The Isolation of Brucella abortus from the Blood-Stream of Cattle. ∗

Abstract

Soule 1 has perhaps done the most extensive work on this subject. His method consisted of adding 50 cc. of whole blood to 450 cc. of glycerol-infusion-broth and then incubating under 10% carbon dioxide. More than 5000 ordinary “herd-run” cattle were studied. Two series of tests were made on each animal at intervals of approximately 6 months. In the first series of tests agglutinins were present in the blood of 2,237 of the animals but only 299 gave positive blood-cultures. In the second series agglutinins were present in 2,607 cases with 206 positive blood-cultures. There were 40 positive blood-cultures without concomitant agglutinins.

This cultural method evidently requires extreme aseptic precautions in order to prevent contamination of the cultures with extraneous organisms. In our use of this technic, contaminations with molds, spore-forming bacteria, and gram-positive cocci occurred in nearly two-thirds of our cultures and we isolated Brucella abortus but once in 75 attempts.

Because of these discouraging results we studied 4 other methods, using the blood of cattle and more extensively the blood of guinea pigs. Rainsford's 2 method of removing all the serum from a clot of blood and then culturing the clot was used as well as the technic of Boez and Robin. 3 The latter authors maintain that the bactericidal action of the blood is destroyed by mixing it with an acid citrate which prevents clotting and reduces the pH to approximately 5.5. This acidity will not kill the bacteria but does not allow their multiplication. The acidity is counteracted by cultivation in an alkaline medium at pH 8.3. The mixed blood and medium attain a pH of approximately 7.5 which is suitable for the growth of the majority of bacteria. Haring 4 has had success drawing blood into sufficient sodium citrate to prevent clotting, heating to 56°C. for 15 minutes and inoculating blood agar slants with the heated mixture. Stewart et al. 5 recommend the method of Massa and Battistini 6 : The blood is collected in liquoïde “Roche” (sodium polyanetholsulphonate), which is anticoagulant and destructive of leukocytes but not bacteria. Ten cc. of blood are drawn into 2 cc. of 1% aqueous solution of liquoïde. The mixture is incubated for 10 days and then transplanted to agar slants. Continued incubation of the blood and liquoïde mixture may be allowed and subcultures made at intervals.