Evaluation of a Dual-Staining Method for Acid-Fast Bacilli

We describe here a procedure that significantly enhances the intensity and method-specificity of immunocytochemical staining in large mounted semi-thin plastic Epon sections. The procedure was developed for the detection of the neurotransmitter gamma-aminobutyric acid (GABA) in rat brain tissue fixed with glutaraldehyde, but it may also be helpful in unmasking other antigens under different conditions. In addition to some practical suggestions for improving the reproducibility of the staining procedure, we demonstrate that the crucial step in the procedure is pre-treatment of the deplasticized sections with proteinase-K before exposure to the first antibody. This leads to a high morphological resolution and an excellent immunocytochemical signal.

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Morphometric dimensions of the stallion sperm head depending on the staining method used

Bulletin of the Veterinary Institute in Pulawy ◽

10.1515/bvip-2015-0039 ◽

2015 ◽

Vol 59 (2) ◽

pp. 263-270 ◽

Cited By ~ 10

Author(s):

Dorota Banaszewska ◽

Magdalena Czubaszek ◽

Renata Walczak-Jędrzejowska ◽

Katarzyna Andraszek

Keyword(s):

Surface Area ◽

Sperm Morphology ◽

Staining Method ◽

Sperm Head ◽

Morphometric Measurements ◽

Morphological Evaluation ◽

Length Width ◽

Staining Techniques ◽

Control Samples

AbstractSemen was collected from Polish Halfbred stallions. Twenty individuals from 3 to 4 years of age were selected for the study. At least one ejaculate from each stallion was collected and assessed. Sperm morphology was evaluated using Papanicolaou stain, SpermBlue®, and a complex of eosin and gentian stain, whereas unstained slides were used as control samples. Morphometric measurements were performed on 100 randomly selected sperm heads in each ejaculate. The length, width, perimeter, and surface area of the sperm head were measured. The frequency of nuclear vacuoles was determined as well. Tygerberg’s strict criteria, which most precisely characterise the sperm head, were used in the morphological evaluation of the sperm. The results obtained indicate that in the case of staining with SpermBlue®and with eosin+gentian complex, the morphometry of the sperm head seems to be most similar to that observed in unstained smears. It also seems that neither shrinking nor swelling is uniform in the various staining techniques (Papanicolaou stain). Moreover, it appears that in comparison with unstained sperm, all methods caused the width of the head to increase as a result of swelling.

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Discrimination between Viable and Dead Encephalitozoon cuniculi (Microsporidian) Spores by Dual Staining with Sytox Green and Calcofluor White M2R

Journal of Clinical Microbiology ◽

10.1128/jcm.38.10.3811-3814.2000 ◽

2000 ◽

Vol 38 (10) ◽

pp. 3811-3814 ◽

Cited By ~ 26

Author(s):

L. C. Green ◽

P. J. LeBlanc ◽

E. S. Didier

Keyword(s):

Systemic Disease ◽

Organ Transplant ◽

Spore Wall ◽

Staining Procedure ◽

Excitation Wavelength ◽

Encephalitozoon Cuniculi ◽

Calcofluor White ◽

Sytox Green ◽

Intracellular Parasites ◽

Dual Staining

Microsporidia are obligate intracellular parasites, recognized as causing chronic diarrhea and systemic disease in AIDS patients, organ transplant recipients, travelers, and malnourished children. Species of microsporidia that infect humans have been detected in drinking-water sources, and methods are needed to ascertain if these microsporidia are viable and capable of causing infections. In this study, Calcofluor White M2R and Sytox Green stains were used in combination to differentiate between live (freshly harvested) and dead (boiled)Encephalitozoon cuniculi spores. Calcofluor White M2R binds to chitin in the microsporidian spore wall. Dual-stained live spores appeared as turquoise-blue ovals, while dead spores appeared as white-yellow ovals at an excitation wavelength of 395 to 415 nm used for viewing the Calcofluor stain. Sytox Green, a nuclear stain, is excluded by live spores but penetrates compromised spore membranes. Dual-stained dead spores fluoresced bright yellow-green when viewed at an excitation wavelength of 470 to 490 nm, whereas live spores failed to stain with Sytox Green. After live and dead spores were mixed at various ratios, the number of viably stained spores detected in the dual-staining procedure correlated (P = 0.0025) with the expected numbers of viable spores. Spore mixtures were also assayed for infectivity in a focus-forming assay, and a correlation (P = 0.0002) was measured between the percentage of focus-forming microsporidia and the percentage of expected infectious spores in each mixture. By analysis of variance, no statistically significant differences were measured between the percentage of viably stained microsporidia and the percentage of infectious microsporidia (P = 0.964) in each mixture. These results suggest that Calcofluor White M2R and Sytox Green stains, when used together, may facilitate studies to identify viable microsporidia.

