Standardized Method for HPLC Identification of Mycobacteria

The objective of this method is to identify mycobacteria by analysis of mycolic acids, using high performance liquid chromatography (HPLC). A suspension of acid-fast bacteria is saponified to cleave the mycolic acids bound to the cell wall. Mycolic acids are then separated by acidification and extraction into chloroform. After conversion to ultraviolet(UV)-absorbing p-bromophenacyl esters the mycolic acids are analyzed on a reverse-phase C18 column using HPLC2. A gradient of methanol and dichloromethane (methylene chloride) generated by microprocessor-controlled pumps is used to separate the mycolic acid esters, which are detected with a UV spectrophotometer. Reproducible chromatographic patterns containing combinations of different diagnostic peaks are formed. Pattern recognition is by visual comparison of sample results with mycolic acid patterns from reference species of known mycobacteria. Correct pattern interpretation requires training. Computer-assisted pattern recognition technology and high-sensitivity fluorescence detection are being evaluated.