284 STATE BOARD OF AGRICULTURE. 



milk, a comparatively simple process, the following factors must be con- 

 sidered: The medium and its proper preparation; the proper H-ion 

 concentration of the medium; the employment of an agent which will 

 eliminate fast-growing organisms ; the method of incubation. 



The proper medium for the isolation and cultivation of Bad. abortus 

 is liver or spleen (bovine or swine) infusion agar. This medium was de- 

 veloped by Stafseth (5), formerly of this laboratory. The technique 

 for preparing the medium is similar to the method described by Giltner 

 (6) for the preparation of meat infusion agar except that glass wool 

 sliould be used in filtering, and excessive heating should be avoided in the 

 preparation and sterilization. Huntoon (7) has shown that about half 

 of the initial growing value of media is removed by over-heating, and 

 employing cotton, cloth, or paper in filtration. The writer has found that 

 the above-named factors must be considered in the isolation and culti- 

 vation of Bad. abortus. 



The H-ion concentration of the medium is the most important fac- 

 tor in the cultivation of this organism. The H-ion concentration 

 for obtaining the optimum growth lies between 6.6 and 6.4. The limit- 

 ing H-ion concentratiun in which growth may be obtained lies be- 

 tween 6 and 7.6. The organism grows very slowly at the extreme 

 limits of this range. In fact, it is very difficult to get newly isolated 

 strains to grow on a medium having a different H-ion concentration 

 than the optimum stated above. The H-ion concentrations were in 

 each case determined colorimetrically, using Brom Thymol Blue as 

 indicator. The titratable acidity (using phenophthaleiu as indicator) 

 for obtaining the optimum growth should be about +1.2. This method, 

 however, is not to be relied upon owing to the fact that a medium 

 giving a titratable reaction of +1.2 may not correspond to the proper 

 H-ion concentration that is necessary for the growth of Bad. abortus. 



The agent necessary lor the partial elimination of other organisms 

 was found to be gentian violet. The selective bactericidal action of 

 gentian violet and its practical application Avas first studied by Church- 

 man (8). This dye, when incorporated in a medium, will inhibit the 

 growth of a large per cent, of gram positive and a small per cent, of 

 gram negative organisms. Churchman applied the term "violet posi- 

 tive," to those organisms whose growth was inhibited by the action of 

 the dye, and ''violet negative" to those which were not afl'ected by the 

 dye. The action of the dye appears to simulate the bactericidal action 

 of various other bactericides in that its action is fundamentally a 

 quantitative one. 



In this work, gentian violet was prepared in a saturated aqueous solu- 

 tion and incoii3orated in the media in sufficient quantity to give the 

 dye a final dilution of 1 :10,000. The growth of Bad. abortus was not 

 in the least affected by the presence of the dye. The developing colonies 

 take up the color of the dye and present a clear violet blue color to 

 transmitted light and a dark violet color to reflected light. With a 

 little practice, one may easily differentiate colonies of Bad. abortus 

 from colonies of other organisms on the media by macroscopic examina- 

 tion alone. 



From previous researches there have been developed two satisfactory 

 methods for growing Bad. abortus from naturally infected material, 

 namely, in a closed chamber in symbiosis with cultures of Bacillus sub^ 



