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Microbiologists are supposed to identify the flora obtained from environmental samples, and it is not limited to clean room environments. In this article, I am going to focus on bacteria identification solely.
There are several methods of bacteria identification available, which can be classified into two large groups: the phenotypic group and the genotypic group. The phenotypic group includes:
- Biochemical tests
- Biotyping
- Serotyping
- Phagetyping
- Antimicrobial susceptibility
- Multi-Locus Enzyme Electrophoresis (MLEE)
- Electrophoretic protein typing and immunoblotting
- Gas Chromatography of Fatty Acid Methyl Ester
The genotypic group includes:
- Plasmid typing
- Restriction Enzyme Analysis (REA)
- Pulsed-Field Gel Electrophoresis (PFGE)
- Ribotyping
- RAPD
- Rep-PCR
- PCR-ribotyping
The only commercially available gas chromatography (GC) system dedicated to bacteria identification by fatty acid methyl ester (FAME) analysis is the Sherlock Microbial Identification System (MIS), developed by Microbial ID, Inc. (MIDI). The original database for aerobic bacteria identification was developed by M. Sasser, in 1990.
The principle of the FAME method rests upon the assumption that some microorganisms have typical cellular FAME compositions, which can be compared with the mean FAME composition of the strains used to create the library. After comparison, the identities of unknown microorganisms are determined.
For many years, analysis of short chain fatty acids (volatile fatty acids, VFAs) has been routinely used in identification of anaerobic bacteria. In numerous scientific papers, the fatty acids between 9 and 20 carbons in length have also been used for bacteria identification, especially nonfermentative Gram negative organisms. With the advent of fused silica capillary columns (which allows recovery of hydroxy acids and resolution of many isomers), it has become practical and easier to use GC of whole cell FAMEs to identify isolated and pure microbial cultures, bacterial of medical importance, and in taxonomic studies.
The FAME method uses a specific sample preparation procedure and a sophisticated chromatographic system to yield qualitatively and quantitatively reproducible fatty acid composition profiles. This system was developed for microbiologists and it does not require extensive knowledge of gas chromatography.
Sample Preparation Procedure
Bacteria selected for identification by FAME analysis are subcultured twice on Trypticase Soy Broth solidified with 1.5% agar and then incubated aerobically at 28 ºC for 24 h. Growth is examined for the presence of pure culture and submitted to the fatty acid extraction, in simple, five, basic steps:
- Removal of cells from culture media
- Lysis of the cells to liberate fatty acids from the cellular lipids
- Formation of FAMEs
- Transfer of the FAMEs from the aqueous phase to the organic phase
- Aqueous wash of the organic extract prior to chromatographic analysis
Chromatographic System
Recognition of fatty acid profiles is performed using the MIS system along with a standard library. The MIS consists of a gas chromatograph equipped with a fused silica capillary column, a flame ionization detector, an integrator and an automatic sampler coupled to a computer system. The Sherlock computer software automatically sets the operating parameters of the gas chromatograph each time a sample is processed. Fatty acids are separated because of different retention times, using synthetic air, hydrogen as the carrier and nitrogen as the makeup gas. Coupled to Sherlock is the ChemStation software used for operating sampling, analysis, and integration of the chromatographic samples. The fatty acid percentages are automatically calculated and after comparison with the MIDI Standard library, the bacterial identifications are expressed on the basis of genus, species and sub-species level.
Fabio L. C. Pacheco invites you to visit Microbiology Online. If you liked this article, you will also enjoy Microbiology Online. It is all about great tutorials in industrial microbiology, focusing on pharmaceutical microbiology labs.
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