Microbiologists are supposed to identify the flora obtained from environmental samples, and it is not restricted to clean room environments. In this report, concentrate on bacteria identification exclusively. There are several Methods of bacteria identification available, which may be classified into two large categories: the phenotypic group and the genotypic group. The phenotypic group comprises:
- Biochemical tests
- Biotyping
- Multi-Locus Enzyme Electrophoresis MLEE
- Electrophoretic protein typing and immunoblotting
- Gas Chromatography of Fatty Acid Methyl Ester
The Genotypic group comprises:
- RAPD
- Rep-PCR
- PCR-ribotyping
The Only commercially available gas chromatography GC system specializing in bacteria identification by fatty acid methyl ester FAME analysis is the Sherlock Microbial Identification System MIS, developed by Microbial ID, Inc… MIDI. The database for aerobic bacteria identification was designed by M. Sasser, in 1990.The principle of the FAME method rests upon the premise that some germs have typical cellular FAME compositions, which is contrasted with the expression FAME composition of the breeds used to produce the library. After contrast, the identities of unknown germs are determined.
For many years, Evaluation of short chain fatty acids volatile fatty acids, VFAs has been routinely utilized in identification of anaerobic bacteria. In numerous scientific papers, the fatty acids between 9 and 20 carbons in length also have been utilized for bacteria identification, particularly no fermentative Gram negative organisms. With the arrival of fused silica capillary columns which allows recovery of hydroxyl acids and resolution of several isomers, it is now practical and easier to use GC of cell FAMEs to identify isolated and pure microbial cultures, bacterial of medical significance, and in taxonomic studies.
The FAME method uses a Specific sample preparation procedure and a complex chromatogram method 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 subculture twice on Trypticase Soy Broth solidified with 1.5 percent agar and then incubated aerobically at 28 ºC for 24 h. Development is analyzed for the existence of pure culture and submitted to the fatty acid extraction, in easy, five, fundamental steps:
- Removal of cells from culture media
- Lysis of the cells to liberate fatty acids in the mobile lipids
- Formation of FAMEs
- Transfer of the FAMEs in the aqueous phase to the organic phase
- Aqueous wash of the natural extract before chromatographic analysis
Chromatographic System
Recognition of fatty Acid profiles is done using the MIS system alongside a normal library. The MIS is made up of a gas chromatograph equipped with a fused silica capillary column, a flame ionization detector, an integrator and an automated sampler coupled to a computer system. The Sherlock computer software automatically sets the operating parameters of the gas chromatograph every time a sample is processed. Fatty acids are split due to different retention times, using artificial atmosphere, and hydrogen as the carrier and nitrogen as the makeup gas. Coupled to Sherlock is the ChemStation software used for managing sampling, evaluation, and integration of the chromatographic samples.
