As stated in my previous post “The Importance of Bacteria Identification in Clean Rooms”, we are supposed to identify the flora obtained from environmental samples, and it is not limited to clean room environments. In this post, 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 fatty acid compositions, which can be compared with the mean fatty acid 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 fatty acid methyl esters 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, summarized in the picture below.

Reagent # 1: NaOH P.A. in Methanol HPLC
Reagent # 2: HCl 6,0 N P.A. in Methanol HPLC
Reagent # 3: hexane HPLC in methyl tert-butyl ether HPLC
Reagent # 4: aqueous solution of NaOH P.A.
All glassware should be new and only Teflon and glass should come in contact with reagents.

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.
If you want to know more about bacteria identification using gas chromatography, read chapter 11 of this book or visit the official site.