A simple chemical test to distinguish mycobacteria from other mycolic-acid-containing actinomycetes

Two hundred and fifty-two representatives of the general Corynebacterium, Gordona, Mycobacterium, Nocardia, Rhodococcus and Tsukamurella were degraded by alkaline hydrolysis and their mycolic acids extracted as methyl esters following phase-transfer-catalysed esterification. When the mycolic acid methyl esters were treated with a mixture of acetonitrile and toluene all mycobacterial mycolates formed copious white precipitates whereas all but 5 out of the 106 non-mycobacterial mycolates remained in solution. The precipitated methyl mycolates and the dried soluble mycolates were compared by pyrolysis gas chromatography and silica gel thin-layer chromatography. On pyrolysis, the precipitated methyl mycolates from mycobacteria yielded fatty acid methyl esters with 20 to 26 carbon atoms whereas those from the remaining taxa produced shorter-chain esters. Mycobacteria and Tsukamurella paurometabola gave multispot mycolic acid patterns on thin-layer chromatography of their methyl esters whereas those from the remaining strains gave single spots. Our results indicate that Rhodococcus chlorophenolicus strains contain mycolic acids atypical of mycobacteria. It can be concluded that the mycolic acid precipitation test provides a simple and reliable way of distinguishing mycobacteria from all other prokaryotes, notably from other mycolic-acid-containing taxa.