218 DIFGO MANUAL 



from time to time. Using Bacto-Thiamine Assay Medium we have found the 

 most effective assay range to be between 0.005 and 0.03 microgram thiamine. 



The concentrations of thiamine required for the preparation of the standard 

 curve may be prepared by dissolving 0.1 gram of thiamine in 1000 ml. of distilled 

 water, giving a stock solution of 100 micrograms per ml. Dilute the stock solution 

 by adding 1 ml. to 99 ml. of distilled water. This solution is further diluted by 

 adding 1 ml. to 99 ml. distilled water to give the final solution. Use 0.0, 0.5, 1.0, 

 1.5, 2.0, 3, 4 and 5 ml. of this final solution per tube. The stock solution of 

 thiamine used for preparing the standard curve is stable for 2 months when stored 

 at 2-6° C. under toluene. 



After 20-24 hours incubation at 35-3 7 °G., L. jermentum 36 is capable of 

 utilizing the pyrimidine and thiazole moieties of the thiamine molecule. It is 

 essential, therefore, that the growth response be measured turbidimetrically prior 

 to this time. The tubes should be incubated at 35-3 7 °C. for not longer than 

 16-18 hours, and then placed in the refrigerator for 15-30 minutes in order to 

 stop growth. The growth can then be measured by any suitable nephelometric 

 method. 



To rehydrate the basal medium, suspend 85 grams of Bacto-Thiamine Assay 

 Medium in 1000 ml. distilled water and heat to boiling for 2-3 minutes. The 

 slight precipitate which forms should be evenly distributed by shaking. Five (5) 

 ml. of the medium are added to each tube in the preparation of the tubes for the 

 Standard curve and to each tube containing material under assay. For the assay, 

 each tube must contain 5 ml. of rehydrated medium, increasing amounts of the 

 standard or the unknown and sufficient distilled water to give a total volume of 

 10 ml. per tube. The tubes should not be autoclaved because of possible destruc- 

 tion of thiamine, but are steamed at 100°C. for 15 minutes. Overheating of the 

 medium will give unsatisfactory results. 



One hundred grams of Bacto-Thiamine Assay Medium will make 2.2 liters of 

 final medium. 



1 J. Biol. Chem., 155:153:1944. 



