58 



Mr. E. W. A. Walker. 



firstly, that in the case of the motile bacilli, the non-motile phase, when 

 first obtained, often shows a tendency to revert very quickly to the motile 

 eu-agglutinable phase in bouillon culture ; and, secondly, because, when 

 rapid reversion does not occur, organisms in this group, and also in the 

 dysentery group, which have been grown in a succession of cultures in 

 dilutions of the homologous agglutinating serum, most often grow in early 

 bouillon subcultures in the form of a deposit with clear supernatant fluid. 

 Although these deposits may often shake up to form perfectly good sus- 

 pensions for formolisation and dilution to standard opacity, they yield on 

 other occasions suspensions in which many of the bacilli remain more or less 

 agglomerated. The latter suspensions are always undesirable for use in any 

 kind of observations on serum agglutination, and they were put aside as 

 quite unsuitable for experimentation, if, after the usual period of heating in 

 the water-bath, followed by 24 hours' standing at room temperature, there 

 was any deposit whatever at the foot of the control tube, or more than a 

 faint granulation to be seen (with a lens) in the fluid. 



B. typhosus. 



An actively motile agglutinable bouillon culture of B. typhosus (T.e.)* 

 was taken from a strain then in use, and a stock of formolised agglu- 

 tinable culture of standardised opacity was prepared from it. A subculture 

 was preserved in agar stab, and a subculture was made in the agglutinating 

 serum of rabbits immunised with T.e., diluted 1 to 9 bouillon. The 

 latter culture grew in the form of flocculi of agglutinated bacilli, which 

 sank to the foot of the tube. It was carried on at intervals of 48 hours in 

 small tubes in a succession of cultures in the diluted serum through nine 

 passages. The serum dilutions were tested for sterility before use by 

 incubation for 48 hours at 37° C. 



The successive subcultures were always made from the top of the fluid, 

 care being taken not to disturb the deposit. In the first and second 

 cultures this fluid looked clear ; but, in the later cultures, an increasing 

 turbidity of the fluid appeared, in addition to the flocculent deposit at the 

 foot of the tube. This appearance suggested that an increasing number 

 of the bacilli present were insusceptible to the agglutinating action of 

 the serum bouillon medium. Nevertheless, the microscope often showed 

 the presence of small clumps among the free bacilli. 



From the fifth and ninth serum bouillon culture ordinary bouillon cultures 

 were made, and, from these, subcultures (T.e. 5 and T.e. 9) were prepared 



* The same strain as that denoted T (E) in the paper by Gardner and myself already 

 referred to (4). 



