DEHYDRATED CULTURE MEDIA 111 



or plates as desired, under aseptic conditions. Tubes are slanted in the desired 

 position and medium allowed to solidify. 



One pound of Bacto-Peizer TB Medium Base will make 11.8 liters of medium. 



^Am. J. Clin. Path., 20:682:1950. 2 Am. J. Clin. Path., 21:982:1951. 



BACTO 



TRYPTOSE AGAR (B64) 



DEHYDRATED 



Bacto-Tryptose 20 g. 



Bacto-Dextrose 1 g. 



Sodium Chloride 5 g. 



Bacto-Agar 15 g. 



Thiamine Hydrochloride 0.005 g. 



Bacto-Tryptose Agar is recommended for the isolation, cultivation and differ- 

 entiation of the Brucella. "Standard Methods for the Examination of Dairy 

 Products"^ and "Diagnostic Procedures and Reagents"^ of the American Public 

 Health Association, recommend this medium for this purpose. Bacto-Tryptose 

 Agar is also recommended as a general medium for the cultivation of a large 

 variety of pathogenic organisms, especially the streptococci. 



Isolation or cultivation of organisms of the Brucella group was originally ac- 

 complished by means of Liver Infusion Agar as recommended by Stafseth,^ and 

 Huddleson, Hasley and Torrey.* Completely satisfactory results were not con- 

 sistently obtained on this medium due to variations of the infusions from liver 

 tissue. Because of these irregular results on Liver Infusion Agar, an extensive 

 investigation of media for the propagation of Brucella was undertaken in our 

 laboratories in cooperation with Dr. Huddleson. This investigation led to the 

 development of a new medium^ which is excellently adapted to the nutritional 

 requirements of the Brucella, and which is satisfactory for both primary isolation 

 and routine propagation of these organisms.^ This medium is prepared with 

 Bacto-Tryptose as the sole source of nitrogen. The suitability of this peptone for 

 cultivation of the Brucella is demonstrated by the fact that, in a 2 per cent 

 solution of Bacto-Tryptose containing 0.5 per cent sodium chloride and 0.1 per 

 cent Bacto-Dextrose, growth is obtained from small inocula in 24 hours, w^hile 

 a much larger inoculum and an incubation period of 7-10 days are required to 

 obtain similar growth in Liver Broth. 



For isolation of Brucella strains from infected milk, where Gram-positive con- 

 taminants are present, crystal violet (gentian violet) is used in a final concen- 

 tration of 1 to 700,000 in Tryptose Agar. This amount of dye is sufficient to 

 suppress practically all Gram-positive organisms which might otherwise inter- 

 fere. To secure the proper concentration, 1.4 ml. of a one-tenth per cent solution 

 of Bacto-Crystal Violet should be added to each liter of Tryptose Agar before 

 sterilization of the medium. The sterile medium is poured into sterile petri 

 dishes, solidified and then inoculated. Each plate receives 0.1-0.2 ml. gravity 

 cream spread upon the surface of the medium by means of a sterile glass rod 

 with a right angle bend. The spreading of the inoculum is readily accomplished 

 by rotating the plate in a horizontal plane. 



The inoculated plates are incubated for 5 days at 37 °C. in an atmosphere of 

 10 per cent carbon dioxide. At the end of this period Brucella generally appear 

 as purplish, smooth, hemispherical colonies about 1-5 mm. in diameter, and are 

 transparent to transmitted light. Occasionally the colonies may be flat instead of 