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Hasil diagnostik Mycobacterium tuberculosis pada penderita batuk ≥2 minggu dengan pewarnaan Ziehl-Neelsen di Puskesmas Ranomuut dan Puskesmas Kombos Manado

Jurnal e-Biomedik ◽

10.35790/ebm.4.2.2016.14264 ◽

2016 ◽

Vol 4 (2) ◽

Author(s):

Ramalia P. Mohamad ◽

John Porotu'o ◽

Heriyannis Homenta

Keyword(s):

Mycobacterium Tuberculosis ◽

Pulmonary Tuberculosis ◽

Positive Result ◽

Staining Method ◽

Descriptive Study ◽

Health Centers ◽

Common Symptom ◽

Acid Fast Bacilli ◽

Primary Health ◽

The Common

Abstract: The common symptom of lung disease is cough which is usually recovered after 2-3 weeks. However, if the cough persists continually then it should be paid attention seriously ith detect Mycobacterium tuberculosis in the sputum of patients with cough ≥2 weeks at Puskesmas Ranomuut and Puskesmas Kombos (primary health centers) in Manado by using the Ziehl-Neelsen staining method. This was a descriptive study using total sampling from August to November 2016. The results showed that there were 25 patients with cough ≥2 weeks; 7 patients at Puskesmas Ranomuut and 18 patients at Puskesmas Kombos. The majority of patients were male (68%) and aged 15-34 years old (44%). The Ziehl-Neelsen staining of patients’ sputum revealed that at Puskesmas Kombos there were 2 patients (11,1%) with positive acid-fast bacilli meanwhile at Puskesmas Ranomuut there was no positive result. Conclusion: Acid-fast bacilli were detected in 11.1% patients with cough ≥2 weeks at Puskesmas Kombos only. Keywords: Mycobacterium tuberculosis, pulmonary tuberculosis, Ziehl-Neelsen Abstrak: Gejala penyakit paru yang paling sering ditemukan ialah batuk. Batuk biasanya akan membaik dalam 2-3 minggu namun bila batuk terjadi terus-menerus perlu diselidiki lebih lanjut karena merupakan gejala utama dari penyakit tuberkulosis paru. Penelitian ini bertujuan untuk mendeteksi adanya kuman Mycobacterium tuberkulosis dalam dahak penderita batuk ≥2 minggu di Puskesmas Ranomuut dan Puskesmas Kombos dengan pemeriksaan mikrobiologi menggunakan teknik pewarnaan Ziehl-Neelsen. Jenis penelitian ialah deskriptif. Pengambilan sampel menggunakan cara total sampling dalam kurun waktu Agustus-November 2016. Hasil penelitian mendapatkan 25 pasien batuk ≥2 minggu terdiri dari 7 pasien di Puskesmas Ranomuut dan 18 pasien di Puskesmas Kombos. Jumlah pasien laki-laki (68%) lebih banyak dibandingkan perempuan (32%) dengan usia terbanyak 15-34 tahun (44%). Pada pemeriksaan BTA di Puskesmas Ranomuut tidak ditemukan pasien dengan BTA positif sedangkan di Puskesmas Kombos terdapat 2 pasien dengan BTA positif (11,1%). Simpulan: Kuman BTA positif dalam sputum penderita batuk ≥2 minggu sebanyak 2 orang (11,1%) hanya di Puskesmas Kombos.Kata kunci: Mycobacterium tuberculosis, tuberkulosis paru, Ziehl-Neelsen

