The medium and inoculum were thoroughly mixed and incubated for 72 

 hours at 37°C. The tubes were then cooled, and the acid production 

 was measured by titrating with approximately 0,1 normal sodium hydrox- 

 ide to pH 6.8 using a Beckman -pH meter, model G. The same sodium hy- 

 droxide solution was always used for both the standard and the samples. 

 The micrograms of riboflavin per tube were then determined for each 

 level by reading the amount on the standard curve. The average value 

 per cc. was calculated and any result which was not within £ 10 percent of th« 

 average was not used. If at least three of the levels were not within 

 this range, the assay was not included in the final average for the 

 material. The formula for the calculation was: 



Average microgram Micrograms 



per cc. X cc X dilution x 1 - per gram 



wei^t of 



sample 



In order to determine the accuracy of the assay, a recovery deter- 

 mination VBS run with each batch of samples by adding a definite amount 

 of riboflavin to one of the unknown samples. The recovery sample and 

 the unknown were assayed at the same time. The amount of riboflavin 

 found in the recovery sample minus the ajnount found in the unknown was 

 compared with the amount added. Ninety to 110 percent recovery was us- 

 ually obtained. 



Niacin 



For the niacin assays Lactobacillus arabinosus was grown on the med- 

 ium recommended by Roberts and Snell (2). Good growth, low blanks, a 

 strai^t line standard curve, and excellent recoveries were obtained us- 

 ing this medium. The onlj^ modification was that 0.1 gram of additional 

 cystine and 0.1 gram of tryptophane per liter of medium were added. This 

 seemed to increase the growth of the organism. The culture was kept as 

 a stab culture on agar medium as recommended in (l): 2.5 percent yeast 

 extract, 0.5 percent dextrose, 0.5 percent anhydrous sodium acetate and 

 1,5 percent agar. The broth medium for the daily inoculum was the same 

 as that used for the riboflavin assay and the inoculum suspension was 

 prepared in the same manner. 



The samples for the niacin assays were extracted by the same method 

 as that used for the riboflavin assay so that both assays could be run. 

 on the same sample l/. Niacin samples extracted by the enzymatic method 

 were kept in the refrigerator for as long as five days to test the des- 

 truction of the vitamin during storage and there was no observable de- 

 crease in the niacin content during this time. Consequently, the niacin 



1/ Results on the unknown and recovery samples indicated that it was 

 also possible to extract the niacin by autoclaving with 100 cc. of 1 

 normal sulfuric acid or v;ith 100 cc.of water for 1/2 hour at 15# pressure. 



83 



