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To get insight into the sensitivity of fluorescence microscopy for diagnostic and follow-up sputum samples from pulmonary tuberculosis patients in clinical trials, the yield of smear positivity – among culture positive sputum samples – encountered in diagnostic and follow-up samples was retrospectively analyzed from the data available in a mycobacteriology laboratory in India. The sensitivity of fluorescence microscopy for diagnostic and follow-up samples respectively was found to be 94.3% and 60.7%. With these values as guidelines, the performance of fluorescence microscopy in the treatment of multi-drug resistant tuberculosis under DOTS plus program remains to be monitored and studied.