Evaluation of acridine orange staining for rapid detection of mycobacteria in primary specimen

Background - Tuberculosis continues to be a major health problem worldwide. The emergence of drug-resistant organisms threatens to make this disease once again incurable. So, timely isolation and identification of M.tuberculosis becomes important. The acid fast bacilli in the sputum can be detected microscopically by Ziehl- Neelsen stain and fluorescent stain.Objectives - Evaluation and comparison of Phenolic-Acridine orange fluorescent staining method for detection of M.tuberculosis in suspected samples with the traditional Ziehl-Neelsen stain. Materials and Methods - One hundred sputum samples were collected from suspected cases of pulmonary tuberculosis from both IPOP patients attending PSG hospitals. Samples were decontaminated by using N -acetyl- LcysteineNaOH method. Smears were prepared and stained by Phenolicacridine orange fluorescent stain and Ziehl -Neelsen stain. Samples were also inoculated on Lowenstein Jensen medium (gold standard).Results - Out of 100 samples 20 were positive by fluorescent phenolic- acridine orange stain and 22 were positive by Ziehl- Neelsen stain. Culture was positive on Lowenstein Jensen medium for 31 of the samples. Conclusion - In fluorescence microscopy smears are examined under 40x objective, so it is less time consuming and the fluorescing bacilli easily identified. The differentiation of the bacilli was better in phenolic-Acridine orange staining as the dull green background enables easy visualization. The fluorescence intensity of the Acridine orange-stained AFB was stable over several days when the stained smears were kept in the dark. Phenolic- Acridine orange can be considered as an alternative fluorescent stain for demonstrating acid fast bacilli in clinical samples. Keyword :phenolic-Acridine orange, Ziehl -Neelsen stain, M.tuberculosis