ASSAY METHODS 53 



substances present in the extracts, aside from riboflavin, will have a 

 negligible effect. 



Subsequent investigations dealing with the nutrition of the test organ- 

 ism have shown that the basal medium recommended by Snell and Strong 

 is far from ideal in the sense of the previous paragraph. Roberts and 

 Snell 50 have developed a vastly improved medium in which the organism 

 responds to riboflavin to a much higher degree. Nevertheless, the original 

 medium is such that it has given very satisfactory results comparable to 

 those obtained using the improved medium, and it serves for practical 

 purposes. If one were assaying materials never assayed before, the 

 results could be accepted with greater certainty using the more complete 

 medium of Roberts and Snell. 



The most serious extraneous factors that can complicate riboflavin 

 assay by this method are the fatty substances, 51 - 52 - 53 the effects of which 

 are sometimes stimulatory and sometimes inhibitory depending upon the 

 agents, their concentration and upon the presence or absence of other 

 agents in the extract. These disturbing factors can be eliminated by 

 preliminary solvent extraction, or more simply by careful filtration of 

 the extract at pH 4.5 to obtain a clear solution. The latter procedure is 

 the one used in the U. S. Pharmacopoeia method. 



In the application of the method to urine, urea can be present in 

 sufficient amount to introduce an error due to its inhibitory effect. This 

 effect can be corrected for if necessary. 54 



There are two valid methods for evaluating the response of the organism 

 to riboflavin: turbidimetric measurement of growth after 24 hours or less 

 (16 hours in case the Roberts-Snell medium is used) and titration of the 

 acidity developed after 72 hours. Either method gives wholly satisfactory 

 results; the titration method possibly is a little less exacting, requires no 

 special apparatus and is specified in the U. S. Pharmacopoeia. The tur- 

 bidity method, however, gives results overnight which for some purposes 

 may be a tremendous advantage. It requires a turbidity measuring 

 device; a suitable photoelectric colorimeter will serve, but even better, 

 however, is a thermoelectric turbidimeter such as has been in use in the 

 author's laboratory for twenty years. 55 



Biological Methods. In view of the satisfactory assay of riboflavin by 

 the two methods discussed, the animal assay methods are mostly of 

 historical interest. This does not mean, of course, that animal experiments 

 involving riboflavin are outmoded. 



The rat growth method of Bourquin and Sherman 56 has been used 

 with minor modifications for a number of years and was the standard 

 with which the newer methods were initially compared. These investi- 

 gators were fortunate in preparing an alcoholic extract (80 per cent) of 



