Percoll discontinuous density gradient centrifugation method for the fractionation of the subpopulations of Mycobacterium smegmatis and Mycobacterium tuberculosis from in vitro cultures

Bacterial populations in the in vitro laboratory cultures, environment, and patients contain metabolically different subpopulations that respond differently to stress agents, including antibiotics, and emerge as stress tolerant or resistant strains. To contain the emergence of such strains, it is important to study the features of the metabolic status and response of the subpopulations to stress agents. For this purpose, an efficient method is required for the fractionation and isolation of the subpopulations from the cultures. Here we describe in detail the manual setting up of a simple, easy-to-do, reproducibly robust Percoll discontinuous density gradient centrifugation for the fractionation of subpopulations of short-sized cells (SCs) and normal/long-sized cells (NCs) from Mycobacterium smegmatis and Mycobacterium tuberculosis cultures, which we had reported earlier. About 90-98% enrichment was obtained respectively for SCs and NCs for M. smegmatis and 69-67% enrichment was obtained respectively for the SCs and NCs for M. tuberculosis. • The Percoll discontinuous density gradient centrifugation helps the fractionation and isolation of mycobacterial subpopulations that differ in density. • The method offers a consistently reproducible high enrichment of the subpopulations of SCs and NCs from the in vitro cultures of M. smegmatis and M. tuberculosis. • Our earlier reports on the consistency in the differential response of the subpopulations, enriched using the method, to oxidative, nitrite, and antibiotic stress proves its validity.