'^,2 LOCAL ILSSLE REACIIVLIY 



inucli as 3 c;.c. pei kilo ol body wcighl, ol llic same material was 

 injected intravenously. When the preparations ^vere re-titrated 

 after short intervals ot time, in many instances there was observed 

 either a partial or comjDlete disappearance of the potency. The 

 irregularity of the results with the above descriljed methods of 

 ]:)rcparation, whilst demonstrating the possil:)ility of eliciting the 

 phenomenon, necessitated the development of a more reliable 

 jjrocedure. With this purpose in mind, the "agar washings" ( ul- 

 ture filtrates of meningococc us described below ^vere used. 



Meningococcus "agar washings" culture filtrates: 



Meningococcus is inoculated into one per cent rabbit blood 

 broth of pH '7.2-7.4. After t^venty to twenty-two hours' incuba- 

 tion, the supernatant broth cidtme, free of red blood cells, serves 

 as the inoculum. Three to 4 c.c. of the inoculum are pomed on 

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

 flasks. After twenty to twenty-two hoins' incid)ation, the growth 

 of each flask is washed off 'vvith 2-4 c.c. of 0.9 per cent NaCl solu- 

 tion containing 0.4 per cent phenol. The washings are then 

 pooled, centrifugated within the following one to two hours and 

 the clear supernatant fluid is filtered through Berkefeld "V" can- 

 dles, shortly after centrifugalization (Shwartzman, 1929/;) . 



In vie^v of the possibility of contamination during the process 

 of washing of the agar it seems advisable to pool the washings 

 of not more than 5 flasks, make culture controls on blood and 

 glucose agar plates and filter the collected ^vashings of each 5 

 K(jlle flasks through a separate candle. The following day the 

 filtrates of washings proxen as imcontaminated are pooled. These 

 precautions are especially warranted in the prej^aration of men- 

 ingococcus toxic substances because of more frequent contamina- 

 tions observed with this microorganism. The reasons for the 

 occurrence of the contamination is probably due to the use of 

 rabbit blood in the inocidum. The sterility of the filtrates shoidd 

 be controlled by subcultures on enriched media and incubated 

 for at least forty-eight hours, inasmuch as meningococcus may take 

 this period of time for its appearance. If the xvork is done cm 

 masse and many candles are used for filtration, whereby it may 

 pro\e diflicult to check the permeability of the filters, it is advis- 

 able to heat the fUtrates at 56° c. for fifteen miniues. thus de- 

 stroying the meningococcus cells that may pass through the fdters. 

 This temperature does not appreciably reduce the toxic titer. 



