* To whom all correspondence should be addressed. Tel.: +91 9886616197; E-mail:vishnumanav@gmail.com JOURNAL OF PURE AND APPLIED MICROBIOLOGY, Sept. 2018. Vol. 12(3), p. 1405-1409 Evaluating Different Counter Stains in Fluorescent Staining Technique for Detecting Acid Fast Bacilli: Best Amongst The Better Shameema Mol N, Vishnu Prasad Shenoy*, Seema Shetty and Kiran Chawla Department of Microbiology, Kasturba Medical College Manipal Manipal Academy of Higher Education, Manipal, Karnataka, India. http://dx.doi.org/10.22207/JPAM.12.3.43 (Received: 20 June 2018; accepted: 12 August 2018) 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. Keywords: Tuberculosis, Acid Fast Bacilli, Light-Emitting Diode, Fluorescence Microscope, Counter stain. Tuberculosis (TB) is one of the deadliest menace of the developing world 1 . In 1993, World Health Organization (WHO) declared Tuberculosis as a Global Emergency 2 . Accounting to the depredations caused by the disease, especially over the last several hundred years, it has earned the epithet, ‘The captain of all these men of death’. As reported by WHO in the year 2016, 10.4 million people around the world were infected with TB and 1.7 million cases attributed to TB related deaths, 95% cases from low and middle income countries. 64% of new cases have been reported from high burden countries like India, Indonesia, China, Philippines, Pakisthan, Nigeria and South Africa, with India taking a lead in the number of cases 3 . Tuberculosis control aims at reducing the transmission rate thereby decreasing the number of cases, which eventually lessens morbidity and mortality 4 . Accurate and efficient diagnosis of infection plays a significant role in managing the disease burden. Despite the recent development of more sensitive technologies, most of the developing countries still continue to rely on sputum smear microscopy which is an uncomplicated, economical and accessible tool for