ASSAY METHODS 71 



materials, but has been only briefly described. 221 A number of changes 

 result from lack of the vitamin, 221, 222 including failure to grow, but it is 

 not apparent how Bird and co-workers 220 calculated their results. In the 

 assay technic recommended by Day and Totter, 223 weights of the chicks 

 at the end of four weeks are the basis for calculating the results. 



The chick assay method as applied by Bird and co-workers yielded 

 results which in general agreed substantially with microbiological assays 

 when the materials for the latter were treated enzymatically with prepara- 

 tions from hog kidney and almonds. 220 Plant extracts, however, gave 

 higher results with chick assays than were obtainable by microbiological 

 assay. It may be that plants contain combined forms of folic acid which 

 are not hydrolyzed by any of the treatments used. Chicks respond equally 

 to folic acid, the tri- and heptaglutamate forms, and presumably to other 

 combined forms which may occur naturally (p. 31). 



/>-Aminobenzoic Acid 



While p-aminobenzoic acid can be determined colorimetrically by 

 diazotizing and coupling with dimethyl-a-napthylamine 224 and by the 

 use of thiamine as a reagent, 225 such methods are neither highly specific 

 nor are they sensitive in comparison with microbiological methods and 

 have not been used as general assay procedures. 



Animal experiments have demonstrated the physiological effect of feed- 

 ing p-aminobenzoic acid under prescribed conditions to chicks, 226, 227 

 mice, 228, 229 and rats, 226, 230 but the effects may be indirect and not always 

 reproducible, 231 and no assay method has resulted from these observations. 



Microbiological Assays. Four microbiological assays for p-amino- 

 benzoic acid have been developed. They utilize respectively, Acetobacter 

 suboxydans 232 Lactobacillus arabinosus 17-5, 233 a mutant strain (amino- 

 benzoicless) of Neurospora 23 * and CI. acetobutylicum. 235 In the table 

 below is given certain crucial information regarding these tests. 



Microbiological Assays for p-Aminobenzoic Acid 

 Growth 



It is probable that each of the four methods is capable of yielding 

 satisfactory results and for the most part they embody methods which 

 are now common in microbiological assays. In method 3, the size of the 

 mold cultures is measured with calipers which is something of an innova- 



