102 LOCAL TLSSUE REACTIVIIY 



gave rise to easily broken up ilakcs. The C^., slock strain grew only on the 

 l)oIloni ol ihe test tnl)e. the supernatant broth remaining perfectly clear. 



All the />'. t\l>li<).su.s stock stiains showed well pronounced motility. 

 IWeiity-lour hour cultmes of various strains on plain agar were washed 

 ill distilled water by centrifugalizalion and resuspended in 0.85 per cent and 

 1.4 per cent NaCl solutions. The suspensions were incubated in a water bath 

 at 37.5° c. for two hours and then placed in the refrigerator for twenty-two 

 hours. The C^r, stock suspension was completely agglutinated but the remain- 

 ing strains stayed in suspension. 



The various strains were cultivated in series in mixtures of homologous 

 immune sera with plain broth until rough variants were obtained. Some 

 rough variants employed here were recovered from cultures cultivated for a 

 number of generations in the presence of bacteriophage. 



Ihe various B. typhosus stock strains were also passed through mice in 

 order to increase the virulence of the strains and possibly enhance their 

 smoothness. The technique adopted for the purpose was as follows: 



A twenty-four hold old cgar slant of a given strain was emulsified in 0.85 

 per cent NaCl solution to a turbidity representing approximately one billion 

 organisms per cubic centimeter. One c.c. of the emulsion was injected intra- 

 jjeritoneally into two mice. Twenty-four hours later cultures were made on 

 Endo agar plates from the heart's blood of the dead or killed mice. The 

 twenty-four hour old growth on the plates was suspended to the above in- 

 dicated tuibidity in 0.85 per cent NaCl solution. One-half of 1 c.c. of the 

 emulsion was used for injection into a mouse. Only heart's blood cultures 

 were employed for passages. The strains passed through mice were desig- 

 nated "mouse" strains, with the name of the stock strain from ^\'hich they were 

 derived and a number indicating the number of passages. None of the stock 

 strains (including the Ty Tl strain) iniderwent any change in the appear- 

 ance of the colonies in spite of numerous passages. 



The different stock strains of B. typhosus and B. coli, the vari- 

 ants obtained by means of immune sera and bacteriophage and 

 the strains passed through mice were designated accordingly and 

 utilized as follows: 



Serum neutralizability of reacting factors of variants of 

 typhosus and B.coli : 



The toxic filtrates necessary for the plienomenon of local skin 

 reactivity -^vere "agar washings" filtrates. These filtrates were des- 

 ignated by their nimiber and the name of the strain employed 

 for their preparation. The "motise" strains did nc^t luidergo more 

 than one passage through artificial media previous to their use for 

 the preparation of toxic filtrates. The t^venty-four hour old 

 heart's blood cultmes on Endo agar plates were seeded into plain 

 broth. The plain brcjth properly diluted served as the inocidum 

 for the Kolle flask agar cultures necessary for the preparation of 

 the filtrates. 



