbing are smeared onto microscope slides and the methylene blue solution is dropped on the slide. The slide is covered with a glass cover slip and examined under a microscope. The entire time from the collection to the viewing is about 30 seconds. Histopathological images of the conjunctival epithelial cells and neutrophils in eye discharges were dyed blue and the nuclei were stained more intensely blue. Bacterial infections consisted mainly of neutrophils, and viral infections consisted mainly of lymphocytes. Löffler's alkaline methylene blue staining can be done in about 30 seconds for diagnosis. Even though this is a one color stain, it is possible to infer the cause of the infection by detection of the absence of bacteria and/or fungi in context of the differential distribution of neutrophils and lymphocytes.

Comparison of algorithms for blood stain detection applied to forensic hyperspectral imagery

NASA Astrophysics Data System (ADS)

Yang, Jie; Messinger, David W.; Mathew, Jobin J.; Dube, Roger R.

2016-05-01

Blood stains are among the most important types of evidence for forensic investigation. They contain valuable DNA information, and the pattern of the stains can suggest specifics about the nature of the violence that transpired at the scene. Early detection of blood stains is particularly important since the blood reacts physically and chemically with air and materials over time. Accurate identification of blood remnants, including regions that might have been intentionally cleaned, is an important aspect of forensic investigation. Hyperspectral imaging might be a potential method to detect blood stains because it is non-contact and provides substantial spectral information that can be used to identify regions in a scene with trace amounts of blood. The potential complexity of scenes in which such vast violence occurs can be high when the range of scene material types and conditions containing blood stains at a crime scene are considered. Some stains are hard to detect by the unaided eye, especially if a conscious effort to clean the scene has occurred (we refer to these as "latent" blood stains). In this paper we present the initial results of a study of the use of hyperspectral imaging algorithms for blood detection in complex scenes. We describe a hyperspectral imaging system which generates images covering 400 nm - 700 nm visible range with a spectral resolution of 10 nm. Three image sets of 31 wavelength bands were generated using this camera for a simulated indoor crime scene in which blood stains were placed on a T-shirt and walls. To detect blood stains in the scene, Principal Component Analysis (PCA), Subspace Reed Xiaoli Detection (SRXD), and Topological Anomaly Detection (TAD) algorithms were used. Comparison of the three hyperspectral image analysis techniques shows that TAD is most suitable for detecting blood stains and discovering latent blood stains.

Fluorescein-labeled β-Glucosidase as a Bacterial Stain

PubMed Central

Pital, Abe; Janowitz, Sheldon L.; Hudak, Charles E.; Lewis, Evelyn E.

1967-01-01

Fluorescein isothiocyanate-labeled β-glucosidase was used as a simple staining reagent with selected gram-positive and gram-negative organisms. Staining in situ appeared to be dependent on the presence of accessible glycosidic-type linkages in the bacterial cell wall. Extensive wall damage or lysis did not occur when stained cells were suspended in washing and mounting solutions. The apparent specificity of labeled enzyme for wall substance was tested by blocking reactions, staining of isolated cell walls, and failure to stain substances lacking appropriate glycosidic linkages. Severe cell wall lesions were produced after prolonged contact with labeled enzyme, and this phenomenon may also be related to staining specificity. Gram-negative organisms and spores were poorly stained unless protected glycopeptide substrate was previously exposed by treatment of cells with thioglycolic acid or dilute alkaline sodium hypochlorite solution. A potential for staining tissues and cell lines may also exist. Some possible applications of labeled enzymes are briefly discussed. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:4169543

Better detection of Demodex mites by Löffler's alkaline methylene blue staining in patients with blepharitis.

PubMed

Kiuchi, Katsuji

2018-01-01

To determine whether the Löffler's alkaline methylene blue staining method is better than no staining in detecting Demodex mites in the eyelashes of patients with blepharitis. Eyelashes were collected from 22 patients with blepharitis. The mean age of the patients was 82.5±6.2 years (± SD) with a range from 71 to 93 years. Eyelashes were epilated by forceps and placed individually on microscope slides. The number of Demodex mites was determined by conventional optical microscopy before and immediately after the addition of the methylene blue staining solution. The mean Demodex count before the addition of the methylene blue solution was 2.9±2.9, and it was 4.4±3.9 after the addition of the methylene blue solution ( P <0.01, Wilcoxon test). The methylene blue staining method is a simple and useful method in detecting the presence and quantifying the number of Demodex mites. We recommend the methylene blue staining method not only for the diagnosis of the presence of Demodex mites but also to evaluate the therapeutic effects of medications to eliminate the mite infestation.

Use of the gram stain in microbiology.

PubMed

Beveridge, T J

2001-05-01

The Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria that retain the initial crystal violet stain (purple) are said to be "gram-positive," whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be "gram-negative." This staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria. Gram-positives have a thick, relatively impermeable wall that resists decolorization and is composed of peptidoglycan and secondary polymers. Gram-negatives have a thin peptidoglycan layer plus an overlying lipid-protein bilayer known as the outer membrane, which can be disrupted by decolorization. Some bacteria have walls of intermediate structure and, although they are officially classified as gram-positives because of their linage, they stain in a variable manner. One prokaryote domain, the Archaea, have such variability of wall structure that the Gram stain is not a useful differentiating tool.

Aureobasidium pullulans morphology: two adapted polysaccharide stains.

PubMed

Oller, Anna R

2005-12-01

Morphological stages of Aureobasidium pullulans were investigated utilizing different media ingredients and were visualized by bright-field microscopy. A polysaccharide stain was developed to stain chlamydospores, cell walls, hyphae, and conidia, since current staining techniques do not reveal subcellular details to identify fungi, especially those that exhibit polysaccharide secretions.

Multicenter Assessment of Gram Stain Error Rates

PubMed Central

Balada-Llasat, Joan-Miquel; Harrington, Amanda; Cavagnolo, Robert

2016-01-01

Gram stains remain the cornerstone of diagnostic testing in the microbiology laboratory for the guidance of empirical treatment prior to availability of culture results. Incorrectly interpreted Gram stains may adversely impact patient care, and yet there are no comprehensive studies that have evaluated the reliability of the technique and there are no established standards for performance. In this study, clinical microbiology laboratories at four major tertiary medical care centers evaluated Gram stain error rates across all nonblood specimen types by using standardized criteria. The study focused on several factors that primarily contribute to errors in the process, including poor specimen quality, smear preparation, and interpretation of the smears. The number of specimens during the evaluation period ranged from 976 to 1,864 specimens per site, and there were a total of 6,115 specimens. Gram stain results were discrepant from culture for 5% of all specimens. Fifty-eight percent of discrepant results were specimens with no organisms reported on Gram stain but significant growth on culture, while 42% of discrepant results had reported organisms on Gram stain that were not recovered in culture. Upon review of available slides, 24% (63/263) of discrepant results were due to reader error, which varied significantly based on site (9% to 45%). The Gram stain error rate also varied between sites, ranging from 0.4% to 2.7%. The data demonstrate a significant variability between laboratories in Gram stain performance and affirm the need for ongoing quality assessment by laboratories. Standardized monitoring of Gram stains is an essential quality control tool for laboratories and is necessary for the establishment of a quality benchmark across laboratories. PMID:26888900

Propidium iodide (PI) stains Nissl bodies and may serve as a quick marker for total neuronal cell count.

PubMed

Niu, Junfei; Li, Chunman; Wu, Haihui; Feng, Xianling; Su, Qingning; Li, Shihe; Zhang, Lihong; Yew, David Tai Wai; Cho, Eric Yu Pang; Sha, Ou

2015-03-01

Propidium iodide (PI) reacts with both DNA and RNA and is a commonly used fluorescent reagent for nucleic acid staining. The aim of the study was to compare the cellular staining patterns of PI with that of Nissl staining in rat nervous tissues and to report a modified staining method that selectively labels Nissl bodies in neurons. Cryosections and paraffin sections of different tissues of normal Sprague-Dawley rats, including trigeminal ganglia, dorsal root ganglia, spinal cord, liver, and small intestine, were stained by either PI or the hematoxylin and eosin method. Some sections were treated with RNase or DNase before the above staining, and some were double stained with PI and a Nissl stain. The sections were observed by light, fluorescence or confocal microscopy. Results showed strong PI signals detected as patterns of granules in the neuronal cytoplasm of all nervous tissues, whereas the staining of neuronal nuclei was weaker. In contrast, nuclei of neuroglial cells were strongly stained by PI, while the cytoplasm was not obviously stained. Pretreatment of the neural tissue with RNase abolished the PI signals. Furthermore, the PI positive granules in neuronal cytoplasm co-localized with Nissl bodies stained by the fluorescent Nissl stain. When the tissue was pretreated with DNase, PI only stained the cytoplasmic granules of neurons, but not that of glial cells. Our results show that PI stains Nissl bodies and may serve as an economical and convenient neuron marker for neuronal cell counting when specific neural markers such as antibodies are not readily available. Copyright © 2015. Published by Elsevier GmbH.

