DEHYDRATED CULTURE MEDIA 209 



the autoclave for 15 minutes at 15 pounds pressure (121°C.). The final reaction 

 of the medium is pH 7.9. 



One pound of Bacto-Mycin Assay Agar will make 19.6 liters of medium. 



1 The Compilation of Tests and Methods of 

 Assay for Antibiotic Drugs, Federal Security 

 Agency, Food and Drug Administration. 



BACTO 



TYROTHRIGIN ASSAY BROTH (B415) 



DEHYDRATED 



Bacto-Tryptone 10 g. 



Bacto-Dextrose 5 g. 



Disodium Phosphate 1-6 g, 



Bacto-Tyrothricin Assay Broth is prepared according to the U. S. Pharmacopeia 

 formula^ for the inoculum broth used in the assay of tyrothricin. The test or- 

 ganism, streptococcus sp., Lancefield group D, strain No. H69D5 (ATCC ^9854) 

 is carried in stock on Blood Agar prepared with Bacto-Tyrothricin Assay Agar. 

 Inoculation is made from the slant into Bacto-Tyrothricin Assay Broth and tubes 

 are incubated at 37°C. for 18-20 hours. The cultures are examined micro- 

 scopically at this time to check their purity. Bacto-Tyrothricin Assay Broth is 

 also recommended as a buffered Dextrose Broth for the cultivation of a large 

 variety of saprophytic and pathogenic microorganisms. 



In the assay of tyrothricin by the U. S. Pharmacopeia method, 80 ml. of the 

 sterile Tyrothricin Assay Broth is enriched with 20 ml. of a filter sterilized 5 

 per cent solution of bovine serum albumin at pH 6.8-7.2 to give the assay 

 medium. To 100 ml. of this mixture is added 1 ml. of an 18-20 hour culture of 

 the test organism. The Tyrothricin Reference Standard, and the sample under 

 assay properly diluted, are mixed with propylene glycol diluting solution in 

 5 ml. amounts to give 10 tubes with 0.0, 0.1, 0.15, etc., to 0.5 microgram tyro- 

 thricin. To each tube containing 5 ml. of the mixture of tyrothricin and propylene 

 glycol is added 5 ml. of the 1:100 dilution of the test organism in the assay 

 medium. Tubes are prepared in triplicate. The tubes are thoroughly shaken and 

 incubated at 37 °G., for 16-20 hours. The amount of growth is determined by any 

 suitable photometric method. The average value of the readings of each set of 

 triplicate tubes is used to obtain a single value for each tube. Prepare a standard 

 curve for the U. S. P. Tyrothricin Reference Standard by plotting the average 

 density measurements against the concentration of tyrothricin in micrograms per 

 ml. Similarly, prepare a curve from the average values obtained from the tyro- 

 thricin being assayed. From the average density of the control tubes, determine 

 the photometric reading equivalent to one-half the density of the controls and 

 interpolate this point on both of the above curves. Intercepts drawn from these 

 points to the base line will indicate the quantity of tyrothricin. 



To rehydrate the medium, dissolve 16.6 grams Bacto-Tyrothricin Assay Broth 

 in 1000 ml. of distilled water. Distribute in tubes and sterilize in the autoclave 

 for 15 minutes at 15 pounds pressure (121°C.). Avoid excessive heat during 

 the sterilization period and cool the medium rapidly. Final reaction of the 

 medium will be pH 7.0. To prepare the complete liquid medium for the assay of 

 tyrothricin add 20 ml. of a 5 per cent filter sterilized solution of bovine serum 

 albumin, adjusted to pH 6.8-7.2 with normal sodium hydroxide solution to each 

 80 ml. of sterile Bacto-Tyrothricin Assay Broth cooled to 45 °G., or less. 



One pound of Bacto-Tyrothricin Assay Broth will make 27.4 liters medium. 



1 Pharmacopeia of the United States, XIV Re- 

 vision: 650: 1950. 



