Analysis of Cellular Fatty Acids of Bacteria 

 by Gas-Liquid Chromatography 



C. Wayne Moss 



INTRODUCTION 



Cliemical analyses of cell structure of bacteria and of products from their metabolism 

 provide useful additional information for identification and classification. Extensive studies with 

 cellular fatty acids have shown that certain closely related species can be distinguished on the 

 basis of qualitative differences in their fatty acids {/, 2). A recent example is the Legionnaires' 

 disease bacterium (LDB), which is different from other gram-negative bacteria because it contains 

 large amounts (> 779c ) of branched-chain fatty acids (5). 



The lipids of bacteria are found in the cell wall/cell membrane fraction, where the unit fatty 

 acids are chemically bonded to other cellular components. The acids are liberated or cleaved from 

 these components and subsequently analyzed by gas-liquid chromatography (GLC). GLC is the 

 method of choice for fatty acids because of its speed, sensitivity, and excellent separating effi- 

 ciency. The following is a protocol for analysis of bacterial cellular fatty acids by GLC techniques 

 with special reference to the LDB. A detailed review on GLC and its application to microbiology 

 appeared recently {2). 



PREPARATION OF SAMPLES 



A. Saponification - to detemiine "total" cellular fatty acids, bound lipids must be freed by a 

 saponification or hydrolysis procedure. 



1. Add 0.5 ml sterile distilled water to the surface of an agar slant (16 x 150 mm). 



2. Gently scrape the cells off the agar, and then transfer the turbid cell suspension to an 18 

 or 20 X 1 50 mm test tube with a TetTon-lined screw cap. 



3. Add 4 ml of 5% NaOH in 50% aqueous methanol, and tighten the Tetlon-lined cap. 



4. Heat tiie cell suspension for 30 min in a boiling water batii ( 100°C). 



5. Remove the tube from the water bath and cool to room temperature. If necessary, tubes 

 may be held overniglit in a refrigerator. 



B. Methylation - before conversion to esters, the sodium salts of the acids from saponification 

 must be converted to the free fomi. 



1. Add approximately 1.0 ml of 6 N HCl to the cool saponificate to lower the pH to 2.0. 



2. Mix well and check with pH indicator paper. If needed, add more 6 N HCl drop by drop 

 until pH is 2.0. 



3. Add 4 ml of 10% boron trichloride-methanol reagent (Applied Science Laboratories, 

 State College, PA) and mix well. Tighten Tetlon-lined cap. 



4. Heat for 5 min in a water bath maintained at 80°C-85°C. 



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