Better detection of Demodex mites by Löffler’s alkaline methylene blue staining in patients with blepharitis

PubMed Central

Kiuchi, Katsuji

2018-01-01

Purpose To determine whether the Löffler’s alkaline methylene blue staining method is better than no staining in detecting Demodex mites in the eyelashes of patients with blepharitis. Materials and methods Eyelashes were collected from 22 patients with blepharitis. The mean age of the patients was 82.5±6.2 years (± SD) with a range from 71 to 93 years. Eyelashes were epilated by forceps and placed individually on microscope slides. The number of Demodex mites was determined by conventional optical microscopy before and immediately after the addition of the methylene blue staining solution. Results The mean Demodex count before the addition of the methylene blue solution was 2.9±2.9, and it was 4.4±3.9 after the addition of the methylene blue solution (P<0.01, Wilcoxon test). Conclusion The methylene blue staining method is a simple and useful method in detecting the presence and quantifying the number of Demodex mites. We recommend the methylene blue staining method not only for the diagnosis of the presence of Demodex mites but also to evaluate the therapeutic effects of medications to eliminate the mite infestation. PMID:29713140

Adaptive segmentation of nuclei in H&S stained tendon microscopy

NASA Astrophysics Data System (ADS)

Chuang, Bo-I.; Wu, Po-Ting; Hsu, Jian-Han; Jou, I.-Ming; Su, Fong-Chin; Sun, Yung-Nien

2015-12-01

Tendiopathy is a popular clinical issue in recent years. In most cases like trigger finger or tennis elbow, the pathology change can be observed under H and E stained tendon microscopy. However, the qualitative analysis is too subjective and thus the results heavily depend on the observers. We develop an automatic segmentation procedure which segments and counts the nuclei in H and E stained tendon microscopy fast and precisely. This procedure first determines the complexity of images and then segments the nuclei from the image. For the complex images, the proposed method adopts sampling-based thresholding to segment the nuclei. While for the simple images, the Laplacian-based thresholding is employed to re-segment the nuclei more accurately. In the experiments, the proposed method is compared with the experts outlined results. The nuclei number of proposed method is closed to the experts counted, and the processing time of proposed method is much faster than the experts'.

Automated single-slide staining device. [in clinical bacteriology

NASA Technical Reports Server (NTRS)

Wilkins, J. R.; Mills, S. M.

1975-01-01

An automatic single-slide Gram staining device is described. A timer-actuated solenoid controls the dispensing of gentian violet, Gram iodine solution, decolorizer, and 1% aqueous safranin in proper sequence and for the time required for optimum staining. The amount of stain or reagent delivered is controlled by means of stopcocks below each solenoid. Used stains and reagents can be flushed automatically or manually. Smears Gram stained automatically are equal in quality to those prepared manually. The time to complete one Gram cycle is 4.80 min.

Multicenter Assessment of Gram Stain Error Rates.

PubMed

Samuel, Linoj P; Balada-Llasat, Joan-Miquel; Harrington, Amanda; Cavagnolo, Robert

2016-06-01

Gram stains remain the cornerstone of diagnostic testing in the microbiology laboratory for the guidance of empirical treatment prior to availability of culture results. Incorrectly interpreted Gram stains may adversely impact patient care, and yet there are no comprehensive studies that have evaluated the reliability of the technique and there are no established standards for performance. In this study, clinical microbiology laboratories at four major tertiary medical care centers evaluated Gram stain error rates across all nonblood specimen types by using standardized criteria. The study focused on several factors that primarily contribute to errors in the process, including poor specimen quality, smear preparation, and interpretation of the smears. The number of specimens during the evaluation period ranged from 976 to 1,864 specimens per site, and there were a total of 6,115 specimens. Gram stain results were discrepant from culture for 5% of all specimens. Fifty-eight percent of discrepant results were specimens with no organisms reported on Gram stain but significant growth on culture, while 42% of discrepant results had reported organisms on Gram stain that were not recovered in culture. Upon review of available slides, 24% (63/263) of discrepant results were due to reader error, which varied significantly based on site (9% to 45%). The Gram stain error rate also varied between sites, ranging from 0.4% to 2.7%. The data demonstrate a significant variability between laboratories in Gram stain performance and affirm the need for ongoing quality assessment by laboratories. Standardized monitoring of Gram stains is an essential quality control tool for laboratories and is necessary for the establishment of a quality benchmark across laboratories. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Say goodbye to coffee stains

NASA Astrophysics Data System (ADS)

Burak Eral, H.; van den Ende, Dirk; Mugele, Frieder

2012-04-01

Discussing ideas over a mug of coffee or tea is the lifeblood of science, but have you ever thought about the stains that can be inadvertently left behind? H Burak Eral, Dirk van den Ende and Frieder Mugele explain how these stains, which can be a major annoyance in some biology techniques, can be altered for the better using a technique called electrowetting.

Practical selection methods for rat and mouse round spermatids without DNA staining by flow cytometric cell sorting.

PubMed

Hayama, Tomonari; Yamaguchi, Tomoyuki; Kato-Itoh, Megumi; Ishii, Yumiko; Mizuno, Naoaki; Umino, Ayumi; Sato, Hideyuki; Sanbo, Makoto; Hamanaka, Sanae; Masaki, Hideki; Hirabayashi, Masumi; Nakauchi, Hiromitsu

2016-06-01

Round spermatid injection (ROSI) into unfertilized oocytes enables a male with a severe spermatogenesis disorder to have children. One limitation of the application of this technique in the clinic is the identification and isolation of round spermatids from testis tissue. Here we developed an efficient and simple method to isolate rodent haploid round spermatids using flow cytometric cell sorting, based on DNA content (stained with Hoechst 33342 or Dye Cycle Violet) or by cell diameter and granularity (forward and side scatter). ROSI was performed with round spermatids selected by flow cytometry, and we obtained healthy offspring from unstained cells. This non-invasive method could therefore be an effective option for breeding domestic animals and human male infertility treatment. Mol. Reprod. Dev. 83: 488-496, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Offset-sparsity decomposition for enhancement of color microscopic image of stained specimen in histopathology: further results

NASA Astrophysics Data System (ADS)

Kopriva, Ivica; Popović Hadžija, Marijana; Hadžija, Mirko; Aralica, Gorana

2016-03-01

Recently, novel data-driven offset-sparsity decomposition (OSD) method was proposed by us to increase colorimetric difference between tissue-structures present in the color microscopic image of stained specimen in histopathology. The OSD method performs additive decomposition of vectorized spectral images into image-adapted offset term and sparse term. Thereby, the sparse term represents an enhanced image. The method was tested on images of the histological slides of human liver stained with hematoxylin and eosin, anti-CD34 monoclonal antibody and Sudan III. Herein, we present further results related to increase of colorimetric difference between tissue structures present in the images of human liver specimens with pancreatic carcinoma metastasis stained with Gomori, CK7, CDX2 and LCA, and with colon carcinoma metastasis stained with Gomori, CK20 and PAN CK. Obtained relative increase of colorimetric difference is in the range [19.36%, 103.94%].

Performance of the Xpert MTB/RIF assay in the diagnosis of tuberculosis in formalin-fixed, paraffin-embedded tissues.

PubMed

Polepole, Pascal; Kabwe, Mwila; Kasonde, Mpanga; Tembo, John; Shibemba, Aaron; O'Grady, Justin; Kapata, Nathan; Zumla, Alimuddin; Bates, Matthew

2017-01-01

Extrapulmonary tuberculosis (EPTB), which accounts for 10%-40% of the global burden of TB, with the highest incidence in Sub-Saharan Africa, is strongly associated with human immunodeficiency virus infection. Diagnosing EPTB is challenging, and recently, there has been a concerted effort to evaluate the latest molecular diagnostics for diagnosing TB in a range of specimen types. The Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA, USA) is one such technology, which simultaneously detects Mycobacterium tuberculosis and rifampicin resistance. Our objective was to evaluate the accuracy of the Xpert MTB/RIF assay for the diagnosis of EPTB and detection of rifampicin resistance in routinely processed formalin-fixed, paraffin-embedded (FFPE) tissues, compared with histological detection of TB as the gold standard. A convenience set of 100 biobanked FFPE tissues, including lymph nodes (n = 64), male genital tract tissue (n = 10), abdominal tissue (n = 8), female genital tissue (n = 5), breast tissue (n = 5), synovial tissue (n = 4), skin (n = 2), tongue tissue (n = 1), and thyroid (n = 1), from routine cases of clinically suspected EPTB admitted to the University Teaching Hospital, Lusaka, Zambia, were analyzed using the Xpert MTB/RIF assay and in-house polymerase chain reaction (PCR) assay targeting IS6110, in parallel with Ziehl-Neelsen (ZN) staining, against histology as the gold standard. Some 66% of specimens had histological evidence of TB infection. ZN staining was positive for TB in 8% of cases, and Xpert MTB/RIF was positive for TB in 25% of cases. Taking histology as the gold standard, the sensitivity and specificity were as follows: In lymph tissue the accuracy of the Xpert MTB/RIF assay was 41% (95%CI 27-57), not significantly better than ZN or the in-house PCR assay. In non-lymph tissue the sensitivity of the in-house PCR assay was 82% (95%CI: 56%-95%), significantly higher than the Xpert MTB/RIF assay (P = 0.004). The Xpert MTB/RIF assay indicated rifampicin

A tale of two communities: intestinal polyparasitism among Orang Asli and Malay communities in rural Terengganu, Malaysia.

PubMed

Elyana, Fatin Nur; Al-Mekhlafi, Hesham M; Ithoi, Init; Abdulsalam, Awatif M; Dawaki, Salwa; Nasr, Nabil A; Atroosh, Wahib M; Abd-Basher, Mohamad Hafiz; Al-Areeqi, Mona A; Sady, Hany; Subramaniam, Lahvanya R; Anuar, Tengku Shahrul; Lau, Yee Ling; Moktar, Norhayati; Surin, Johari

2016-07-16

Intestinal parasitic infections (IPIs) are still major health problems in many developing countries including Malaysia, particularly in the poor and socioeconomically deprived rural and remote communities in Peninsular Malaysia. This study was conducted to determine the prevalence of IPIs and to identify the key factors associated with intestinal polyparasitism as well as to evaluate the knowledge, attitude and practices (KAP) on IPIs among rural Orang Asli and Malay communities in Terengganu, Malaysia. A cross-sectional study was conducted among 340 participants (165 Orang Asli and 175 Malay) aged ≤ 15 years from the Hulu Terengganu and Kemaman districts of Terengganu. Faecal samples were examined for the presence of intestinal parasites by using direct smear, formalin-ether sedimentation, trichrome stain, modified Ziehl Neelsen stain, in vitro cultivation in Jones' medium, Kato Katz and Harada Mori techniques. Demographic, socioeconomic, environmental and behavioural information of the participants and their KAP for IPIs were collected by using a pre-tested questionnaire. Overall, 149 (90.3 %) Orang Asli and 43 (24.6 %) Malay children were infected by at least one parasite species. The overall prevalences of intestinal polyparasitism among the Orang Asli and Malay were 68.5 % (113/165) and 14.3 % (25/175), respectively. Multiple logistic regression analysis showed that using unsafe water supply as a source for drinking water, the presence of domestic animals, not wearing shoes when outside, not washing vegetables before consumption, not washing hands after playing with soil, indiscriminate defecation and the low level of mother's education were the key risk factors for intestinal polyparasitism among the Orang Asli, while working mothers and the presence of domestic animals were the risk factors among the Malay children. Almost all the Malays were well aware about the IPIs while Orang Asli respondents had a poor level of related awareness. This study

Identification of the 1B vaccine strain of Chlamydia abortus in aborted placentas during the investigation of toxaemic and systemic disease in sheep.

