70 THE BIOCHEMISTRY OF B VITAMINS 



A number of the apparent defects mentioned above are relatively easily 

 remedied. The basal media can be adjusted to take care of the effects of 

 alanine and other amino acids, and with less satisfaction the unknown 

 stimulatory substances. 



A drawback so far as L. casei is concerned is the relatively high activity 

 of thymine — as little as 1 /*g in a sample would be enough to introduce 

 serious error. 209 For S. faecalis, however, folic acid is at least 5000 times as 

 active as thymine. 213 This discrepancy is not as important as might be 

 supposed, however, since L. casei responds to smaller concentrations of 

 folic acid than does S. faecalis. Stokstad and Hutchings 216 suggest that 

 with S. faecalis the total response minus the residual response after selec- 

 tively destroying the folic acid could be used as a measure of the true 

 folic acid content. Whether such a procedure is necessary or practical has 

 not been demonstrated. 



The most serious defect of the S. faecalis assay is the response to a 

 simpler compound, rhizopterin. The degree of importance of this observa- 

 tion depends upon the distribution of rhizopterin in nature. If it is wide- 

 spread and relatively abundant, the defect is quite serious. Whether or 

 not this is so can be determined only by comparative assays on completely 

 digested samples, using both L. casei and S. faecalis. Because the two 

 organisms do not respond equally to combined forms of folic acid, 215 one 

 would have to be sure that digestion had been complete before valid con- 

 clusions could be drawn. 



From the foregoing discussion, it is clear that no well standardized and 

 completely satisfactory microbiological method for folic acid assay has 

 been devised. Stokstad and Hutchings wisely suggest the use of both L. 

 casei and S. faecalis. 216 Complete digestion of the combined forms without 

 destruction constitutes one crucial problem (p. 40). 



Animal Assay Methods. 



Folic acid assays may be performed using chicks, rats or monkeys. The 

 use of rats involves the feeding of sulfa drugs to prevent intestinal syn- 

 thesis 217 and involves so many complications and uncertainties that it 

 does not appear to be a useful assay method at the present time. Use can 

 be made of rhesus monkeys 21S but the expense and inconvenience could 

 hardly be justified on the basis of an assay method. 



Chick Assay. While O'Dell and Hogan 219 and their co-workers used 

 the cure of anemia in chicks in connection with concentrating "vitamin 

 B c ," this method of assay has apparently not been applied to the deter- 

 mination of the vitamin in foods and tissues. 



The preferred chick assay method involves the use of a 4-week prophy- 

 lactic test period. It was applied successfully by Bird et al. 220 to natural 



