How to Perform Bacteria Identification Using Gas Chromatography of Fatty Acid Methyl Ester

Microbiologists are supposed to identify the floraidentify isolated and pure microbial cultures, bacterial
obtained from environmental samples, and it is notof medical importance, and in taxonomic studies.
limited to clean room environments. In this article, IThe FAME method uses a specific sample preparation
am going to focus on bacteria identification solely.procedure and a sophisticated chromatographic
There are several methods of bacteria identificationsystem to yield qualitatively and quantitatively
available, which can be classified into two largereproducible fatty acid composition profiles. This
groups: the phenotypic group and the genotypicsystem was developed for microbiologists and it
group. The phenotypic group includes:does not require extensive knowledge of gas
- Biochemical testschromatography.
- BiotypingSample Preparation Procedure
- SerotypingBacteria selected for identification by FAME analysis
- Phagetypingare subcultured twice on Trypticase Soy Broth
- Antimicrobial susceptibilitysolidified with 1.5% agar and then incubated
- Multi-Locus Enzyme Electrophoresis (MLEE)aerobically at 28 ºC for 24 h. Growth is
- Electrophoretic protein typing and immunoblottingexamined for the presence of pure culture and
- Gas Chromatography of Fatty Acid Methyl Estersubmitted to the fatty acid extraction, in simple, five,
The genotypic group includes:basic steps:
- Plasmid typing
- Restriction Enzyme Analysis (REA)1. Removal of cells from culture media
- Pulsed-Field Gel Electrophoresis (PFGE)2. Lysis of the cells to liberate fatty acids from the
- Ribotypingcellular lipids
- RAPD3. Formation of FAMEs
- Rep-PCR4. Transfer of the FAMEs from the aqueous phase
- PCR-ribotyping The only commercially available gasto the organic phase
chromatography (GC) system dedicated to bacteria5. Aqueous wash of the organic extract prior to
identification by fatty acid methyl ester (FAME)chromatographic analysis
analysis is the Sherlock Microbial Identification SystemChromatographic System
(MIS), developed by Microbial ID, Inc. (MIDI). TheRecognition of fatty acid profiles is performed using
original database for aerobic bacteria identificationthe MIS system along with a standard library. The
was developed by M. Sasser, in 1990.MIS consists of a gas chromatograph equipped with
The principle of the FAME method rests upon thea fused silica capillary column, a flame ionization
assumption that some microorganisms have typicaldetector, an integrator and an automatic sampler
cellular FAME compositions, which can be comparedcoupled to a computer system. The Sherlock
with the mean FAME composition of the strains usedcomputer software automatically sets the operating
to create the library. After comparison, the identitiesparameters of the gas chromatograph each time a
of unknown microorganisms are determined.sample is processed. Fatty acids are separated
For many years, analysis of short chain fatty acidsbecause of different retention times, using synthetic
(volatile fatty acids, VFAs) has been routinely used inair, hydrogen as the carrier and nitrogen as the
identification of anaerobic bacteria. In numerousmakeup gas. Coupled to Sherlock is the ChemStation
scientific papers, the fatty acids between 9 and 20software used for operating sampling, analysis, and
carbons in length have also been used for bacteriaintegration of the chromatographic samples. The
identification, especially nonfermentative Gramfatty acid percentages are automatically calculated
negative organisms. With the advent of fused silicaand after comparison with the MIDI Standard library,
capillary columns (which allows recovery of hydroxythe bacterial identifications are expressed on the
acids and resolution of many isomers), it has becomebasis of genus, species and sub-species level.
practical and easier to use GC of whole cell FAMEs to