PubMed

Sargison, N D; Truyers, I G R; Howie, F E; Thomson, J R; Cox, A L; Livingstone, M; Longbottom, D

2015-09-01

One hundred and forty Cheviot and 100 Suffolk cross Mule primiparous 1-2-year-old ewes, from a flock of about 700 ewes, were vaccinated with an attenuated live 1B strain Chlamydia abortus vaccine about 4 weeks before ram introduction (September 2011). Between 08 March and 01 April 2012, 50 2-year-old ewes aborted and 29 of these died, despite antimicrobial and anti-inflammatory treatment and supportive care. Seven fetuses and three placentae from five 2-year-old ewes were submitted for pathological investigation. The aborted fetuses showed stages of autolysis ranging from being moderately fresh to putrefaction. Unusual, large multifocal regions of thickened membranes, with a dull red granular surface and moderate amounts of grey-white surface exudate were seen on each of the placentae. Intracellular, magenta-staining, acid fast inclusions were identified in Ziehl Neelsen-stained placental smears. Immunohistochemistry for Chlamydia-specific lipopolysaccharide showed extensive positive labelling of the placental epithelia. Molecular analyses of the aborted placentae demonstrated the presence of the 1B vaccine-type strain of C. abortus and absence of any wild-type field strain. The vaccine strain bacterial load of the placental tissue samples was consistent with there being an association between vaccination and abortion. Initial laboratory investigations resulted in a diagnosis of chlamydial abortion. Further investigations led to the identification of the 1B vaccine strain of C. abortus in material from all three of the submitted aborted placentae. Timely knowledge and understanding of any potential problems caused by vaccination against C. abortus are prerequisites for sustainable control of chlamydial abortion. This report describes the investigation of an atypical abortion storm in sheep, and describes the identification of the 1B vaccine strain of C. abortus in products of abortion. The significance of this novel putative association between the vaccine strain

CGP stain: An inexpensive, odorless, rapid, sensitive, and in principle in vitro methylation-free Coomassie Brilliant Blue stain.

PubMed

Yasumitsu, Hidetaro; Ozeki, Yasuhiro; Kawsar, Sarkar M A; Toda, Tosifusa; Kanaly, Robert

2010-11-01

Coomassie Brilliant Blue (CBB) protein stains are inexpensive but detect proteins at only at microgram levels. Because of acetic acid and methanol, they cause skin irritation and reduce work motivation by malodor. Recent mass spectrometric (MS) analyses demonstrated that nanogram-sensitive colloidal CBB staining resulted in in vitro methylations of proteins. We propose a rapid, inexpensive, sensitive, odorless, less harsh, and in vitro methylation-free CBB stain. CGP uses three components: citric acid, CBB G-250, and polyvinylpyrrolidone. CGP detects proteins at 12ng within 45min, and because it is nonalcohol, in principle in vitro methylation would be eliminated. Indeed, MS analysis of CGP-stained bands confirmed a lack of methylation. 2010 Elsevier Inc. All rights reserved.

Real-time PCR assay for the diagnosis of pleural tuberculosis

PubMed Central

Cárdenas Bernal, Ana María; Giraldo-Cadavid, Luis Fernando; Prieto Diago, Enrique; Santander, Sandra Paola

2017-01-01

Abstract Introduction: The diagnosis of pleural tuberculosis requires an invasive and time-consuming reference method. Polymerase chain reaction (PCR) is rapid, but validation in pleural tuberculosis is still weak. Objective: To establish the operating characteristics of real-time polymerase chain reaction (RT-PCR) hybridization probes for the diagnosis of pleural tuberculosis. Methods: The validity of the RT-PCR hybridization probes was evaluated compared to a composite reference method by a cross-sectional study at the Hospital Universitario de la Samaritana. 40 adults with lymphocytic pleural effusion were included. Pleural tuberculosis was confirmed (in 9 patients) if the patient had at least one of three tests using the positive reference method: Ziehl-Neelsen or Mycobacterium tuberculosis culture in fluid or pleural tissue, or pleural biopsy with granulomas. Pleural tuberculosis was ruled out (in 31 patients) if all three tests were negative. The operating characteristics of the RT-PCR, using the Mid-P Exact Test, were determined using the OpenEpi 2.3 Software (2009). Results: The RT-PCR hybridization probes showed a sensitivity of 66.7% (95% CI: 33.2%-90.7%) and a specificity of 93.5% (95% CI: 80.3%-98.9%). The PPV was 75.0% (95% CI: 38.8%-95.6%) and a NPV of 90.6% (95% CI: 76.6%-97.6%). Two false positives were found for the test, one with pleural mesothelioma and the other with chronic pleuritis with mesothelial hyperplasia. Conclusions: The RT-PCR hybridization probes had good specificity and acceptable sensitivity, but a negative value cannot rule out pleural tuberculosis. PMID:29021638

Utility of Modified Ultrafast Papanicolaou Stain in Cytological Diagnosis.

PubMed

Sinkar, Prachi; Arakeri, Surekha Ulhas

2017-03-01

Need for minimal turnaround time for assessing Fine Needle Aspiration Cytology (FNAC) has encouraged innovations in staining techniques that require lesser staining time with unequivocal cell morphology. The standard protocol for conventional Papanicolaou (PAP) stain requires about 40 minutes. To overcome this, Ultrafast Papanicolaou (UFP) stain was introduced which reduces staining time to 90 seconds and also enhances the quality. However, reagents required for this were not easily available hence, Modified Ultrafast Papanicolaou (MUFP) stain was introduced subsequently. To assess the efficacy of MUFP staining by comparing the quality of MUFP stain with conventional PAP stain. FNAC procedure was performed by using 10 ml disposable syringe and 22-23 G needle. Total 131 FNAC cases were studied which were lymph node (30), thyroid (38), breast (22), skin and soft tissue (24), salivary gland (11) and visceral organs (6). Two smears were prepared and stained by MUFP and conventional PAP stain. Scores were given on four parameters: background of smears, overall staining pattern, cell morphology and nuclear staining. Quality Index (QI) was calculated from ratio of total score achieved to maximum score possible. Statistical analysis using chi square test was applied to each of the four parameters before obtaining the QI in both stains. Students t-test was applied to evaluate the efficacy of MUFP in comparison with conventional PAP stain. The QI of MUFP for thyroid, breast, lymph node, skin and soft tissue, salivary gland and visceral organs was 0.89, 0.85, 0.89, 0.83, 0.92, and 0.78 respectively. Compared to conventional PAP stain QI of MUFP smears was better in all except visceral organ cases and was statistically significant. MUFP showed clear red blood cell background, transparent cytoplasm and crisp nuclear features. MUFP is fast, reliable and can be done with locally available reagents with unequivocal morphology which is the need of the hour for a cytopathology set-up.

Standardized method to produce tetracycline-stained human molar teeth in vitro.

PubMed

Chan, Daniel C N; Rozier, Gregory Shayne; Steen, Angela; Browning, William D; Mozaffari, Mahmood S

2006-09-01

This study tested the hypothesis that exposure of human molar teeth to tetracycline (TCN) derivatives in vitro results in tooth discoloration resembling the clinical presentation of TCN staining. The effects of exposure of 20 extracted human molar teeth to distilled water, chlortetracycline, doxycycline, or minocycline were compared. The baseline color of each tooth was analyzed with a dental spectrophotometer. The pulp chambers were each filled with a TCN derivative solution and then sealed. The teeth were placed in a centrifuge tube and then centrifuged at 2800 rpm for 20 minutes. Color change was monitored weekly for 7 weeks. Digital images of the surfaces were recorded. For each specimen at every evaluation period, color change from baseline was calculated using Commission Internationale d'Eclairage (CIE) Delta E 2000 (deltae00). There was a significant association between the type of derivative used and deltae00, as well as between the evaluation period and deltae00. There was also a significant association between the interaction term, derivative x evaluation period, and deltae00. Results of the Holm-Sidak post hoc test demonstrated that all 3 TCN derivatives were associated with significantly larger deltae00 than the control group (P < or = .05). All 3 TCN derivative solutions produced significant color changes as time progressed. Different TCN derivatives produced a different L* (lightness), C* (chroma), and H* (hue), with minocycline behaving distinctly differently from chlortetracycline and doxycycline. The model could be used to study the underlying mechanisms of TCN staining as well as many aspects of vital tooth

Sperm viability assessment in marine invertebrates by fluorescent staining and spectrofluorimetry: A promising tool for assessing marine pollution impact.

