820 



BRUCELLA 



to fit the numerous observations on variation in the distance 

 of the band from the surface, and on the so-called double 

 zone phenomenon, in which two bands of growth, separated 

 from each other by apparently unaltered medium, are 

 visible. 



The demand for an increased partial pressure of CO 2 

 is particularly characteristic of abortus strains. Not all 

 strains, however, of this organism require it (see Smith 

 1924, 19266). The Southern Rhodesian strains (Bevan 

 1930), for example, grow quite well under ordinary 

 aerobic conditions. Moreover, even strains that need 

 extra CO 2 on isolation frequently become adapted, 

 after a variable time in the laboratory, to do without 

 it. The growth of many strains of Br. melitensis is 

 greatly benefited by incubation in an atmosphere of 

 5-10 per cent. CO 2, though some growth always occurs 

 in ordinary air. The porcine strains, both American 

 and Danish, appear to be least dependent upon CO 2. 

 The addition of this gas to the air never improves their 

 growth and sometimes actually inhibits it. How the 

 CO 2 acts is not definitely known. Alteration in the 

 H-ion concentration of the medium does not seem to 

 be the explanation. It has been suggested that the 

 gas passes through the cell wall and brings about a 

 change in the intracellular H-ion concentration or 

 oxidation-reduction potential, which is necessary for 

 the initiation of growth. As Gladstone, Fildes, and 

 Richardson (1935) have shown, CO2 seems to be required in a greater or less degree 

 by practically all bacteria, and presumably plays an important part in their 

 metabolism (see Chapter 3). 



Cultivation in the Presence of Dyes.— To Huddleson and Abell (1928) and 

 Huddleson (1929, 1931) we owe a valuable method of distinguishing between the 

 melitensis, abortus, and suis types, depending on their ability to grow in the presence 

 of certain aniline dyes. Without entering into the detailed technique of the method, 

 we may say that the general procedure is to prepare plates of liver agar, pH 6-6, 

 containing 1/30,000 and 1/60,000 thionin, 1/25,000 and 1/50,000 basic fuchsin, 

 1/50,000 and 1/100,000 methyl violet, and 1/100,000 and 1/200,000 pyronin. The 

 dyes used must be obtained from the National Aniline Chemical Company of New 

 York, or standardized against these dyes. The organisms are inoculated rather 

 heavily on to the plates, which are then incubated for 3 days aerobically, or in 10 per 

 cent. CO 2, according to the probable nature of the strains under examination. 

 Strains oiBr. melitensis usually grow to some extent in the presence of all four dyes ; 

 Br. abortus strains are inhibited by thionin, but grow freely in the presence of 

 the other three ; Br. suis strains grow well in the presence of thionin, but are in- 

 hibited by basic fuchsin, methyl violet, and pyronin. Though this is the 

 general behaviour of the three types, there is considerable variation between different 

 strains of the same type, especially those coming from different localities (Meyer and 

 Zobell 1932, Wilson 1933). Some strains oi melitensis, for example, may grow very 

 poorly on the thionin, methyl violet, or pyronin plates. Southern Rhodesian strains 



Fig. 174. — Br. abortus. 



Growth in form of band 

 situated 6-8 mm. below 

 the surface. Bang's 

 gelatin agar serum 

 medium, pH 7-0, incu- 

 bated aerobically. 



