Analysis of Cellular Fatty Acids of Bacteria by Gas-Liquid Chromatography 



6. Turn off the stirrer, open the stopcock in the upper part of the tube, and bubble a small 

 vokime of hydrogen gas into the tube for approximately 5 sec. CAUTION: Hydrogen is a 

 highly tlammable gas and must be handled with extreme caution. 



7. Close the stopcock and mix the contents for 15 min with the magnetic stirrer. Add a 

 small volume of hydrogen gas two (more) times, and allow 15-min intervals for stirring. 



8. Remove the catalyst by filtration through a small Whatman paper filter (#1) and collect 

 the hydrogenated sample in a 13- x 100-mm screw-capped tube. 



9. Reduce the sample volume to about 0. 1 ml under a gentle fiow of nitrogen gas in the 

 hood. 



10. Analyze 1-3 /.tl of sample by GLC under identical conditions to those described for the 

 original methyl ester sample. 



B. Acetylation of Methyl Ester 



1. After analysis by GLC, add 5 drops of trifluoroacetic anhydride to the methyl ester 

 sample tube with a Pasteur pipette. 



2. Mix gently and leave at room temperature for 30 min. 



3. Add 0.3 ml distilled water and mix gently to wash the sample. 



4. Allow the layers to separate. Carefully transfer the hexane layer (top) to a clean 13- x 

 100-mm screw-capped tube. 



5. Analyze 1-3 /j1 of the acetylated sample by GLC under identical conditions to those 

 described for the original methyl ester sample. 



QUANTITATION 



As noted previously, the bases for differentiation among bacteria are the presence of specific 

 fatty acids and their relative concentration compared to those of other acids in the sample. 

 Therefore, in addition to observing the overall general fatty acid profile, it is desirable to deter- 

 mine the relative concentration of peaks in the chromatogram. Peak areas are obtained from the 

 chromatogram (DISC or electronic integrator), and the percentage of each acid is calculated from 

 the ratio of the area of its peak to the total area of all peaks. In this laboratory, small peaks with 

 areas less than 2% of the total are disregarded. 



CONTROLS 



Because of the extreme sensitivity of the technique, highly purified chemicals, solvents, 

 reagents, and gases are used in GLC studies. Glassware is a potential source of contamination and 

 should be checked for cleanness and cracks. Tefion-lined caps should be used, and these and all 

 glassware should be rinsed with solvents (hexane, acetone) before use. A bacterium of known 

 fatty acid composition should be included along with the unknown organism. Also, a "reagent 

 blank" (i.e., distilled water) should be processed occasionally as a check on all reagents, solvents, 

 and the GLC column. 



121 