PubMed

Gallo, Alessandra; Boni, Raffaele; Tosti, Elisabetta

2018-01-01

The viability of spermatozoa is a crucial parameter to evaluate their quality that is an important issue in ecotoxicological studies. Here, a new method has been developed to rapidly determine the viability of spermatozoa in three marine invertebrates: the ascidian Ciona intestinalis, the sea urchin Paracentrotus lividus and the mollusc Mytilus galloprovincialis. This method employed the dual DNA fluorescent staining coupled with spectrofluorimetric analysis. The dual fluorescent staining used the SYBR-14 stained live spermatozoa and propidium iodide stained degenerated cells that had lost membrane integrity. Stain uptake was assessed by confocal microscopy and then the percentage of live and dead spermatozoa was quantified by spectrofluorimetric analysis. The microscopic examination revealed three populations of spermatozoa: living-SYBR-14 stained, dead-PI stained, and dying-doubly stained spermatozoa. The fluorescence emission peak values recorded in a spectrofluorimeter provide the portion of live and dead spermatozoa showing a significant negative correlation. The stain combination was further validated using known ratios of live and dead spermatozoa. The present study demonstrated that the dual DNA staining with SYBR-14 and propidium iodide was effective in assessing viability of spermatozoa in marine invertebrates and that spectrofluorimetric analysis can be successfully employed to evaluate the percentage of live and dead spermatozoa. The method develop herein is simple, accurate, rapid, sensitive, and cost-effective, so it could be a useful tool by which marine pollutants may be screened for spermiotoxicity. Copyright © 2017 Elsevier Inc. All rights reserved.

Prevalence and risk factors associated with endoparasitic infection in dogs from Transylvania (Romania): A retrospective study.

PubMed

Mircean, Viorica; Dumitrache, Mirabela Oana; Mircean, Mircea; Colosi, Horațiu Alexandru; Györke, Adriana

2017-08-30

During six years (April 2010-April 2016) we examined individual feces samples collected from 1314 dogs located in Center and Northwest Romania (Transylvania). Stool samples were analyzed by saturated salt flotation, sedimentation technique and modified Ziehl-Nielsen staining method. The overall prevalence of endoparasitic infections was 66.6% (n=875). Sixteen species/genera of endoparasites were identified. The most prevalent species were Ancylostoma caninum/Uncinaria stenocephala (33.0%) (p=0.0001) followed by Trichocephalus vulpis (25.0%). Mixed infections, were significantly more frequent (p=0.0001) than single species infections. The age and the living condition/service of dogs were identified as the main risk factors for infection with endoparasites. Copyright © 2017 Elsevier B.V. All rights reserved.

An optimized staining technique for the detection of Gram positive and Gram negative bacteria within tissue.

PubMed

Becerra, Sandra C; Roy, Daniel C; Sanchez, Carlos J; Christy, Robert J; Burmeister, David M

2016-04-12

Bacterial infections are a common clinical problem in both acute and chronic wounds. With growing concerns over antibiotic resistance, treatment of bacterial infections should only occur after positive diagnosis. Currently, diagnosis is delayed due to lengthy culturing methods which may also fail to identify the presence of bacteria. While newer costly bacterial identification methods are being explored, a simple and inexpensive diagnostic tool would aid in immediate and accurate treatments for bacterial infections. Histologically, hematoxylin and eosin (H&E) and Gram stains have been employed, but are far from optimal when analyzing tissue samples due to non-specific staining. The goal of the current study was to develop a modification of the Gram stain that enhances the contrast between bacteria and host tissue. A modified Gram stain was developed and tested as an alternative to Gram stain that improves the contrast between Gram positive bacteria, Gram negative bacteria and host tissue. Initially, clinically relevant strains of Pseudomonas aeruginosa and Staphylococcus aureus were visualized in vitro and in biopsies of infected, porcine burns using routine Gram stain, and immunohistochemistry techniques involving bacterial strain-specific fluorescent antibodies as validation tools. H&E and Gram stain of serial biopsy sections were then compared to a modification of the Gram stain incorporating a counterstain that highlights collagen found in tissue. The modified Gram stain clearly identified both Gram positive and Gram negative bacteria, and when compared to H&E or Gram stain alone provided excellent contrast between bacteria and non-viable burn eschar. Moreover, when applied to surgical biopsies from patients that underwent burn debridement this technique was able to clearly detect bacterial morphology within host tissue. We describe a modification of the Gram stain that provides improved contrast of Gram positive and Gram negative microorganisms within host

Port-Wine Stains

MedlinePlus

... or surgery. Port-wine stains can also develop grape-like growths of small blood vessels called vascular ... another part of themselves — like their height or eye color. It's also important, emotionally, for kids to ...

The application of visible wavelength reflectance hyperspectral imaging for the detection and identification of blood stains.

PubMed

Li, Bo; Beveridge, Peter; O'Hare, William T; Islam, Meez

2014-12-01

Current methods of detection and identification of blood stains rely largely on visual examination followed by presumptive tests such as Kastle-Meyer, Leuco-malachite green or luminol. Although these tests are useful, they can produce false positives and can also have a negative impact on subsequent DNA tests. A novel application of visible wavelength reflectance hyperspectral imaging has been used for the detection and positive identification of blood stains in a non contact and non destructive manner on a range of coloured substrates. The identification of blood staining was based on the unique visible absorption spectrum of haemoglobin between 400 and 500 nm. Images illustrating successful discrimination of blood stains from nine red substances are included. It has also been possible to distinguish between blood and approximately 40 other reddish stains. The technique was also successfully used to detect latent blood stains deposited on white filter paper at dilutions of up to 1 in 512 folds and on red tissue at dilutions of up to 1 in 32 folds. Finally, in a blind trial, the method successfully detected and identified a total of 9 blood stains on a red T-shirt. Copyright © 2014 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved.

Organic staining on bone from exposure to wood and other plant materials.

PubMed

Pollock, Corey R; Pokines, James T; Bethard, Jonathan D

2018-02-01

Determining the depositional environment and the postmortem alterations to a set of remains are necessary aspects of a forensic investigation to explain the circumstances surrounding the death of an individual. The present study examines organic staining as a method for reconstructing the depositional environment of skeletal remains and the taphonomic agents with which they came into contact. Organic staining results largely from tannins leaching from plant materials and therefore can be seen on bone deposited in wooden coffin environments or on terrestrial surfaces. The present study examines the hypothesis that the degree of staining observed on skeletal elements would increase as the length of exposure to the organic matter increased and that different plant materials and environments would leave different patterns or colorations of staining. The sample consisted of 165 pig (Sus scrofa) femora divided into four groups exposed to differing experimental conditions, including burial in direct contact with soil or burial in a simulated coffin environment, immersion in water with wood samples, and surface deposition with plant matter contact. The bones were removed once a month from their experimental environments and the level of staining was recorded qualitatively using the Munsell Soil Color Chart. In all of the experimental environments, staining was present after two months of exposure, and the color darkened across the bone surface with each episode of data collection. The results from the present study indicate that staining can manifest on bone within a relatively short time frame once skeletonization occurs and a variety of colorations or patterns of staining can manifest based on the plant material. The present research also demonstrates the potential of organic staining to aid in estimations of the postmortem interval as well as a depositional environmental reconstruction through plant species identification. Copyright © 2017 Elsevier B.V. All rights

Correction of stain variations in nuclear refractive index of clinical histology specimens

PubMed Central

Uttam, Shikhar; Bista, Rajan K.; Hartman, Douglas J.; Brand, Randall E.; Liu, Yang

2011-01-01

For any technique to be adopted into a clinical setting, it is imperative that it seamlessly integrates with well-established clinical diagnostic workflow. We recently developed an optical microscopy technique—spatial-domain low-coherence quantitative phase microscopy (SL-QPM) that can extract the refractive index of the cell nucleus from the standard histology specimens on glass slides prepared via standard clinical protocols. This technique has shown great potential in detecting cancer with a better sensitivity than conventional pathology. A major hurdle in the clinical translation of this technique is the intrinsic variation among staining agents used in histology specimens, which limits the accuracy of refractive index measurements of clinical samples. In this paper, we present a simple and easily generalizable method to remove the effect of variations in staining levels on nuclear refractive index obtained with SL-QPM. We illustrate the efficacy of our correction method by applying it to variously stained histology samples from animal model and clinical specimens. PMID:22112118

Comparison and evaluation of mitotic figures in oral epithelial dysplasia using crystal violet and Feulgen stain.

PubMed

Rao, Roopa S; Patil, Shankargouda; Agarwal, Anveeta

2014-05-01

Routine staining procedures often pose a problem in differentiating a mitotic cell from an apoptotic cell, deteriorating the reliability of histology grading. Although various new methods have been recommended for identifying mitotic figures (MFs) in tissues, the time factor and cost makes them less feasible. Thus, an attempt was made to evaluate the efficacy of crystal violet and Feulgen reaction in identifying MFs and also to see for any variation in the number of MFs in various grades of Epithelial dysplasia. 1. Using crystal violet and Feulgen stain in the identification and counting of MFs on diagnosed cases of epithelial dysplasia and thereby to evaluate their efficacy. 2. To evaluate the variation in the number of MFs in various grades of epithelial dysplasia. The study sample includes retrieval of 30 formalin fixed paraffin embedded tissue sections diagnosed for different grades of epithelial dysplasia (WHO grading system, 2005) from the archives, Department of Oral Pathology, MSRDC, Bengaluru. Ten tissue sections each of mild, moderate and severe epithelial dysplasia were stained with H&E, Feulgen and 1% crystal violet stains and the number of MFs were counted. Five cases of cervical carcinoma were taken as control. Stained sections were compared, and data obtained was statistically analyzed using the Kruskal-Wallis test. A significant increase in the number of MFs (p = 0.02) was observed in Feulgen stained sections as compared to H&E stain. Feulgen stain can be considered as a simple, reliable, cost-effective and reproducible method of staining MFs.

