Studies on thin-layer chromatography after incubation with [35S]methionine as an aid to identification of mycobacteria.

Abstract

Thin-layer chromatography (TLC) of ethyl ether-ethanol extracts of mycobacteria obtained after incubation with [35S]methionine is useful for differentiation among mycobacterial species, as the distribution of radioactive spots in TLC shows a characteristic pattern except for a few species, including M. intracellulare and M. gordonae. Some supplementary studies have been carried out in the present investigation and the following results have been obtained. 1. Pretreatment of bacterial cells with trichloroacetic acid (TCA) is required to remove radioactive sulfur-containing amino acids. 2. The radioactive spots that appear in TLC of the ethyl ether-ethanol extract are divided into the following groups: (1) Petroleum ether-soluble fraction (the spot at Rf value 0.95); (2) acetone-soluble (petroleum ether-insoluble) fraction (the spots at Rf values 0.50, 0.30, and 0.13--0.20); (3) ethanol-soluble (petroleum ether- and acetone-insoluble) fraction (the spot at Rf value 0.10); (4) petroleum ether-, acetone-, and ethanol-insoluble fraction (the spot at Rf value 0.00). The spot which appears in the region of Rf values 0.13--0.20 (it may appear as two spots) in the TLC of ethyl ether-ethanol extracts is composed of two compounds, one soluble in acetone and the other insoluble in acetone but soluble in ethanol. 3. Strains of the M. avium-M. intracellulare complex have been divided into three subgroups by the pattern of radioactive spots B, C, and D, which show Rf values of 0.13--0.20, 0.30, and 0.50, respectively, in the TLC of the ethyl ether-ethanol extracts. The first group shows none of these spots, and strains of the serotypes 1, 2, and 8 belong in this group. The second group shows spot B, and strains of the serotypes 4, 18, and 20 belong in this group. The third group shows spots B, C, and D, and strains of the serotype 16 belong in it.