Standarized Method for HPLC Identification of Mycobacteria

摘要

The objective of this method is to identify mycobacteria by analysis of mycolic211u001eacids using high performance liquid chromatography (HPLC). A suspension of acid-211u001efast bacteria is saponified to cleave the mycolic acids bound to the cell wall . 211u001eMycolic acids are then separated by acidification and extraction into chloroform. 211u001eAfter conversion to ultraviolet(UV)-absorbing p-bromophenacyl esters the mycolic 211u001eacids are analyzed on a reverse-phase C18 column using HPLC. A gradient of 211u001emethanol and dichloromethane (methylene chloride) generated by microprocessor-211u001econtrolled pumps is used to separate the mycolic acid esters, which are detected 211u001ewith a UV spectrophotometer. Reproducible chromatographic patterns containing 211u001ecombinations of different diagnostic peaks are formed. Pattern recognition is by 211u001evisual comparison of sample results with mycolic acid patterns from reference 211u001especies of known mycobacteria. Correct pattern interpretation requires training. 211u001eComputer-assisted pattern recognition technology and high-sensitivity 211u001efluorescence detection are being evaluated.