222 DIFCC MANUAL 



ditions and the supernatant liquid decanted. Suspend the cells from this culture in 

 10 ml. of sterile B12 Assay Medium USP in single strength, prepared by dis- 

 solving 4.75 grams of Bacto-Bi2 Assay Medium USP in 100 ml. of distilled 

 water. Centrifuge and decant the supernatant liquid. Again, suspend the cells in 

 10 ml. of the medium, centrifuge and decant the supernatant liquid. Repeat this 

 process a third time. Finally, resuspend the cells in 10 ml. of the sterile single 

 strength medium. Add 0.1 ml. of this suspension to 10 ml. of sterile suspension 

 medium and mix. The cell suspension so obtained is the inoculum. Inoculate 

 each tube aseptically with one drop of the inoculum. The use of such a diluted 

 inoculum gives more uniform results than a heavier inoculum. 



It is essential that a standard curve be constructed each time an assay is run, 

 since conditions of autoclaving, temperature of incubation, etc., which influence 

 the standard curve readings, cannot be duplicated exactly from time to time. A 

 standard curve is obtained by using Bacto-Bi2 Assay Medium USP with levels 

 of 0.0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 and 0.25 millimicro- 

 grams Vitamin B12 per assay tube (10 ml.) for the turbidimetric and acidimetric 

 determinations. Determinations are made in triplicate. 



Using Bacto-B22 Assay Medium USP the most effective range has proved to 

 be between 0.01 millimicrograms and 0.1 millimicrograms. 



The concentration of Vitamin B12 required for the preparation of the standard 

 curve may be prepared by adding the contents of one ampul containing 20 

 micrograms per ml. (1 ml.) in 99 ml. of 25 per cent alcohol in distilled water 

 giving the stock solution of 200 millimicrograms per ml. Further dilutions are 

 made as follows: 



A. Add 1 ml. stock solution to 99 ml. distilled water 

 (1 ml. = 2 millimicrograms) 



B. Add 2.5 ml. of A to 97.5 ml. distilled water 



(1 ml. = 0.05 millimicrograms). 



C. Add 1 ml. of A to 99 ml. distilled water 

 (1 ml. =z 0.02 millimicrograms). 



Use 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5 ml. dilution C per tube and for 

 the last tube use 5 ml. dilution B. If preferred, Vitamin Bjg may be supplied in 

 the form of a 0.1 per cent trituration of crystalline Vitamin B12 with man- 

 nitol (1 gram = 1 mg. Vitamin B12). Dissolve 0.2 grams in 1000 ml. dis- 

 tilled water to obtain the stock solution of 200 millimicrograms per ml. The 

 stock solution of Vitamin Bjg is stable for 2 months if stored at 2-6° C. under 

 toluene. 



Acidimetric determinations are made electrometrically using pH 7.0 as the end 

 point after 72 hours incubation at 30-37°C., but held constant to within 0.5°. We 

 recommend an incubation temperature of 35-3 7 °G. Turbidimetric determina- 

 tions are made after 20-24 hours incubation. The standard curve is then con- 

 structed from the values obtained. 



To rehydrate the basal medium, dissolve 95 grams of Bacto-B^g Assay Medium 

 USP in 1000 ml. of 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. Tubes are sterilized by autoclaving for 5 minutes at 15 lbs., 

 pressure (121°G.). Overheating of the medium will give unsatisfactory results. 



One hundred grams of Bacto-Bi2 Assay Medium USP will make 2.1 liters final 

 medium. 



1 Pharmacopeia of the United States, Third Supplement, XIV, Revision: 15:1951. 