Establishment of rat model of silicotuberculosis and its pathological characteristic

PubMed Central

Dong, Hu; Jing, Wu; Yabo, Yang; Xiaokang, Yang; Wan, Wang; Min, Mu; Wenyang, Wang; Zhaoquan, Chen; Yingru, Xing; Rongbo, Zhang

2014-01-01

Objective: To establish different stages of silicosis rat model complicated with tuberculosis infection, and compare the pathological characteristics and analyze the impact of silicosis on tuberculosis infection. Methods: SD rats were subjected to intratracheal administration of silica with non-exposure method at the 1st, 30th, or 60th day. At the 50th day, the rats were injected with the suspension of H37Rv (a virulent standard strain of Mycobacterium tuberculosis) via tail-vein. After 40 days post-infection, rats were sacrificed, the lung tissues were isolated, and paraffin was embedded and sectioned. The sections were treated using HE staining for structure observation, acid fast stain of Ziehl–Neelsen for bacterial detection, and Warthin–Starry silver staining for displaying the distribution of dust particles. The bacterial load was quantified by colony counting. Results: Primary to tertiary silicosis could be discovered at the 30th, 60th, and 90th day of post-infection. The rats could be infected by injection of M. tuberculosis via tail vein, with tuberculosis load and the degree of lung tissue lesions positively correlated with silicosis. Conclusion: The rat model of silicotuberculosis was established successfully, which facilitated understanding the ‘cross-talk’ of silicosis and tuberculosis during the process they drive each other. PMID:25355545

Cytochemical evaluation of the Guard procedure a regressive staining method for demonstrating chromosomal basic proteins. I. Effects of fixation, blocking reactions, selective extractions, and polyacid "differentiation".

PubMed

Cowden, R R; Rasch, E M; Curtis, S K

1976-08-12

Appropriately fixed preparations stained by a modification of the Guard (1959) reaction for "sex chromatin" display selective staining of interphase chromatin and mitotic or meiotic chromosomes. This is a regressive staining method which seems to depend on the selective displacement of an acidic dye from less basic structures, and retention of the dye at more basic sites. The results obtained with the reaction can be controlled by the length of time that the preparations are "differentiated" in solutions containing phosphomolybdic and phosphotungstic acids (polyacids). After three- or four-hour exposures to polyacid solutions, all chromatin is stained. However, with longer differentiation, "condensed" chromatin can be stained preferentially. Of a number of fixatives investigated, only 10% formalin, ethanol-acetic acid (3:1), and Bouin's solution proved useful. Others resulted in diminished specificity or a total loss of selectivity. The most intense staining was obtained after formalin fixation. Less intense dyebinding was observed after fixation in 3:1 - probably due to extraction of some histone fractions-and the least amount of dye was bound in Bouin's-fixed chromatin - probably due to blockage of arginine residues by picric acid. The reaction was not affected by enzymatic removal of nucleic acids or the extraction of lipids. It was diminished by treatment with trypsin or weak acetylation, and it was completely prevented by strong acetylation, deamination, or extraction of basic proteins with HCl. The results presented suggest that the modified Guard (1959) procedure selectively demonstrates basic nucleoproteins. Further, by the use of regressive differentiation in polyacid solutions, the retention of dye in more condensed chromatin can be favored.

Control of brown stain: in Eastern white pine

Treesearch

Robert E. Stutz; Peter Koch; Millard L. Oldham

1961-01-01

Degrade caused by brown stain and blue stain in eastern white pine was virtually eliminated by the use of sap stain chemicals and sodium azide. Combinations of buffered sodium azide with both sodium pentachlorophenate plus borax and buffered ethyl mercury phosphate were effective.

Detection of Wolbachia endobacteria in Culex quinquefasciatus by Gimenez staining and confirmation by PCR.

PubMed

Muniaraj, M; Paramasivan, R; Sunish, I P; Arunachalam, N; Mariappan, T; Jerald Leo, S Victor; Dhananjeyan, K J

2012-12-01

Wolbachia are common intracellular bacteria that are found in arthropods and nematodes. These endosymbionts are transmitted vertically through host eggs and alter host biology in diverse ways, including the induction of reproductive manipulations, such as feminization, parthenogenesis, male killing and sperm-egg incompatibility. Since they can also move horizontally across species boundaries, Wolbachia is gaining importance in recent days as it could be used as a biological control agent to control vector mosquitoes or for paratransgenic approaches. However, the study of Wolbachia requires sophisticated techniques such as PCR and cell culture facilities which cannot be affordable for many laboratories where the diseases transmitted by arthropod vectors are common. Hence, it would be beneficial to develop a simple method to detect the presence of Wolbachia in arthropods. In this study, we described a method of staining Wolbachia endobacteria, present in the reproductive tissues of mosquitoes. The reliability of this method was compared with Gram staining and PCR based detection. The microscopic observation of the Gimenez stained smear prepared from the teased ovary of wild caught and Wolbachia (+) Cx. quinquefasciatus revealed the presence of pink coloured pleomorphic cells of Wolbachia ranging from cocci, comma shaped cells to bacillus and chain forms. The ovaries of Wolbachia (-) cured mosquito did not show any cell. Although Gram's staining is a reliable differential staining for the other bacteria, the bacterial cells in the smears from the ovaries of wild caught mosquitoes did not take the stain properly and the cells were not clearly visible. The PCR amplified product from the pooled remains of wild caught and Wolbachia (+) Cx. quinquefasciatus showed clear banding, whereas, no banding was observed for the negative control (distilled water) and Wolbachia (-) Cx. quinquefasciatus. The Gimenez staining technique applied, could be used to detect the members of

Chromosome-specific staining to detect genetic rearrangements associated with chromosome 3 and/or chromosome 17

DOEpatents

Gray, Joe W.; Pinkel, Daniel; Kallioniemi, Olli-Pekka; Kallioniemi, Anne; Sakamoto, Masaru

2002-01-01

Methods and compositions for staining based upon nucleic acid sequence that employ nudeic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML), retinoblastoma, ovarian and uterine cancers, and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.

Chromosome-specific staining to detect genetic rearrangements associated with chromosome 3 and/or chromosome 17

DOEpatents

Gray, Joe W.; Pinkel, Daniel; Kallioniemi, Olli-Pekka; Kallioniemi, Anne; Sakamoto, Masaru

2008-09-09

Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML), retinoblastoma, ovarian and uterine cancers, and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.

Chromosome-specific staining to detect genetic rearrangements associated with chromosome 3 and/or chromosome 17

DOEpatents

Gray, Joe W [San Francisco, CA; Pinkel, Daniel [Lafayette, CA; Kallioniemi, Olli-Pekka [Turku, FI; Kallioniemi, Anne [Tampere, FI; Sakamoto, Masaru [Tokyo, JP

2009-10-06

Methods and compositions for staining based upon nucleic acid sequence that employ .[.nudeic.]. .Iadd.nucleic .Iaddend.acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML), retinoblastoma, ovarian and uterine cancers, and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.

Chromosome-Specific Staining To Detect Genetic Rearrangements Associated With Chromosome 3 And/Or Chromosone 17

DOEpatents

Gray; Joe W.; Pinkel; Daniel; Kallioniemi; Olli-Pekka; Kallioniemi; Anne; Sakamoto; Masaru

2002-02-05

Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML), retinoblastoma, ovarian and uterine cancers, and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.

Bulbar conjunctival fluorescein staining in hydrogel contact lens wearers.

PubMed

Lakkis, C; Brennan, N A

1996-07-01

To evaluate the clinical usefulness of bulbar conjunctival staining in assessing hydrogel contact lens patients. Overall staining, as well as staining at five separate sites (limbal, nasal band, temporal band, superior, and inferior) was graded on an analog scale in 48 contact lens wearing subjects and 50 controls. The degree to which subjects experienced sensations of dryness, wateriness, itchiness, grittiness, and comfort was also assessed using analog scales. Some measure of conjunctival staining was noted in 98% of subjects, with contact lens wearers showing statistically significant greater staining than controls. Only 12% of controls showed staining of greater than grade 1 (equivalent), whereas 62% of contact lens wearers were above this level. Regression analysis showed that overall staining was a function of whether contact lenses were worn, the degree of dryness, and the amount of itchiness. Conjunctival fluorescein staining appears to serve some clinical usefulness as a composite indicator for certain factors and symptoms and, in addition, provides information which is unique for each individual.

Proposals for best-quality immunohistochemical staining of paraffin-embedded brain tissue slides in forensics.

PubMed

Trautz, Florian; Dreßler, Jan; Stassart, Ruth; Müller, Wolf; Ondruschka, Benjamin

2018-01-03

Immunohistochemistry (IHC) has become an integral part in forensic histopathology over the last decades. However, the underlying methods for IHC vary greatly depending on the institution, creating a lack of comparability. The aim of this study was to assess the optimal approach for different technical aspects of IHC, in order to improve and standardize this procedure. Therefore, qualitative results from manual and automatic IHC staining of brain samples were compared, as well as potential differences in suitability of common IHC glass slides. Further, possibilities of image digitalization and connected issues were investigated. In our study, automatic staining showed more consistent staining results, compared to manual staining procedures. Digitalization and digital post-processing facilitated direct analysis and analysis for reproducibility considerably. No differences were found for different commercially available microscopic glass slides regarding suitability of IHC brain researches, but a certain rate of tissue loss should be expected during the staining process.

Acridine orange staining reaction as an index of physiological activity in Escherichia coli

NASA Technical Reports Server (NTRS)

McFeters, G. A.; Singh, A.; Byun, S.; Callis, P. R.; Williams, S.

