Evaluating different counter stains in fluorescent staining technique for detecting acid fast bacilli: Best amongst the better

N. Shameema Mol, Vishnu Prasad Shenoy, Seema Shetty, Kiran Chawla

Research output: Contribution to journalArticle

Abstract

Tuberculosis is a global emergency needing an early and accurate diagnosis for management and control. Smear microscopy is a rapid diagnostic method for detecting acid fast bacilli (AFB) and improvising this method would contribute in better detection rates. The present study compares the efficacy of various counter stains used in Auramine O staining method in examining sputum smears under fluorescence microscope. This was a prospective study conducted in the Department of Microbiology from January to March, 2017. 102 sputum specimens were collected during this period from patients clinically suspected with pulmonary tuberculosis. Five smears were prepared from each specimen, where each smear was stained with Auramine O staining technique using five different counter stains, 0.1% potassium permanganate, 0.1% methylene blue, 0.1% toluidine blue, 0.1% malachite green and undiluted blue ink. Among the 102 sputum specimens, 50(49.01%), 50(49.01%), 49(48.03%), 44(43.13%) and 48(47.05%) smears showed positive results using potassium permanganate, methylene blue, toluidine blue, malachite green and blue ink respectively. On comparing the degree of fluorescence exhibited by debris in these smears, using potassium permanganate, methylene blue, toluidine blue, malachite green and blue ink, fluorescence was observed in 22(21.6%), 11(10.8%), 46(45.1%), 0(0%) and 31(30.4%) smears respectively. When the counter stains were analyzed, there was a statistically significant difference for debris getting fluoresced (p<0.001). In conclusion, methylene blue as a counter stain has almost equivalent effect compared to routinely used potassium permanganate. It has shown to have the best sensitivity and specificity, reduced debris fluorescence and better contrast in appreciating AFB.

Original languageEnglish
Pages (from-to)1405-1409
Number of pages5
JournalJournal of Pure and Applied Microbiology
Volume12
Issue number3
DOIs
Publication statusPublished - 01-09-2018

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Potassium Permanganate
Methylene Blue
Tolonium Chloride
Bacillus
Ink
Benzophenoneidum
Coloring Agents
Fluorescence
Sputum
Staining and Labeling
Acids
Microbiology
Pulmonary Tuberculosis
Early Diagnosis
Microscopy
Tuberculosis
Emergencies
Prospective Studies
Sensitivity and Specificity
malachite green

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Microbiology
  • Applied Microbiology and Biotechnology

Cite this

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title = "Evaluating different counter stains in fluorescent staining technique for detecting acid fast bacilli: Best amongst the better",
abstract = "Tuberculosis is a global emergency needing an early and accurate diagnosis for management and control. Smear microscopy is a rapid diagnostic method for detecting acid fast bacilli (AFB) and improvising this method would contribute in better detection rates. The present study compares the efficacy of various counter stains used in Auramine O staining method in examining sputum smears under fluorescence microscope. This was a prospective study conducted in the Department of Microbiology from January to March, 2017. 102 sputum specimens were collected during this period from patients clinically suspected with pulmonary tuberculosis. Five smears were prepared from each specimen, where each smear was stained with Auramine O staining technique using five different counter stains, 0.1{\%} potassium permanganate, 0.1{\%} methylene blue, 0.1{\%} toluidine blue, 0.1{\%} malachite green and undiluted blue ink. Among the 102 sputum specimens, 50(49.01{\%}), 50(49.01{\%}), 49(48.03{\%}), 44(43.13{\%}) and 48(47.05{\%}) smears showed positive results using potassium permanganate, methylene blue, toluidine blue, malachite green and blue ink respectively. On comparing the degree of fluorescence exhibited by debris in these smears, using potassium permanganate, methylene blue, toluidine blue, malachite green and blue ink, fluorescence was observed in 22(21.6{\%}), 11(10.8{\%}), 46(45.1{\%}), 0(0{\%}) and 31(30.4{\%}) smears respectively. When the counter stains were analyzed, there was a statistically significant difference for debris getting fluoresced (p<0.001). In conclusion, methylene blue as a counter stain has almost equivalent effect compared to routinely used potassium permanganate. It has shown to have the best sensitivity and specificity, reduced debris fluorescence and better contrast in appreciating AFB.",
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AB - Tuberculosis is a global emergency needing an early and accurate diagnosis for management and control. Smear microscopy is a rapid diagnostic method for detecting acid fast bacilli (AFB) and improvising this method would contribute in better detection rates. The present study compares the efficacy of various counter stains used in Auramine O staining method in examining sputum smears under fluorescence microscope. This was a prospective study conducted in the Department of Microbiology from January to March, 2017. 102 sputum specimens were collected during this period from patients clinically suspected with pulmonary tuberculosis. Five smears were prepared from each specimen, where each smear was stained with Auramine O staining technique using five different counter stains, 0.1% potassium permanganate, 0.1% methylene blue, 0.1% toluidine blue, 0.1% malachite green and undiluted blue ink. Among the 102 sputum specimens, 50(49.01%), 50(49.01%), 49(48.03%), 44(43.13%) and 48(47.05%) smears showed positive results using potassium permanganate, methylene blue, toluidine blue, malachite green and blue ink respectively. On comparing the degree of fluorescence exhibited by debris in these smears, using potassium permanganate, methylene blue, toluidine blue, malachite green and blue ink, fluorescence was observed in 22(21.6%), 11(10.8%), 46(45.1%), 0(0%) and 31(30.4%) smears respectively. When the counter stains were analyzed, there was a statistically significant difference for debris getting fluoresced (p<0.001). In conclusion, methylene blue as a counter stain has almost equivalent effect compared to routinely used potassium permanganate. It has shown to have the best sensitivity and specificity, reduced debris fluorescence and better contrast in appreciating AFB.

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