112 DIFGO MANUAL 



convex. Streptococci, which are the usual interfering contaminants, form large, 

 opaque, spreading, rough colonies slightly purple in color and not at all to be 

 confused with those of the Brucella. 



If the original plate contains colonies of other bacteria or molds, typical 

 Brucella colonies should be purified by transfer to Tryptose Agar slants or to 

 another plate of the crystal violet medium. The organisms should be properly 

 identified and the species determined. If aerobic types of Brucella such as suis 

 or melitensis, are present in the milk, their growth will not be inhibited by in- 

 cubating the inoculated plates in an atmosphere of 10 per cent carbon dioxide. 



Bacto-Tryptose and Bacto-Tryptose Agar are also particularly well suited to 

 the isolation of Brucella from the blood. During 1937, Huddleson^ had an oppor- 

 tunity to study the use of Bacto-Tryptose in an enrichment medium, and Bacto- 

 Tryptose Agar in the isolation of Brucella melitensis from 55 cases of undulant 

 fever on the Island of Malta. Of the total number of cases, 38 were febrile and 

 17 were afebrile at the time the blood was cultured. Positive cultures were ob- 

 tained in 32 cases of the former group and 5 of the latter. Growth appeared 

 within 4 days in 23 of the cultures. One culture required 18 days of incubation 

 before a positive subculture was obtained. 



Briefly, the procedure recommended by Huddleson^ is as follows: An enrich- 

 ment broth, composed of 2 per cent Bacto-Tryptose, 0.5 per cent sodium chlo- 

 ride and 1 per cent sodium citrate, or Bacto-Tryptose Broth, page 102, with 1 

 per cent sodium citrate added, is prepared and distributed in 20 ml. amounts 

 in 50 ml. serum vials closed with rubber diaphragm stoppers. The vials are then 

 autoclaved for 15 minutes at 15 pounds pressure (121°C.). The air in the vial 

 is replaced with carbon dioxide by puncturing the diaphragm with a 23-gauge 

 needle, removing the air and replacing it with carbon dioxide before introducing 

 the blood. In a personal communication Huddleson recommended the use of 

 10 per cent carbon dioxide rather than the 25 per cent originally specified. 

 Immediately after collection from the patient, the medium is inoculated with 

 2 to 5 ml. of blood by puncturing the stopper wdth the same needle used in col- 

 lecting the blood sample. The vial is shaken vigorously to prevent clotting. 



The vials are then incubated at 37 °G. At the end of every fourth day the 

 culture is mixed by shaking, and 0.5 ml. of the contents is removed by means 

 of a sterile 1 ml. syringe and needle and inoculated on a petri plate of Tryptose 

 Agar. The plate is incubated under 10 per cent carbon dioxide for 4 days. If no 

 growth is obtained from the blood culture within 20 days, it may be discarded. 



A convenient method for establishing an atmosphere of 10 per cent carbon 

 dioxide is that described by Thompson'^ in which a solution of sodium bicar- 

 bonate is mixed with sulfuric acid directly in the container. When a molar solu- 

 tion (84 grams per liter) of sodium bicarbonate is mixed in equal parts with 

 dilute sulfuric acid (1 ml. concentrated acid in 29 ml. distilled water), 22.4 ml. 

 carbon dioxide are liberated for each milliliter of bicarbonate solution. Calcula- 

 tion of the cubic contents of the container in which the cultures are incubated 

 will indicate the quantity of each solution required to create a carbon dioxide 

 concentration of approximately 10 per cent. For example, for a container having 

 a net volume of 1000 ml., one would use 4.5 ml. of each reagent. The solutions 

 are preferably introduced separately into the container and are mixed after it 

 has been sealed. A similar procedure is also described in detail by Shaughnessy.^ 



A satisfactory carbon dioxide tension can also be supplied by one of the fol- 

 lowing procedures: 



1. Replace about 10 per cent of the air in the container with the gas from a 

 tank of liquid carbon dioxide. 



2. Place a lighted smokeless candle near the top of the container with the 

 plates, and seal the container. 