1991-01-01

The assumption that the acridine orange (AO) color reaction may be used as an index of physiological activity was investigated in laboratory grown Escherichia coli. Spectrofluorometric observations of purified nucleic acids, ribosomes and the microscopic color of bacteriophage-infected cells stained with AO confirmed the theory that single-stranded nucleic acids emit orange to red fluorescence while those that are double-stranded fluoresce green in vivo. Bacteria growing actively in a rich medium could be distinguished from cells in stationary phase by the AO reaction. Cells from log phase appeared red, whereas those in stationary phase were green. However, this differentiation was not seen when the bacteria were grown in a minimal medium or when a variation of the staining method was used. Also, shifting bacteria in stationary phase to starvation conditions rapidly changed their AO staining reaction. Boiling and exposure to lethal concentrations of azide and formalin resulted in stationary-phase cells that appeared red after staining but bacteria killed with chlorine remained green. These findings indicate that the AO staining reaction may be suggestive of physiological activity under defined conditions. However, variables in staining and fixation procedures as well as uncertainties associated with mixed bacterial populations in environmental samples may produce results that are not consistent with the classical interpretation of this reaction. The importance of validating the putative physiological implications of this staining reaction is stressed.

Prophylactic cefazolin in amnioinfusions administered for meconium-stained amniotic fluid.

PubMed Central

Edwards, R K; Duff, P

1999-01-01

OBJECTIVE: To determine if amnioinfusion with an antibiotic solution decreased the rate of clinical chorioamnionitis and puerperal endometritis in patients with meconium-stained amniotic fluid. METHODS: Patients in labor at 36 weeks of gestation or greater with singleton pregnancies and meconium-stained amniotic fluid were randomized to receive either cefazolin, 1 g/1,000 mL, of normal saline (n = 90) or normal saline (n = 93) amnioinfusion. Rates of clinically diagnosed chorioamnionitis and endometritis and of suspected and culture-proven neonatal infection were determined. RESULTS: Between the study and control groups, the incidences of clinical chorioamnionitis (7.8% vs. 8.6%), endometritis (2.4% vs. 3.5%), aggregate intrauterine infection (10.0% vs. 11.8%), suspected neonatal infection (17.8% vs. 21.5%), and proven neonatal infection (0.0% vs. 2.2%) were not significantly different. CONCLUSIONS: Prophylactic use of cefazolin in amnioinfusions did not significantly reduce rates of maternal or neonatal infection in patients with meconium-stained amniotic fluid. PMID:10371474

A rapid method for counting nucleated erythrocytes on stained blood smears by digital image analysis

USGS Publications Warehouse

Gering, E.; Atkinson, C.T.

2004-01-01

Measures of parasitemia by intraerythrocytic hematozoan parasites are normally expressed as the number of infected erythrocytes per n erythrocytes and are notoriously tedious and time consuming to measure. We describe a protocol for generating rapid counts of nucleated erythrocytes from digital micrographs of thin blood smears that can be used to estimate intensity of hematozoan infections in nonmammalian vertebrate hosts. This method takes advantage of the bold contrast and relatively uniform size and morphology of erythrocyte nuclei on Giemsa-stained blood smears and uses ImageJ, a java-based image analysis program developed at the U.S. National Institutes of Health and available on the internet, to recognize and count these nuclei. This technique makes feasible rapid and accurate counts of total erythrocytes in large numbers of microscope fields, which can be used in the calculation of peripheral parasitemias in low-intensity infections.

A novel washing algorithm for underarm stain removal

NASA Astrophysics Data System (ADS)

Acikgoz Tufan, H.; Gocek, I.; Sahin, U. K.; Erdem, I.

2017-10-01

After contacting with human sweat which comprise around 27% sebum, anti-perspirants comprising aluminium chloride or its compounds form a jel-like structure whose solubility in water is very poor. In daily use, this jel-like structure closes sweat pores and hinders wetting of skin by sweat. However, when in contact with garments, they form yellowish stains at the underarm of the garments. These stains are very hard to remove with regular machine washing. In this study, first of all, we focused on understanding and simulating such stain formation on the garments. Two alternative procedures are offered to form jel-like structures. On both procedures, commercially available spray or deo-stick type anti-perspirants, standard acidic and basic sweat solutions and artificial sebum are used to form jel-like structures, and they are applied on fabric in order to get hard stains. Secondly, after simulation of the stain on the fabric, we put our efforts on developing a washing algorithm specifically designed for removal of underarm stains. Eight alternative washing algorithms are offered with varying washing temperature, amounts of detergent, and pre-stain removal procedures. Better algorithm is selected by comparison of Tristimulus Y values after washing.

21 CFR 864.1850 - Dye and chemical solution stains.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Dye and chemical solution stains. 864.1850 Section... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1850 Dye and chemical solution stains. (a) Identification. Dye and chemical solution stains for medical purposes are mixtures of...

21 CFR 864.1850 - Dye and chemical solution stains.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Dye and chemical solution stains. 864.1850 Section... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1850 Dye and chemical solution stains. (a) Identification. Dye and chemical solution stains for medical purposes are mixtures of...

21 CFR 864.1850 - Dye and chemical solution stains.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Dye and chemical solution stains. 864.1850 Section... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1850 Dye and chemical solution stains. (a) Identification. Dye and chemical solution stains for medical purposes are mixtures of...

21 CFR 864.1850 - Dye and chemical solution stains.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Dye and chemical solution stains. 864.1850 Section... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1850 Dye and chemical solution stains. (a) Identification. Dye and chemical solution stains for medical purposes are mixtures of...

21 CFR 864.1850 - Dye and chemical solution stains.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Dye and chemical solution stains. 864.1850 Section... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1850 Dye and chemical solution stains. (a) Identification. Dye and chemical solution stains for medical purposes are mixtures of...

7 CFR 28.442 - Middling Yellow Stained Color.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false Middling Yellow Stained Color. 28.442 Section 28.442... Stained Color. Middling Yellow Stained Color is American Upland cotton which in color is deeper than Middling Tinged Color. [57 FR 34498, Aug. 5, 1992] below color grade cotton ...

7 CFR 28.442 - Middling Yellow Stained Color.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false Middling Yellow Stained Color. 28.442 Section 28.442... Stained Color. Middling Yellow Stained Color is American Upland cotton which in color is deeper than Middling Tinged Color. [57 FR 34498, Aug. 5, 1992] below color grade cotton ...

7 CFR 28.442 - Middling Yellow Stained Color.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false Middling Yellow Stained Color. 28.442 Section 28.442... Stained Color. Middling Yellow Stained Color is American Upland cotton which in color is deeper than Middling Tinged Color. [57 FR 34498, Aug. 5, 1992] below color grade cotton ...

7 CFR 28.442 - Middling Yellow Stained Color.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false Middling Yellow Stained Color. 28.442 Section 28.442... Stained Color. Middling Yellow Stained Color is American Upland cotton which in color is deeper than Middling Tinged Color. [57 FR 34498, Aug. 5, 1992] below color grade cotton ...

7 CFR 28.442 - Middling Yellow Stained Color.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false Middling Yellow Stained Color. 28.442 Section 28.442... Stained Color. Middling Yellow Stained Color is American Upland cotton which in color is deeper than Middling Tinged Color. [57 FR 34498, Aug. 5, 1992] below color grade cotton ...

Near-UV laser treatment of extrinsic dental enamel stains.

PubMed

Schoenly, J E; Seka, W; Featherstone, J D B; Rechmann, P

2012-04-01

The selective ablation of extrinsic dental enamel stains using a 400-nm laser is evaluated at several fluences for completely removing stains with minimal damage to the underlying enamel. A frequency-doubled Ti:sapphire laser (400-nm wavelength, 60-nanosecond pulse duration, 10-Hz repetition rate) was used to treat 10 extracted human teeth with extrinsic enamel staining. Each tooth was irradiated perpendicular to the surface in a back-and-forth motion over a 1-mm length using an ∼300-µm-diam 10th-order super-Gaussian beam with fluences ranging from 0.8 to 6.4 J/cm(2) . Laser triangulation determined stain depth and volume removed by measuring 3D surface images before and after irradiation. Scanning electron microscopy evaluated the surface roughness of enamel following stain removal. Fluorescence spectroscopy measured spectra of unbleached and photobleached stains in the spectral range of 600-800 nm. Extrinsic enamel stains are removed with laser fluences between 0.8 and 6.4 J/cm(2) . Stains removed on sound enamel leave behind a smooth enamel surface. Stain removal in areas with signs of earlier cariogenic acid attacks resulted in isolated and randomly located laser-induced, 50-µm-diam enamel pits. These pits contain 0.5-µm diam, smooth craters indicative of heat transfer from the stain to the enamel and subsequent melting and water droplet ejection. Ablation stalling of enamel stains is typically observed at low fluences (<3 J/cm(2) ) and is accompanied by a drastic reduction in porphyrin fluorescence from the Soret band. Laser ablation of extrinsic enamel stains at 400 nm is observed to be most efficient above 3 J/cm(2) with minimal damage to the underlying enamel. Unsound underlying enamel is also observed to be selectively removed after irradiation. Copyright © 2012 Wiley Periodicals, Inc.

Effect of Light-Activated Tooth Whitening on Color Change Relative to Color of Artificially Stained Teeth.

PubMed

Kwon, So Ran; Kurti, Steven R; Oyoyo, Udochukwu; Li, Yiming

2015-01-01

There is still controversy as to the efficacy of light activation used in tooth whitening. The purpose of this study was to evaluate the effect of light activation on tooth color change relative to the artificial dye color. Extracted human third molars (160) were randomly distributed into eight groups of 20 specimens each based on artificial staining and use of light activation. All groups received three 45-minute sessions of in-office whitening at 3-day intervals. Color measurements were performed with an intraoral spectrophotometer at baseline prior to staining (T0), after artificial staining (T1), 1-day--(T2), and 1-week--(T3) post-whitening. Color differences were calculated relative to after artificial staining color parameters (L*1, a*1, b*1) with the use of a software analysis program enabling synchronization of two images. Within the same staining groups, the light-activated samples exhibited a greater color change than their nonlight-activated counterparts. However, only in the case of the yellow-stained samples at 1-day post-whitening was there a significant difference between the nonlight-activated and light-activated groups (Tukey's post hoc multiple comparison test for pairwise comparisons, p < 0.05). Light activation is a valid method for enhancing the efficacy of tooth whitening with respect to overall color change and works best with yellow stains. Light activation is a valid method for enhancing the efficacy of tooth whitening with respect to overall color change and works best with yellow stains.

