DOI: 10.36347/sjams.2019.v07i05.029

Pages: 1829-1836

Apart from genes responsible for drug resistance of Mycobacterium tuberculosis, dramatic reduction of metabolic activity of tubercle bacilli in chronically infected animals reveals long generation time. This could be an additional explanation of drug resistance. Amongst different methods available to determine generation time, colony count method, which is cheap, easily available and affordable one, is compared with a novel method i.e. DNA spectrophotometry in an attempt to cut short turnaround time. Thus, this study was conducted to compare two different methods (Colony count method and DNA spectrophotometry) for determination of generation time of Mycobacterium tuberculosis in vitro. For this, 20 new sputum smear positive patients were placed in the study. Mycobacterium tuberculosis strains were isolated in pure culture by decontaminating, liquefying & concentrating sputum sample & anti-tubercular susceptibility tests were performed. From these, 10 all-drug (Rifampicin and Isoniazid) sensitive isolates were chosen and reference strains were procured. Single mycobacterial cell suspension was prepared from each. After proper standardization, they were incubated in CO2 incubator and 10μl of each culture suspension was inoculated in Middlebrook 7H11 agar plate to measure CFU/ml and from 500 µl of each culture suspension, DNA extraction is done by Phenol-Chloroform method which is followed by Spectrophotometric quantification of OD/ml (at 260nm) at different time-points (Hour 0,18,36,54,72). Generation time was evaluated from graph of the above colony counts using formula. It was observed that serial culturing by counting of CFU is the best method for assessment of generation time while DNA spectrophotometry could not be used for this purpose. In short, colony count method still stands as the gold standard for determination of generation time for mycobacteria whereas DNA spectrophotometry failed poorly to evaluate the same.