INVESTIGATIONS OF AMINO-ACID METABOLISM BY THIN-LAYER CHROMATOGRAPHY FOR DIFFERENTIATION OF BRUCELLA

  • 1 January 1977
    • journal article
    • research article
    • Vol. 237  (4) , 523-529
Abstract
The amino acid metabolism of 23 different Brucella strains was investigated for differentiation purposes. The results were evaluated by TLC, after enzymatic incubation. The organisms were grown on Tryptose blood agar at 37.degree. C for 24 or 48 h. Two mg wet wt of bacteria were incubated with 12.5 .mu.g (0.025 ml) amino acid in small tubes for 16 h at 37.degree. C, and centrifuged for 15 min at 7500 .times. g. For controls, bacterial suspensions were heated for 15 min at 100.degree. C to destroy enzymatic activity, and also centrifuged for 15 min at 7500 .times. g. Usually 4 .mu.l of the supernatant fluids (6 .mu.l for L-asparagine, and 10 .mu.l for L-proline) were pipetted on the thin layer plate. The tests were run in n-butanol acetic acid water, 20:5:5, with a distance of 8 cm. Amino acids were stained with ninhydrin. The tests were repeated 3-5 times with identical results. Amino acid metabolism was indicated by different staining intensities (+ to ++) in comparison to control preparations. All species could be exactly differentiated from each other, with the exception of B, suis, biotype 2, and B. canis, which could not be differentiated by their amino acid metabolism. Biotypes of the same species were mostly identical. The results of these investigations could be reproduced qualitatively, as well as quantitatively. The method described is recommended for routine investigations.

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