Evaluation of gram stain as an alternative in the assessment of human spermatozoa quality.

PubMed

Mantas, D; Msaouel, P; Angelopoulou, R

2006-01-01

During spermiogenesis, protaminosis and sperm chromatin condensation are important prerequisites for the preservation of DNA integrity in spermatozoa. The aim of this study is to assess Gram stain as an alternative technique for the evaluation of human sperm chromatin condensation status. Aniline blue and Gram staining were applied to semen samples from 34 donors in order to determine the relationship between sperm chromatin condensation and infertility. In addition, the possible correlation between morphology and vitality (eosin-Y staining) of spermatozoa compared with their nuclear status (aniline blue and Gram staining) was studied. Chromatin condensation and sperm vitality were significantly higher in fertile men compared to the subfertile. A significant correlation was found between chromatin condensation and (a) sperm vitality (p < 0.01), and (b) nuclear protein status (p < 0.01). Gram staining may be used as a routine method in assisted reproduction laboratories and could assist in the evaluation of sperm quality as well as in the selection of the appropriate fertilization technique.

Rapid multiple immunofluorescent staining for the simultaneous detection of cytokeratin and vimentin in the cytology of canine tumors.

PubMed

Sawa, Mariko; Yabuki, Akira; Kohyama, Moeko; Miyoshi, Noriaki; Yamato, Osamu

2018-06-01

Immunocytochemistry (ICC) is utilized as an advanced technique in veterinary cytology. In tumor diagnosis, cytokeratin and vimentin are markers used to distinguish the origin of tumor cells. Standard enzyme-based ICC has limitations in clinical use; and therefore, more convenient and reliable methods are needed. The purpose of this study was to develop a rapid multiple immunofluorescent (RMIF) detection method for dual cytokeratin and vimentin staining on cytology slides in dogs. Air-dried smear samples from solid tumors and sediments of pleural effusions were prepared from dogs (n = 14) that were admitted to the Veterinary Teaching Hospital, Kagoshima University, Japan. Mouse monoclonal anti-human cytokeratin (AE1/AE3) and rabbit monoclonal anti-human vimentin (SP20) antibodies were used as primary antibodies, followed by staining with Alexa Fluor-conjugated secondary antibodies. Staining using the RMIF method was compared with enzyme-based ICC staining. Rapid multiple immunofluorescent immunostaining was clear and specific in the evaluated smears, whereas the enzyme-based ICC showed nonspecific signals. By using the RMIF staining method, epithelial cells, mesenchymal cells, and mesothelial cells could be classified on a single smear of a pleural effusion. In smears of lymph nodes with epithelial tumor metastases, the RMIF method successfully detected metastatic epithelial tumor cells. The RMIF method might be a useful tool for diagnostic cytology in veterinary medicine. © 2018 American Society for Veterinary Clinical Pathology.

Newer applications of the histological stain prepared from Pterocarpus santalinus.

PubMed

Sen Gupta, P C; Mukherjee, A K

1981-03-01

A histological stain prepared from the heartwood of Pterocarpus santalinus Linn. has been found to be an excellent nuclear stain for various cells of animal and plant origin. As an elastic tissue stain, the results are comparable to standard elastic tissue stains. The striations of voluntary muscle fibers are well shown. The Nissl granules and fibers of cranial nerves in the pons are visualized. When counterstained with light green, it differentially stains muscle and fibrous tissue. The stain can be used as counterstain with certain histochemical procedures with satisfactory results. The preparation and use of this versatile stain are described.

Automated epidermis segmentation in histopathological images of human skin stained with hematoxylin and eosin

NASA Astrophysics Data System (ADS)

Kłeczek, Paweł; Dyduch, Grzegorz; Jaworek-Korjakowska, Joanna; Tadeusiewicz, Ryszard

2017-03-01

Background: Epidermis area is an important observation area for the diagnosis of inflammatory skin diseases and skin cancers. Therefore, in order to develop a computer-aided diagnosis system, segmentation of the epidermis area is usually an essential, initial step. This study presents an automated and robust method for epidermis segmentation in whole slide histopathological images of human skin, stained with hematoxylin and eosin. Methods: The proposed method performs epidermis segmentation based on the information about shape and distribution of transparent regions in a slide image and information about distribution and concentration of hematoxylin and eosin stains. It utilizes domain-specific knowledge of morphometric and biochemical properties of skin tissue elements to segment the relevant histopathological structures in human skin. Results: Experimental results on 88 skin histopathological images from three different sources show that the proposed method segments the epidermis with a mean sensitivity of 87 %, a mean specificity of 95% and a mean precision of 57%. It is robust to inter- and intra-image variations in both staining and illumination, and makes no assumptions about the type of skin disorder. The proposed method provides a superior performance compared to the existing techniques.

Corneal and conjunctival epithelial staining in hydrogel contact lens wearers.

PubMed

Brautaset, Rune L; Nilsson, Maria; Leach, Norman; Miller, William L; Gire, Anisa; Quintero, Sam; Bergmanson, Jan P G

2008-11-01

The purpose of this study was to investigate the prevalence of conjunctival and corneal epithelial staining in soft contact lens wearers and to see if staining could be associated with factors such as type of lens worn, wearing time, care system, age, and sex. The records of 338 adapted hydrogel contact lens wearers were examined retrospectively. Conjunctival staining was found to be present in 32.5% of the subjects and corneal staining was found to be present in 19.5% of subjects. None of the subjects had staining above grade 2 using the Cornea and Contact Lens Research Unit scale. Because of the low prevalence of staining, the low grading of staining found and the large variation in refractive power, lens type worn, wearing modality, and solution used statistical analysis for association between staining and different factors could only be performed for the association between sex and staining and between corneal and conjunctival staining. However, no statistical significant association could be demonstrated. Despite the low prevalence of staining the conjunctiva and cornea should be examined carefully in contact lens wearers and prospective wearers because the conjunctival and corneal epithelium serve as protective barriers for the underlying layers of the cornea and conjunctiva. To allow comparison of data obtained in different studies assessing corneal staining, it is recommended that clinicians develop and adopt a universal standard protocol for this measure.

Gram Stains: A Resource for Retrospective Analysis of Bacterial Pathogens in Clinical Studies

PubMed Central

Srinivasan, Usha; Ponnaluri, Sreelatha; Villareal, Lisa; Gillespie, Brenda; Wen, Ai; Miles, Arianna; Bucholz, Brigette; Marrs, Carl F.; Iyer, Ram K.; Misra, Dawn; Foxman, Betsy

2012-01-01

We demonstrate the feasibility of using qPCR on DNA extracted from vaginal Gram stain slides to estimate the presence and relative abundance of specific bacterial pathogens. We first tested Gram stained slides spiked with a mix of 108 cfu/ml of Escherichia coli and 105 cfu/ml of Lactobacillus acidophilus. Primers were designed for amplification of total and species-specific bacterial DNA based on 16S ribosomal gene regions. Sample DNA was pre-amplified with nearly full length 16S rDNA ribosomal gene fragment, followed by quantitative PCR with genera and species-specific 16S rDNA primers. Pre-amplification PCR increased the bacterial amounts; relative proportions of Escherichia coli and Lactobacillus recovered from spiked slides remained unchanged. We applied this method to forty two archived Gram stained slides available from a clinical trial of cerclage in pregnant women at high risk of preterm birth. We found a high correlation between Nugent scores based on bacterial morphology of Lactobacillus, Gardenerella and Mobiluncus and amounts of quantitative PCR estimated genus specific DNA (rrn copies) from Gram stained slides. Testing of a convenience sample of eight paired vaginal swabs and Gram stains freshly collected from healthy women found similar qPCR generated estimates of Lactobacillus proportions from Gram stained slides and vaginal swabs. Archived Gram stained slides collected from large scale epidemiologic and clinical studies represent a valuable, untapped resource for research on the composition of bacterial communities that colonize human mucosal surfaces. PMID:23071487

Gram stains: a resource for retrospective analysis of bacterial pathogens in clinical studies.

PubMed

Srinivasan, Usha; Ponnaluri, Sreelatha; Villareal, Lisa; Gillespie, Brenda; Wen, Ai; Miles, Arianna; Bucholz, Brigette; Marrs, Carl F; Iyer, Ram K; Misra, Dawn; Foxman, Betsy

2012-01-01

We demonstrate the feasibility of using qPCR on DNA extracted from vaginal Gram stain slides to estimate the presence and relative abundance of specific bacterial pathogens. We first tested Gram stained slides spiked with a mix of 10(8) cfu/ml of Escherichia coli and 10(5) cfu/ml of Lactobacillus acidophilus. Primers were designed for amplification of total and species-specific bacterial DNA based on 16S ribosomal gene regions. Sample DNA was pre-amplified with nearly full length 16S rDNA ribosomal gene fragment, followed by quantitative PCR with genera and species-specific 16S rDNA primers. Pre-amplification PCR increased the bacterial amounts; relative proportions of Escherichia coli and Lactobacillus recovered from spiked slides remained unchanged. We applied this method to forty two archived Gram stained slides available from a clinical trial of cerclage in pregnant women at high risk of preterm birth. We found a high correlation between Nugent scores based on bacterial morphology of Lactobacillus, Gardenerella and Mobiluncus and amounts of quantitative PCR estimated genus specific DNA (rrn copies) from Gram stained slides. Testing of a convenience sample of eight paired vaginal swabs and Gram stains freshly collected from healthy women found similar qPCR generated estimates of Lactobacillus proportions from Gram stained slides and vaginal swabs. Archived Gram stained slides collected from large scale epidemiologic and clinical studies represent a valuable, untapped resource for research on the composition of bacterial communities that colonize human mucosal surfaces.

