DEHYDRATED CULTURE MEDIA 221 



Bacto-Biotin Assay Medium may be used for both turbidimetric and acidi- 

 metric analyses. Turbidimetric readings should be made after 16-20 hours at 

 35-3 7 °C. Acidimetric determinations are made after 72 hours incubation at 35- 

 37°C. The most effective assay range, using Bacto-Biotin Assay Medium, has been 

 found to be between 0.025 millimicrogram and 0.5 millimicrogram biotin. 



To rehydrate the medium, suspend 75 grams of Bacto-Biotin Assay Medium 

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

 cipitate 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 are then autoclaved for 10 minutes at 15 pounds pressure 

 (121°tll.). Oversterilization of the medium will give unsatisfactory results. 



One hundred grams of Bacto-Biotin Assay Medium will make 2.6 liters of 

 final medium. 



BACTO 



Bi2 ASSAY MEDIUM USP (B457) 



DEHYDRATED 



Bacto- Vitamin Free Biotin 0.000008 g. 



Casamino Acids 15 g. Niacin 0.002 g. 



Tomato Juice 10 g. />-Aminobenzoic Acid, Difco . 0.002 g. 



Bacto-Dextrose 40 g. Calcium Pantothenate 0.001 g. 



Bacto-Asparagine 0.2 g. Pyridoxine Hydrochloride . . . 0.004 g. 



Sodium Acetate 20 g. Pyridoxal Hydrochloride .... 0.004 g. 



Ascorbic Acid 4 g. Pyridoxamine Hydrochloride 0.0008 g. 



/-Cystine, Difco 0.4 g. Folic Acid 0.0002 g. 



^/-Tryptophane 0.4 g. Monopotassium Phosphate 1 g. 



Adenine Sulfate 0.02 g. Dipotassium Phosphate 1 g. 



Guanine Hydrochloride 0.02 g. Magnesium Sulfate 0.4 g. 



Uracil 0.02 g. Sodium Chloride 0.02 g. 



Xanthine 0.02 g. Ferrous Sulfate 0.02 g. 



Riboflavin 0.001 g. Manganese Sulfate 0.02 g. 



Thiamine Hydrochloride .... 0.001 g. Sorbitan Monooleate Complex . . 2 g. 



Bacto-Bi2 Assay Medium USP is prepared according to the formula given in 

 the United States Pharmacopeia.^ It is free from Vitamin B^g but contains all 

 the other factors necessary for the growth of Lactobacillus leichmannii ATCC 

 7830. The addition of Vitamin B^a to this medium in specified increasing concen- 

 trations gives a growth response by the test organism which may be measured 

 acidimetrically or turbidimetrically. 



The following procedure, or the procedure specified by USP is recommended 

 for the preparation of the inoculum for the assay of Vitamin 6^2 using Bacto-Bi2 

 Assay Medium USP: 



Stock cultures of L. leichmannii 7830 are prepared by stab inoculation of 3 or 

 more tubes of Bacto-Micro Assay Culture Agar. Transfers should be made bi- 

 monthly for stock culture. Before using a culture in the assay, make at least 10 

 successive transfers of the culture in Bacto-Micro Assay Culture Agar in a two- 

 week period. Incubate 24-48 hours at 35-37°C. Prepare fresh stab cultures every 

 second day and do not use them for preparing the inoculum if more than 4 days 

 old. Inoculum for assay is prepared by subculturing from a 24-48 hour stock 

 culture of L. leichmannii 7830 to 10 ml. of Bacto-Micro Inoculum Broth. After 

 16-24 hours incubation at 35-37°G., the cells are centrifuged under aseptic con- 



