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

Abstract

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