extracts were often kept 2k hours after the extraction was completed 

 because more samples could be run by assaying the two \'ltanilns on sep- 

 arate days. It was not necessarj^ to wash the samples used for the nia- 

 cin assays with ether, but otherwise, they received the same treatment 

 as that described for the riboflavin assays. A greater dilution of 

 the samples was usually necessary for the niacin asoay. The standard 

 curve covered a sli^^tly wider range - 0.00 to 0.5 micrograms. An at- 

 tempt was made to run the levels of sanq^le between 0.05 and O.A-Tiraicro- 

 grams per tube. 



Thiamine 



The samples for the thiamine assay were extracted as follows l/: 

 75 cc. of 0.1 normal hydrochloric acid were added to 5 grams of sample 

 and heated for 1/2 hour on the steam bath. This was cooled and 5 nil. 

 of an enzyme solution containing 6 grams of takadiastase in 100 ml. of 

 2.5 molar sodium acetate were added. The final mixture was adjusted to 

 pH 4.5 - 5.0. A few drops of toluene were added and the samples incu- 

 bated overnight at 37°C. The samples were filtered and diluted to 100 

 cc. 



The fish meal and raw viscera extracts had to be run through 

 decalso tubes for purification and concentration. This step did not 

 have to be included for the meat samples bewuse the latter contained 

 more thiamine and less fluorescing materials than the fish waste ssimples. 

 The samples were prejmred aa described above. An amount of extract con- 

 taining from 3 to 10 micrograms of thiamine was put through decalso. 

 After the decalso was washed with three 10 cc. portions of hot water, ap- 

 proximately 20 cc. of hot acid potassium chloride solution (8.5 ml. of 

 concentrated hydrochloric acid per liter of 25 percent potassium chlor- 

 ide) were added and the eluate was collected in a 25 cc. volumetric flask. 

 This was made up to volume with acid potassivim chloride. Twenty-five cc. 

 of the standard solution containing 0.2 micrograms of thiamine per cc. 

 was also run throu^ the decalso each time and ccmpared with the stand- 

 ard solution which was prepared directly from the stock solution. There 

 Nas never any difficulty in recovering at least 92 percent of the stand- 

 ard from the decalso step. 



The reaction tubes for the thiochrome conversion were test tubes to 

 vrtiich standard tapers and stoppers had been fused so that there was no 

 difficulty shaking the reaction mixtures. Five cc. of the standard or 

 sample were added to each of two tubes. One tube ;vas used as a blank; 

 the other for the analysis. The two tubes were rim at the same time and 

 received the same treatment except that to one 3 cc. of alkaline potas- 

 sium feiricyanide solution (3 cc. of 1.0 percent potassium ferricyanide 

 per 100 cc. of 15 percent sodium hydroxide) were introduced and 15 ml. 

 of redistilled isobutyl alcohol added. To the second tube, the blank. 



1/ It was also possible to use the samples prepared by enzyme extrac- 

 tion for the microbiological assays. 



84 



