220 DIFCO MANUAL 



To rehydrate the basal medium, suspend 75 grams of Bacto-Pantothenate 

 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 are then autoclaved for 10 minutes at 15 

 pounds pressure (121°C.). Oversterilization of the medium will give unsatisfac- 

 tory results. 



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

 of final medium. 

 1 J. Biol. Chem., 156:21:1944. 



BAGTO-BIOTIN ASSAY MEDIUM (B419) 



Bacto-Biotin Assay Medium has the same composition as Bacto-Pantothenate 

 Assay Medium described immediately above, except that biotin has been omitted 

 and 0.002 g. calcium pantothenate included. 



Bacto-Biotin Assay Medium is a complete dehydrated medium for the assay of 

 biotin. It is free from biotin but contains all the other factors necessary for the 

 growth of Lactobacillus arabinosus 17-5 ATCC 8014. The addition of biotin in 

 specified increasing concentrations gives a growth response by L. arabinosus 17-5 

 which may be measured acidimetrically or turbidimetrically. 



The following procedure is recommended for the use of Bacto-Biotin Assay 

 Medium: 



Stock cultures of the test organism, L. arabinosus 17-5, are prepared by stab 

 inoculation of Bacto-Micro Assay Culture Agar. After 24-48 hours incubation at 

 35-3 7 °C., the tubes are stored in the refrigerator. Transplants are made at 

 monthly intervals, in triplicate. 



Inoculum for assay is prepared by subculturing from a stock culture of L. 

 arabinosus 17-5 to 10 ml. of Bacto-Micro Inoculum Broth. After 16-24 hours 

 incubation at 35-3 7 °C., the cells are centrifuged under aseptic conditions, and 

 the supernatant liquid decanted. The cells are resuspended in 10 ml. sterile 

 isotonic sodium chloride solution. The cell suspension is then diluted 1-100 

 with sterile isotonic sodium chloride. One drop of this latter suspension is then 

 used to inoculate each of the assay tubes (10 ml.). 



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 biotin at levels of 0.0, 0.025, 0.05, 0.1, 0.2, 

 0.3, 0.4, 0.5 and 1.0 millimicrogram per assay tube (10 ml.). 



The concentrations of biotin required for the preparation of the standard curve 

 may be prepared by dissolving 0.1 gram of biotin in 1000 ml. of distilled water 

 (100 micrograms per ml.). Dilute this stock solution by adding 2 ml. to 98 ml. 

 of distilled water. This solution is diluted by adding 1 ml. to 999 ml. distilled 

 water giving a solution of 2 millimicrograms of biotin per ml. This solution is 

 further diluted by adding 5 ml. to 95 ml. distilled water giving a final solution of 

 0.1 millimicrogram of biotin per ml. Use 0.0, 0.25, 0.5, 1.0, 2, 3, 4 and 5 ml. of 

 this final solution. The last tube is prepared by adding 0.5 ml. of the standard 

 solution containing 2 millimicrograms of biotin per liter. The stock solution of 

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

 at 2-6°C. under toluene. 



