192 MANUAL OF MICROBIOLOGICAL METHODS 



Ion-exchange columns are useful for batch and analytical scale separa- 

 tions of amino acid (Hirs et at., 1952; Moore and Stein, 1954) but, 

 although quite precise, require more time and attention than the paper 

 method. 



Microbiological assay is employed widely and depends upon strains 

 of microorganisms (commonly lactic acid bacteria) of exacting but known 

 nutrition, capable of growing in chemically defined media provided the 

 amino acid to be assayed is supplied, and responding to graded amounts 

 of this amino acid in a regular fashion. The limits and specificity of the 

 growth response must be checked before unknown samples are assayed. 

 Total growth is determined usually by acidity or turbidity measurements. 

 These methods do not allow separation of amino acids but do permit the 

 quantitative measurement of one amino acid in a mixture. Technics 

 and organisms are available for the microbiological assay of all the amino 

 acids (Snell, 1946; Dunn, 1949; Barton-Wright, 1955). 



Although several enzymatic methods are available, the specific decar- 

 boxylase method is the one most widely used because of simplicity, 

 specificity, accuracy, stability of dried bacterial preparations, and ease of 

 manipulations. The method depends upon the manometric measurement 

 of CO2 production from the amino acid by enzymes from selected strains 

 of microorganisms. Six amino acids may be determined quantitatively 

 in this manner: arginine, lysine, tyrosine, histidine, glutamic acid, and 

 ornithine (Gale, 1948; Umbreit and Gunsalus, 1945; Archibald, 1946). 



The methods employed in the study of three of the major reactions of 

 amino acids will be considered briefly. 



The enzymatic removal of the carboxyl group of an amino acid with the 

 formation of the corresponding amine and CO 2 is known as decarboxyla- 

 tion. Decarboxylase activity may be determined by measuring either 

 the disappearance of the amino acid acted upon by one of the methods 

 listed above or the resulting products. In most instances, CO2 evolution 

 is measured manometrically. In the case of aspartic acid decarboxylase, 

 CO 2 measurements cannot be used because of the low activity. Billen 

 and Lichstein (1949) have therefore employed a microbiological assay 

 method for the /^-alanine produced. 



The enzymatic removal of the amino group of an amino acid with the 

 formation of ammonia and most commonly but not exclusively the cor- 

 responding keto acid is known as deamination. Although the activity 

 may be measured by determining the disappearance of the amino acid 

 acted upon, the most common method is to measure the production of 

 ammonia by nesslerization. 



The enzymatic transfer of the amino group of an a-amino acid to an 

 a-keto acid resulting in the formation of another a-keto acid, the former 

 corresponding in structure to the original a-keto acid and the latter to the 