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A rapid phosphotungstic acid staining method on ultra-thin sections

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100169328 ◽

1994 ◽

Vol 52 ◽

pp. 318-319

Author(s):

K. Chien ◽

R. L. Van de Velde ◽

R.C. Heusser ◽

H. Shiroishi ◽

A.H. Cohen

Keyword(s):

Room Temperature ◽

Phosphotungstic Acid ◽

Staining Method ◽

Tap Water ◽

Petri Dish ◽

Hereditary Disease ◽

Microwave Oven ◽

Staining Procedure ◽

Thin Sections ◽

Nail Patella Syndrome

The Nail-Patella Syndrome (NPS) is an autosomal dominant hereditary disease in which about 60% of the patients have glomerular abnormalities. Phosphotungstic acid (PTA) has been used successfully to demonstrate the specific collagen fibrils of NPS within the glomerular basement membrane and/or mesangial matrix in ultra-thin sections. However, in our laboratory we had difficulty staining Eponate 12 sections with PTA. A modified staining procedure which makes use of the microwave has been developed as follows:1. Glutaraldehyde/osmium fixed kidney specimens were embedded in Eponate 12 and ultra-thin sections collected on Gilder 400 mesh copper grids. Grids were stained using a RTV 700 silicone rubber staining pad. This pad contains shallow, round wells (5mm in diameter and 2mm deep) with slots in the center bottom of each well. By bending the pad, the rim of a grid can be inserted into the slot. On release, the grids adhere securely to the wall of the slot.2. 10% aqueous phosphotungstic acid was dropped into the staining wells around each grid. The staining pad was then placed in a moisture chamber consisting of a pre-heated Petri dish containing wetted filter paper and placed in Pelco 3400 laboratory microwave oven for 110 seconds at 80% power. A Tripour beaker containing 200ml room temperature tap water was placed in the microwave oven as a heat sink.

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A New Perspective on the Status of the Intestinal Parasitic Infections in the Rural Areas of Fars Province South of Iran

Iranian Journal of Public Health ◽

10.18502/ijph.v48i8.2996 ◽

2020 ◽

Author(s):

Mojtaba NOWROZI ◽

Holam Reza MOWLAVI ◽

Mostafa ALISHAVANDI ◽

Gholamreza HATAM

Keyword(s):

Rural Areas ◽

Staining Method ◽

Public Awareness ◽

Influential Factors ◽

Assay Method ◽

Staining Procedure ◽

Parasitic Infections ◽

Fars Province ◽

Intestinal Parasitic Infections ◽

Protozoan Infection

Background: Parasitoses are among the most important problems of most countries especially developing countries. We aimed to detect the situation of intestinal parasitic infections in the Farashband district in Fars Province South of Iran and identify influential factors in the escalation of parasitic diseases and to reduce them. Methods: Overall, 1009 participants from the age of 6 months to 90 years were selected from 3 cities and 15 villages of Farashband district, Fars Province South of Iran from 2015 to 2016. Parasitological methods such as the direct assay method, formalin-ether concentration method, and zinc sulfate flotation were used for diagnosis of worm eggs, cysts, and protozoa trophozoite. Susceptible and protozoan positive samples were stained using the Trichrome staining method. The modified acid-fast staining procedure was conducted for diarrheal samples and the results were used for diagnosis of coccidia. Results: Overall, 313 subjects were infected with at least one intestinal parasite (pathogenic and nonpathogenic). Helminthes infection and protozoan infection were observed in 9 (0.9%) and 304 (30.13%) participants, respectively. Fecal samples of 34 patients with diarrheal feces were used to prepare smears for further examinations using the Ziehl-Neelsen staining method. Examinations showed no infection with coccidia. Conclusion: Helminthes infection has decreased drastically but protozoan infection is still considered a health issue in this region. It is possible to reduce parasitic infections through proper measures such as increasing public awareness and education the public, especially children on health problems with education courses.

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