Spheroplast formation from nine rapidly growing mycobacteria

Abstract

Cell-wall-deficient forms from nine rapidly growing mycobacteria were prepared by treating the cells during exponential growth with D-cycloserine, glycine, lysozyme, EDTA, and lithium chloride for the time corresponding to three cell divisions. The cell-wall-deficient forms were then converted to spheroplasts in the presence of lysozyme and EDTA. The spheroplasts were characterized by their susceptibility to lysis by 0.5% (wt/vol) sodium dodecyl sulfate (SDS), as well as by their response to eight biochemical tests commonly used for mycobacterial identification. Intact cells, on the other hand, could not be lysed even with 2.5% (wt/vol) SDS and, moreover, gave negative responses to certain biochemical tests that turned positive once the cell wall was removed. This implied an exclusion mechanism at the level of the mycobacterial wall. The present method of spheroplast formation was found applicable to various mycobacterial species.