Equivalence of acid alone or acid-alcohol as decolourizing agent in Ziehl-Neelsen method.

BACKGROUND: Microscopists opine that acid-alcohol decolourized slides may enhance acid-fast bacilli (AFB) smear positivity, and published documents on equivalence of acid and acid-alcohol in ZN staining method are not easily accessible. SETTING: National Institute for Research in Tuberculosis, Chennai, India. OBJECTIVE: To document the equivalence of 25% sulphuric acid (ZN-acid method) and 3% hydrochloric acid-alcohol (ZN-alcohol method) as decolourizing agents in ZN method for detection of acid-fast bacilli. METHODS: Two smears from each of 253 sputum samples from pulmonary tuberculosis patients, prepared and allocated, one to ZN-acid method and another to ZN-alcohol method were read blind. All the specimens were cultured for Mycobacterium tuberculosis by modified Petroff's method. Culture of M. tuberculosis was gold standard. RESULTS: The concordance between the methods was 85% (kappa 0.68), and the sensitivity (79%) and specificity (89%) were same for both the methods. CONCLUSION: In conclusion, the common belief that acid-alcohol decolourized slides give enhanced smear positivity stands void.

Increased detection by restaining of acid-fast bacilli in sputum samples transported in cetylpyridinium chloride solution.

SETTING: Seventeen health facilities in a Tuberculosis Unit, and the Tuberculosis Research Centre (TRC), Chennai, India. AIM: Evaluation of restaining by the auramine-phenol method for detection of acid-fast bacilli (AFB) in direct smears of samples transported in cetylpyridinium chloride (CPC) solution. METHODS: Among patients attending the above health facilities, 730 samples were collected in CPC and transported to the TRC. Two direct smears were prepared from each sample, one stained by the usual auraminephenol method (primary staining) and the other stained again by the same method (restaining) for examination by fluorescence microscopy. All the samples were processed for culture of Mycobacterium tuberculosis. RESULTS: A significantly higher proportion (49.6%) of samples were positive by restaining compared to primary staining (32.5%, P < 0.0001). Of 362 samples positive by restaining, 38.7% were negative by primary staining. The yield of different grades of smears was significantly higher with restaining than with primary staining (P < 0.0001). More smear-negative culture-positive smears were observed with the primary than with the restaining method (178/400 [44.5%] vs. 78/ 400 [19.5%], P < 0.001). CONCLUSION: The rate of detection of AFB in direct smears made from sputum samples transported in CPC was higher on restaining than on primary staining.

Comparison of variants of carbol-fuchsin solution in Ziehl-Neelsen for detection of acid-fast bacilli.

To evaluate Ziehl-Neelsen (ZN) staining using variants of carbol-fuchsin solution, duplicate smears from 416 samples were stained with ZN, one set with 1% basic fuchsin and the other 0.3%. Another set of duplicate smears from 398 samples were stained with ZN, one with 1% basic fuchsin and the other 0.1%. The coded smears were read and discrepancies resolved. All samples underwent mycobacterial culture. The sensitivity of ZN using 0.3% (65%) and 1% basic fuchsin (62%) was comparable, while it was reduced using 0.1% (74%) compared to 1% basic fuchsin (83%). Reducing the concentration of basic fuchsin below 0.3% in ZN staining was found to significantly reduce its sensitivity.