Identification of Clinical Isolates of Mycobacteria with Gas-Liquid Chromatography Alone

Abstract

Identification of 18 mycobacterial species was performed by analysis of profiles obtained by using gas-liquid chromatography. Organisms were saponified in methanolic NaOH, and the reaction mixture was treated with BF3 in methanol and extracted with a hexane-chloroform mixture. An identification scheme was developed from 128 stock strains and tested against a collection of 79 clinical isolates. By using gas-liquid chromatographic profiles alone, 58% of specimens were correctly identified to species level, and an additional 41% were correctly identified to a group of two or three organisms. Use in a clinical laboratory over a 2-month period proved chromatography to be as accurate as and more rapid than concurrent biochemical testing. Of 81 isolates tested, 64% were identified to species level by chromatography alone. An additional 35% were differentiated to the same groups of two or three organisms as found in our analysis of stock strains. These groups consisted of: Mycobacterium tuberculosis, M. bovis, and M. xenopi; M. avium complex, M. gastri, and M. scrofulaceum; or M. fortuitum and M. chelonei. Identification to species level from these groups could usually be done by colonial morphology alone and could always be done by the addition of one selected biochemical test. This study demonstrated the practical application of gas-liquid chromatography in the identification of mycobacteria in a clinical laboratory. In particular, all strains of M. gordonae and M. kansasii were identified to species level. M. tuberculosis was definitively identified in 85% of cases. When it could not be definitely identified, the only alternatives were M. bovis and M. xenopi, both of which are rare causes of infection.

Gas chromatography of mycobacterial fatty acids and alcohols: Diagnostic applications

“…Tisdall et al (28,29) demonstrated that fatty acid profiles obtained by gas-liquid chromatography (GLC) could in most cases identify to the level of species or "complex". In comparison with the identification obtained by biochemical profiles they demonstrated that the identity of 320 out of 335 clinical isolates agreed.…”

ACIDIC METHANOLYSIS V. ALKALINE SAPONIFICATION IN GAS CHROMATOGRAPHIC CHARACTERIZATION OF MYCOBACTERIA: DIFFERENTIATION BETWEEN MYCOBACTERIUM AVIUM‐INTRACELLULARE AND MYCOBACTERIUM GASTRI

“…The mycobacteria were saponified in NaOHcontaining (0.5 N) absolute methanol at 70 OC for 30 min, after which the free fatty acids were esterified with boron trifluoride-methanol and extracted with chloroform-hexane ( I :4 vol/vol). The extracts were then evaporated to dryness (9).…”

“…The purpose of the present study was to compare chromatographic results from alkaline saponifica-tion of mycobacteria followed by methylation with results from direct acidic methanolysis. The test organisms were strains of the Mycobacterium avium-intracellulare complex and M. gastri, since these mycobacteria have been reported as difficult to distinguish by their GC fatty acid patterns (9,10).…”

The diagnosis of pulmonary tuberculosis by gaschromatographic detection of tuberculostearic acid using flame ionisation detectors

“…TBSA can be found in mycobacteriae o r nocardiae, but not in other bacteriae or in human tissue [9,10,11]. The detection of TBSA by gas chromatography/mass-spectrometry with selected ion monitoring (GC/MS-SIM) can be used for the diagnosis of pulmonary, as well as extrapulmonary tuberculosis [12][13][14][15][16][17][18][19][20][21][22][23]. This method provides a rapid, highly specific and sensitive diagnostic test for the presence of mycobacteria [20].…”