62 DIFCOMANUAL 



Bartram and Black^ in an investigation of media for the isolation of the coli- 

 form group from raw, pasteurized, and certified milk, found Bacto-Violet Red 

 Bile Agar to be the most satisfactory solid medium for this work. Babel and 

 Parfitt^ studying media for the detection of Escherichia-Aerobacter in ice cream, 

 used Bacto-Violet Red Bile Agar and reported its superiority over other solid 

 media recommended for such work. Yale^-^ reported the use of Bacto-Violet Red 

 Bile Agar in making coli counts of ice cream and pasteurized milk. Fabian and 

 Hook^ used Bacto-Violet Red Bile Agar in establishing the count of Escherichia- 

 Aerobacter in a study of sanitary conditions of ice cream as served at the foun- 

 tain. Miller and Prickett^ published a note on the use of this medium in a prac- 

 tical case concerning recontamination of milk. The Violet Red Bile Agar counts 

 were completed within 24 hours after plating. This is a considerable saving in 

 time in comparison with the confirmed Brilliant Green Bile procedure which 

 requires a minimum period of 48 hours. Quinn and Garnatz^ used Bacto-Violet 

 Red Bile Agar for the coli-aerogenes count of frozen eggs. 



Bacto-Violet Red Bile Agar is a poured plate medium. It is recommended 

 that 15-20 ml. of medium and not more than 1 ml. of milk be used per petri 

 dish of 100 mm. diameter. After pouring the plates, the medium is allowed to 

 solidify. Many investigators prefer to cover the solidified agar with 3-4 ml. of 

 melted medium as recommended by "Standard Methods for the Examination 

 of Dairy Products."^*^ Plates are then incubated at 37°C. for 18-24 hours and 

 at the end of this time are examined by transmitted light. Organisms of the coli- 

 form group, due to their ability to ferment lactose, form purplish red subsurface 

 colonies, 1 to 2 mm. in diameter and are generally surrounded by a reddish zone 

 of precipitated bile. The plates should not be incubated longer than 24 hours, 

 inasmuch as the organisms whose growth has been suppressed may develop and 

 confuse the count. Best results are obtained if plates are not too heavily seeded 

 — the inoculum being diluted so that not more than 150 colonies will develop 

 per plate. 



In using this selective medium best results will be obtained if it is not sub- 

 jected to autoclave sterilization since organisms, not killed by the boiling re- 

 quired to dissolve the medium, will not form colonies during the 24-hour 

 incubation period. Following boiling to dissolve the medium completely, it is 

 ready for use. 



Saccharose in 1 per cent concentration may be added to isolation media, such 

 as Bacto-Violet Red Bile Agar to permit the detection of certain members of the 

 coliform group which ferment saccharose more readily than lactose. This prin- 

 ciple was described by Holt-Harris and Teague^^ and has been employed by 

 many other bacteriologists. 



To rehydrate the medium, suspend 41.5 grams of Bacto-Violet Red Bile Agar 

 in 1000 ml. of cold distilled water and heat to boiling to dissolve the medium 

 completely. Cool to 40-44° C. and pour into plates containing the inoculum. 

 After solidification of the medium a cover layer may be added, if desired. The 

 final reaction of the medium will be pH 7.4. 



One pound of Bacto-Violet Red Bile Agar will make 11 liters of medium. 



^ Standard Methods for the Examination ^ Proc. Intern. Assoc. Ice Cream Manu- 

 of Dairy Products, 9th Edition: 132: 1948. facturers, 2:30:1936. 



2 Standard Methods for the Examination '''Am. J. Pub. Health, 27:564:1937. 

 of Water and Sewage, 9th Edition: 228: 1946. ^ J. Dairy Science, 21:559:1936. 



3 Food Research, 1:551:1936. ^ J. Bact., 45:49:1943. 



* J. Dairy Science, 19:497:1936. 10 Standard Methods for the Examination 



^ Proc. Intern. Assoc. Ice Cream Manu- of Dairy Products, 9th Edition: 135:1948. 



facturers, 2:17:1936. ^ J. Infectious Diseases, 18:596:1916. 