Comparison of Gram stain with DNA probe for detection of Neisseria gonorrhoeae in urethras of symptomatic males.

PubMed Central

Juchau, S V; Nackman, R; Ruppart, D

1995-01-01

The comparison of Gram-stained urethral smears with Gen-Probe for the detection of Neisseria Gonorrhoeae in the urethras of males with symptomatic urethritis revealed a 99.6% correlation between the two methods. A simple Gram stain would appear to be the method of choice for the detection of gonorrhea in symptomatic males, because it is much less expensive and much more rapid than the Gen-Probe method. PMID:8576380

Improved Whole-Blood-Staining Device

NASA Technical Reports Server (NTRS)

Sams, Clarence F.; Crucian, Brian; Paul, Bonnie; Melton, Shannon; Guess, Terry

2012-01-01

Dramatic improvements have been made in NASA s Whole Blood Staining Device (WBSD) since it was last described in "Whole-Blood-Staining Device," NASA Tech Briefs, Vol. 23, No. 10 (October 1999), page 64. The new system has a longer shelf life, a simpler and more effective operational procedure, improved interface with instrumentation, and shorter processing time. More specifically, the improvements have targeted bag and locking clip materials, sampling ports, and air pocket prevention. The WBSD stains whole blood collected during spaceflight for subsequent flow cytometric analysis. In short, the main device stains white blood cells by use of monoclonal antibodies conjugated to various fluorochromes, followed by lysing and fixing of the cells by use of a commercial reagent that has been diluted according to NASA safety standards. This system is compact, robust, and does not require electric power, precise mixing, or precise incubation times. Figure 1 depicts the present improved version for staining applications, which is a poly(tetrafluoroethylene) bag with a Luer-lock port and plastic locking clips. An InterLink (or equivalent) intravenous- injection port screws into the Luer-lock port. The inflatable/collapsible nature of the bag facilitates loading and helps to minimize the amount of air trapped in the fully loaded bag. Some additional uses have been identified for the device beyond whole blood staining. The WBSD has been configured for functional assays that require culture of live cells by housing sterile culture media, mitogens, and fixatives prior to use [Figure 2(a)]. Simple injection of whole blood allows cell-stimulation culture to be performed in reduced gravity conditions, and product stabilization prior to storage, while protecting astronauts from liquid biohazardous materials. Also, the improved WBSD has reconstituted powdered injectable antibiotics by mixing them with diluent liquids [Figure 2(b)]. Although such mixing can readily be performed on

In situ analysis of lung antigen-presenting cells during murine pulmonary infection with virulent Mycobacterium tuberculosis

PubMed Central

Pedroza-González, Alexander; García-Romo, Gina S; Aguilar-León, Diana; Calderon-Amador, Juana; Hurtado-Ortiz, Raquel; Orozco-Estevez, Hector; Lambrecht, Bart N; Estrada-García, Iris; Hernández-Pando, Rogelio; Flores-Romo, Leopoldo

2004-01-01

Scarce information exists about the role of lung antigen-presenting cells (APCs) in vivo during pulmonary tuberculosis. As APCs activate cellular immunity, following intratracheal inoculation with virulent Mycobacterium tuberculosis, we assessed in situ lung APC recruitment, distribution, granuloma involvement, morphology and mycobacterial burden by using MHC-CII, CD14, scavenger receptor class A (SRA), the murine dendritic cell (DC)-restricted marker CD11c and Ziehl–Neelsen staining. CD11c+ DC and CD14+ cell recruitment into lungs appeared by day 14, continuing until day 60. MHC-CII+ cells increased since day 7, persisting until day 60. Thus, virulent mycobacteria delays (14–21 days) lung APC recruitment compared to model antigens and nonvirulent bacilli (24–48 h). Regarding granuloma constitution, highly bacillary CD14+ and SRA+ cells were centrally located. MHC-CII+ cells were more peripheral, with less mycobacteria. CD11c+ cells were heterogeneously distributed within granulomas, with scarce bacilli. When labelling lung suspensions for MHC-CII and classifying cells as macrophages or DC, then staining for Ziehl–Neelsen, a remarkable segregation was found regarding bacillary burden. Most macrophage-like cells contained numerous bacilli, while DC had no or scarce mycobacteria. This implies differential APC contributions in situ during pulmonary tuberculosis regarding mycobacterial uptake, granuloma involvement and perhaps bacillary growth. PMID:15255967

Bone marrow cells stained by azide-conjugated Alexa fluors in the absence of an alkyne label.

PubMed

Lin, Guiting; Ning, Hongxiu; Banie, Lia; Qiu, Xuefeng; Zhang, Haiyang; Lue, Tom F; Lin, Ching-Shwun

2012-09-01

Thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) has recently been introduced as an alternative to 5-bromo-2-deoxyuridine (BrdU) for cell labeling and tracking. Incorporation of EdU into replicating DNA can be detected by azide-conjugated fluors (eg, Alexa-azide) through a Cu(i)-catalyzed click reaction between EdU's alkyne moiety and azide. While this cell labeling method has proven to be valuable for tracking transplanted stem cells in various tissues, we have found that some bone marrow cells could be stained by Alexa-azide in the absence of EdU label. In intact rat femoral bone marrow, ~3% of nucleated cells were false-positively stained, and in isolated bone marrow cells, ~13%. In contrast to true-positive stains, which localize in the nucleus, the false-positive stains were cytoplasmic. Furthermore, while true-positive staining requires Cu(i), false-positive staining does not. Reducing the click reaction time or reducing the Alexa-azide concentration failed to improve the distinction between true- and false-positive staining. Hematopoietic and mesenchymal stem cell markers CD34 and Stro-1 did not co-localize with the false-positively stained cells, and these cells' identity remains unknown.

Candida, fluorescent stain (image)

MedlinePlus

... a fluorescent stain of Candida. Candida is a yeast (fungus) that causes mild disease, but in immunocompromised individuals it may cause life-threatening illness. (Image courtesy of the Centers for ...

Issues in using whole slide imaging for diagnostic pathology: "routine" stains, immunohistochemistry and predictive markers.

PubMed

Taylor, C R

2014-08-01

The traditional microscope, together with the "routine" hematoxylin and eosin (H & E) stain, remains the "gold standard" for diagnosis of cancer and other diseases; remarkably, it and the majority of associated biological stains are more than 150 years old. Immunohistochemistry has added to the repertoire of "stains" available. Because of the need for specific identification and even measurement of "biomarkers," immunohistochemistry has increased the demand for consistency of performance and interpretation of staining results. Rapid advances in the capabilities of digital imaging hardware and software now offer a realistic route to improved reproducibility, accuracy and quantification by utilizing whole slide digital images for diagnosis, education and research. There also are potential efficiencies in work flow and the promise of powerful new analytical methods; however, there also are challenges with respect to validation of the quality and fidelity of digital images, including the standard H & E stain, so that diagnostic performance by pathologists is not compromised when they rely on whole slide images instead of traditional stained tissues on glass slides.

Microvessel prediction in H&E Stained Pathology Images using fully convolutional neural networks.

PubMed

Yi, Faliu; Yang, Lin; Wang, Shidan; Guo, Lei; Huang, Chenglong; Xie, Yang; Xiao, Guanghua

2018-02-27

Pathological angiogenesis has been identified in many malignancies as a potential prognostic factor and target for therapy. In most cases, angiogenic analysis is based on the measurement of microvessel density (MVD) detected by immunostaining of CD31 or CD34. However, most retrievable public data is generally composed of Hematoxylin and Eosin (H&E)-stained pathology images, for which is difficult to get the corresponding immunohistochemistry images. The role of microvessels in H&E stained images has not been widely studied due to their complexity and heterogeneity. Furthermore, identifying microvessels manually for study is a labor-intensive task for pathologists, with high inter- and intra-observer variation. Therefore, it is important to develop automated microvessel-detection algorithms in H&E stained pathology images for clinical association analysis. In this paper, we propose a microvessel prediction method using fully convolutional neural networks. The feasibility of our proposed algorithm is demonstrated through experimental results on H&E stained images. Furthermore, the identified microvessel features were significantly associated with the patient clinical outcomes. This is the first study to develop an algorithm for automated microvessel detection in H&E stained pathology images.

Cost-utility analysis of LED fluorescence microscopy in the diagnosis of pulmonary tuberculosis in Indian settings.

PubMed

Kelly, V; Sagili, K D; Satyanarayana, S; Reza, L W; Chadha, S S; Wilson, N C

2015-06-01

With support from the Stop TB Partnership's TB REACH Wave 2 Grant, diagnostic microscopy services for tuberculosis (TB) were upgraded from conventional Ziehl-Neelsen (ZN) based sputum microscopy to light emitting diode technology-based fluorescence microscopy (LED FM) in 200 high-workload microscopy centres in India as a pilot intervention. To evaluate the cost-effectiveness of LED-FM over conventional ZN microscopy to inform further scale-up. A decision-tree model was constructed to assess the cost utility of LED FM over ZN microscopy. The results were summarised using incremental cost-effectiveness ratio (ICER); one-way and probabilistic sensitivity analyses were also conducted to address uncertainty within the model. Data were analysed from 200 medical colleges in 2011 and 2012, before and after the introduction of LED microscopes. A full costing analysis was carried out from the perspective of a national TB programme. The ICER was calculated at US$14.64 per disability-adjusted life-year, with an 82% probability of being cost-effective at a willingness-to-pay threshold equivalent to Indian gross domestic product per capita. LED FM is a cost-effective intervention for detecting TB cases in India at high-workload medical college settings.