Riboflavin 



The samples were extracted by incubating overnight at 37°C. and 

 pH 4.5 with 30 rag. each of papain and takadiastase and a few drops 

 of toluene l/. The enzymatic method was used for tvro reasons: It 

 eliminated one pH adjustment 6ind the one sample could be used for both 

 the niacin and thiamine assays. The digested samples were filtered at 

 pH 4.5 to remove the fat and then were diluted to 100 cc. Aliquots of 

 the samples, except for the acetone extracted meals, the raw viscera 

 and meat samples, were washed with ether to ronove any remaining fat. 

 The samples were then diluted to approximately 0.1 micrograms per cc. 

 for the assay. 



The medium used for the early riboflavin assays was that recommend- 

 ed in (1) and contained alkali-treated peptone, cystine, yeast supple- 

 ment solution, mineral salts and dextrose. The medium suggested by 

 Roberts and Snell (2) contained enzymatic casein digest, crystallin* 

 vitamins and amino acids, mineral salts and dextrose. This was used for 

 both the riboflavin and niacin assays. The results of the riboflavin 

 analyses checked and the recoveries were cttoparable using both media. 

 Strai^t line standard curves were obtained in both cases. Acid produc- 

 tion in the blanks was neutralized with less than 2 cc. of 0.1 normal 

 sodium hydroxide. 



Lactobacillus easel was the culture used for the riboflavin assays, 

 and was carried as a stab culture on a medixm recommended in (l). This 

 consisted of 1.5 percent agar, 3 percent yeast extract, and 0,5 percent 

 dextrose. After Inoculation of the stab culture, it was incubated over- 

 night at 37°C., and then stored in the refrigerator. The stock culture 

 was transferred once a week. For use in an assay, the culture was in- 

 oculated into a broth tube containing 10 cc. of the complete medixim us- 

 ed for the assay. The broth tubes were incubated overnight at 37 C. The 

 cells were centrifuged down, the liquid decanted, and 10 cc. of sterile 

 physiological saline solution were used to resuspend the cells. A ster- 

 ile hypodermic syringe and needle were used to inoculate each assay tube 

 with this suspension. 



For the assay, 5 cc. of the basal medium of Roberts and Snell (2) 

 minus the riboflavin were put in each tube. The standard tubes were set 

 up Jn duplicate. Eight levels at 0.05 microgram intervals and ranging 

 from 0.00 to 0.4 micrograms of standard riboflavin were used. The aajn- 

 ples were run at five levels, but duplicate tubes were n.ot run. An at- 

 tempt was made to add samples of such a quantity that the tubes would 

 contain from 0.05 to 0.25 micrograms of the vitamin. After the differ- 

 ent amovints of sample were added the volume in each tube was adjusted to 

 10 cc. with water. The tubes were plugged, autoclaved at 15 pounds pres- 

 sure for 15 minutes, cooled, and inoculated with the inoculum suspension. 



1/ Extraction by autoclaving with 0.1 normal hydrochloric acid at 15 

 pounds pressure for 15 minutes was also satisfactory. 



82 



