Gas chromatographic fatty acid profiles for characterisation of mycobacteria: An interlaboratory methodological evaluation

Abstract

Three species of mycobacteria were cultured and processed for cellular fatty acid analysis by capillary gas chromatography in three laboratories to study interlaboratory variations of the resulting Chromatographic profiles. Largely consistent and characteristic fatty acid profiles were obtained, although there were minor quantitative variations in the patterns due to methodological differences (cultivation, hydrolysis, derivatization, gas Chromatographic conditions etc.). The following points were important for achieving informative and reproducible results. A chemically defined growth medium (e.g., Proskauer-Beck) provides more consistent profiles than the lipid-rich Löwenstein-Jensen medium. Harvesting directly into the digesting solution (NaOH or HCl in methanol) followed by heating or autoclaving is a simple and reliable way of releasing fatty acids. Care should be taken to ensure reproducible detection of long-chain alcohols either by using acid methanolysis or including a base-wash step in the procedure following alkaline hydrolysis. The temperature of the gas Chromatographic injector should be at least 325 °C. A capillary column of a minimum length of 10 m coated with a methyl silicone is adequate. Our results indicate the possibility of recommending a practical and reproducible gas Chromatographic procedure for mycobacterial characterisation.