ASSAY METHODS 69 



dust, folic acid yields characteristically a pteridine and an aromatic 

 amine. The latter can be determined colorimetrically after the method of 

 Bratton and Marshall. 204 The presence of aromatic amines in the original 

 solution before reduction is taken care of by the use of a blank, and the 

 amount before reduction subtracted from that obtained after reduction 

 is used as a measure of the folic acid present. 



The specificity of the test has not been explored (in spite of the ten 

 investigators involved) and its sensitivity is not high. It may be used only 

 for concentrates which contain 5 per cent or more of folic acid. 



Microbiological Tests. The two organisms which are most often used 

 for folic acid assays are L. casei, the organism used initially in observa- 

 tions dealing with the "riorite eluate factor" 205 and Streptococcus faecalis 

 R (earlier called Strep, lactis R) which was the principal organism used 

 in connection with first obtaining folic acid in highly concentrated form 20C 

 and in determining its distribution in tissue autolyzates and in enzyme- 

 digested tissues and foods. 



The principal differences between the behaviors of the two organisms 

 in these tests are: (1) L. casei assays are materially affected by the intro- 

 duction of additional alanine into the medium ; S. faecalis assays are not. 207 

 The same general statement appears to apply also to other amino acids 

 (leucine, isoleucine, threonine 208> 209 ) , though direct comparisons appear 

 not to have been made. (2) L. casei requires for maximum growth un- 

 known substances such as occur in enzymatically hydrolyzed casein 50 

 and norite-treated peptone. 210 Such addenda have not been required when 

 S. faecalis is used. 210 (3) Thymine in sufficient concentration has stimulat- 

 ing effect on both organisms as well as certain other lactic acid bac- 

 teria; 211, 212 - 213 however, L. casei is considerably more sensitive to small 

 concentrations. 209 On the other hand, thymine in sufficient amounts has 

 an ultimate effect practically equivalent to folic acid for S. faecalis, but 

 incomplete fermentation only results when excess thymine is furnished 

 L. casei. 213 This latter observed difference may be eliminated when it is 

 possible to include all the stimulatory substances affecting L. casei, except 

 folic acid, in the basal medium. (4) S. faecalis responds to rhizopterin 214 

 (S.L.R. factor) as it does to folic acid, but this glutamic-acid-free relative 

 of folic acid is substantially inactive for L. casei. (5) L. casei responds 

 readily to pteroyl triglutamic acid while for S. faecalis this combined 

 form is only about 6 per cent as active. 215 (Both organisms respond 

 equally to folic acid and fail to respond to pteroyl heptaglutamic acid.) 

 (6) L. casei tests have usually involved relatively long growth periods 

 and titrametric evaluation of the responses. S. faecalis tests, on the other 

 hand, have usually involved short growth periods and turbidimetric meas- 

 urement of the response. 



