292 R. J. GOODLOW, L. A. MIKA, AND W. BRAUN [VOL. 60 



METHODS AND MATERIALS 



Smooth and rough clones isolated from the C0 2 -requiring strain 6232 of B. 

 abortus were used throughout. For the preparation of inocula the growth from 

 a 24-hour modified tryptose agar slant culture (tryptose agar supplemented 

 with glucose, thiamine, and Fe — McCullough et at., 1947) was washed off with 

 sterile Gerhardt and Wilson (1948) synthetic medium (G-W medium) and ad- 

 justed in the Coleman spectrophotometer to 70 per cent light transmittance. 

 To each of a series of flasks containing 100 ml of G-W medium was added 1.0 

 ml of the adjusted suspension of organisms, and the cultures were then incubated 

 at 37 C under approximately 10 per cent C0 2 . At regular intervals throughout 

 the periods of observation samples for the determination of viable cell counts 

 were removed from the cultures, and serial dilutions, prepared in tryptose saline 

 (0.1 per cent tryptose, 0.5 per cent NaCl), were mixed with modified tryptose 

 agar. In addition, samples were streaked on "2-1" agar plates 1 for the deter- 

 mination of the percentage of nonsmooth variants (Braun, 1946). 



In the determination of the effects of the addition of old culture filtrates to 

 freshly inoculated smooth cultures, Seitz filtrates were employed. Ten ml of 

 such filtrates were added to 90 ml of G-W medium, inoculated with 1.0 ml of a 

 tryptose-saline suspension of smooth Brucella abortus, and incubated at 37 C 

 under 10 per cent C0 2 . 



One-dimensional ascending paper chromatograms of Seitz filtrates were pre- 

 pared according to the technique used by Home and Pollard (1948). The strips 

 were suspended in 82 per cent distilled phenol for 24 hours and then developed 

 with 0.1 per cent ninhydrin solution in 85 per cent butanol. 



RESULTS 



The growth of smooth B. abortus, 6232, in G-W synthetic medium (figure 1) 

 was characterized by a steady increase in viable cell counts until the fourth 

 day of incubation, after which the number of viable organisms steadily decreased 

 for a period of 6 to 7 days. After the tenth day of incubation a second rise in 

 viable cells occurred. This increase reached its peak on the twentieth day. The 

 second peak usually exceeded that of the initial maximum viable level. Examina- 

 tion of 2-1 agar plates revealed that during the first 10 days of incubation the 

 population was smooth. Coincidentally with the second rise in viable cells, there 

 was a steady increase of nonsmooth variants (predominantly rough types), 

 which, at the time of the second peak of increase in viable cells, frequently con- 

 stituted over 90 per cent of the population (table 1). 



The effects of adding to freshly inoculated smooth cultures Seitz filtrates of 

 24-day-old cultures of B. abortus, in which the original smooth population had 

 been replaced by nonsmooth variants, were then investigated. The addition of 

 10 per cent nitrate was found to produce a marked effect on the subsequent 



1 Composition of 2-1 agar: 2.5 per cent Difco purified agar; 1 per cent Difco peptone;. 

 0.5 per cent NaCl ; 0.5 per cent Difco beef extract ; 2 per cent glycerol ; and 1 per cent glucose . 

 Adjusted to pH 7.4 before autoclaving. 



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