66 LOCAL TLSSIE REACTIVITY 



before, wasliings ot young cultures on agar may prochue filtrates 

 potent in dilutions as high as 1:4000. 



Burnet (1931) compared the phenomenon-prodticing potencies 

 of "agar washings" filtrates and disintegrates made according to 

 Besredka's meth(jd and found that the latter were stronger. How- 

 ever, his experiments cotdd not be considered conclusive since 

 he did not make any cjuantitative titrations to the end-point for 

 comparative determinations of potencies of the various prepara- 

 tions. 



With this consideration in mind the following experiments 

 were done by me. 



Meningococcus Preparations. Meningococcus Group I, 44D 

 strain (Wadsworth) Avas inoculated into 1 per cent rabbit blood 

 i)roth pH 7.4. After 22 hours of incubation the supernatant broth 

 culture, free from red blood cells, served as the inoculum. On the 

 surface of 0.7 per cent glucose veal infusion agar in Kolle flasks 

 4 c.c. of the inocuhnn ^vere poured. After t^venty-foiu' hoins of in- 

 cubation the gro^vth of 10 Kolle flasks was each washed off with 

 4 c.c. of 0.9 per cent NaCl solution containing 0.4 per cent phenol. 

 The suspension ^vas centrifuged three times, and resuspended each 

 time in the same volume of 0.9 per cent NaCl solution containing 

 0.4 per cent phenol. Washings 1, 2 and <:] were the respective 

 supernatant fluids obtained after each centrifugalization. The 

 washed sediment ^vas then suspended again in 40 c.c. of 0.9 per 

 cent NaCl solution and disintegrated by freezing in dry ice and 

 tha^ving three conseciiti\'e times. The suspension of killed men- 

 ingococcus ^vas the meningococcus disintegrate employed in the 

 experiments about to he described. 



B. typhosus Preparatious. Three washings ^vere obtained from 

 B. typhosus cultures grown on 10 Kolle flasks, in a manner simi- 

 lar to the foreooinsf. The washed sediment, ho^ve\'er, was mixed 

 with 0.4 gm. of NaCl, which had previously been sterilized in 

 the autoclave at 250° f. for fifteen minutes and dried over cal- 

 cium chloride. The mixture '^vas dried in a vacuum desiccator 

 overnight, ground to a fine powder in an agate mortar and then 

 suspended in 40 c.c. of sterile distilled water, containing 0.4 per 

 cent phenol. This ^vas left overnight in the incubator and tiien 

 centriftiged for one hour. The supernatant fluid was called "Bes- 

 redka B. typJiosus disintegrate." 



Rabbits were prepared by single intradermal injections of 0.25 

 c.c. of undiluted material. Various dilutions of each of "agar 



