372 RIBOFLAVIN 



C. MICROBIOLOGICAL METHODS 



E. E. SNELL 



Riboflavin was found to he a growth factor for certain lactic acid bac- 

 teria^"' ^^ shortly after its isolation as a vitamin for rats.^'' Relatively few 

 microorganisms that require this vitamin are known; most of these are 

 lactic acid bacteria or closely related organisms. 



The use of a strain of Lactobacillus casei for the quantitative determina- 

 tion of riboflavin was suggested by Snell and Strong"^ in 1939. The proce- 

 dure recommended has been widely and successfully used since that time, 

 both in its original form and in several slightly modified forms. Indeed, 

 the subsequent widespread use of microorganisms for quantitative assay 

 work was greatly stimulated by the success of this method. The original 

 assay medium is identical with that later recommended by the Association 

 of Official Agricultural Chemists. ^^ The medium adopted by the U. S. 

 Pharmacopeia^^ differs only in the concentration of glucose, which has been 

 increased from 1.0 to 3.0%. Procedural details, precision, reliabihty, and 

 specificity of the method are given in numerous treatises. ^^-^^ 



A medium based on peptone, glucose, yeast extract, acetate, and inor- 

 ganic salts and freed from riboflavin by treatment of the crude ingredients 

 with light, alkali, or lead acetate followed by hydrogen sulfide is used. 

 Growth of L. casei in this medium increases with the riboflavin concentra- 

 tion in the range from to about 0.20 y per 10 ml. of medium. Pure ribo- 

 flavin and samples to supply riboflavin at several levels within this range 

 are added to individual tubes containing 5 ml. of the double-strength me- 

 dium. Each tube is then diluted to 10 ml., capped, autoclaved, cooled, and 

 inoculated. Response of the test organism is customarily determined by 

 acid titration after 72 hours incubation at 37°. Turbidimetric estimations 

 of growth can be made as early as 24 hours. Interpolation of the response 

 obtained to several different known amoinits of sample onto the curve 

 obtained by plotting the responses to increasing amounts of pure riboflavin 



^° S. Orla-Jensen, N. C. Otte, and A. Snog-Kjaer, Zenlr. Bakteriol. Parasitenk., Abt. 



II, M, 434 (1936). 

 61 E. E. Snell, F. M. Strong, and W. H. Peterson, Biochem. J. 31, 1789 (1937). 

 «2 R. Kuhn, P. Gyorgy, and T. Wagner-Jauregg, Ber. 66B, 317, 576, 1034, 1577, 1950 



(1933). 

 63 E. E. Snell and F. M. Strong, Ind. Eng. Chem. Anal. Ed. 11, 346 (1939). 

 6< A. R. Kemmerer, /. Assoc. Offic. A(jr. Cheiuisfs, 27, 541 (1944). 

 6*U. S. Pharmacopeia 14th Revision, p. 752, 1950. 



66 E. E. Snell, in Vitamin Methods, Vol. 1, p. 327. Academic Press, New York, 1950. 

 6'' Association of Vitamin Chemists, Methods of Vitamin Assay, 2nd ed., p. 245. 



Interscience publishers, New York, 1951. 

 68 F. M. Strong, in Biological Symposia, Vol. XII, Estimation of the Vitamins, p. 



143. Jaques Cattell Press, Lancaster, Pa., 1947. 



