TYPHOID TOXIN 379 



in vacua to T Vth or -f^fh its original volume. The product when injected 

 intra-peritoneally in doses of O2 c.c. kills a guinea-pig weighing 300 

 grams. 



8. Toxin of Macfadyen and Rowland. The growth on agar is scraped off 

 and cooled to - 90 C. by means of liquid air, then triturated at a very low 

 temperature in a special apparatus. The product is diluted in normal saline 

 solution and centrifuged. The supernatant liquid is very toxic and is fatal 

 to guinea-pigs when inoculated intra-peritoneally in doses of O'l c.c. 



Bassenge and Mayer obtained a similar but less toxic product by freezing 

 the bacilli with liquid air and grinding them up in a hand mortar. 



9. Toxin of Balthazard. (a) Mode of preparation. The principle is the 

 same as that underlying Macfadyen and Rowland's method. 



A bacillus whose virulence has been increased by growing it in collodion sacs in 

 the peritoneal cavities of guinea-pigs, is sown on large surfaces of agar contained in 

 flat flasks (the agar is prepared with a 3 per cent, solution of Defresne's peptone 

 and contains no meat). After incubating for 24-48 hours the growth is scraped off, 

 mixed with a little normal saline solution and rapidly centrifuged. The deposit 

 is again shaken up with normal saline solution and centrifuged a second time. In 

 this manner all foreign matter is removed. 



The bacilli are then mixed with a 2 per cent, solution of urea or 1 per cent, solution 

 of ammonium chloride (the effect of these solutions is to swell and break the cells 

 and so facilitate the diffusion of the intra- cellular products). The emulsion thus 

 obtained is distributed into tubes which are completely filled and sealed in the 

 flame. 



To facilitate the diffusion of the intra-cellular products, the tubes are now alter- 

 nately frozen and thawed. They are kept at 58 C. for 8 days and daily submitted 

 to a temperature of -21 C. for a couple of hours in a refrigerating machine (the 

 evaporation of methyl chloride being adopted as the cooling agent). At the end 

 of 8 days the tubes are centrifuged for 24 hours. The bacilli collect at the lower 

 end of the centrifuge tube and the supernatant liquid carefully decanted constitutes 

 the toxin. 



(/?) Properties. Balthazard's method though lengthy and expensive 

 yields a very powerful toxin, containing products of the typhoid bacillus 

 unmixed with foreign substances. Inoculated sub-cutaneously in doses of 

 3 c.c. it kills rabbits weighing 2 kg. and in doses of 2 c.c. guinea-pigs weighing 

 200 grams. It is however less toxic than Conradi's toxin, which has the 

 further advantage of being more easily prepared. 



The action of Balthazard's toxin on animals generally is similar to that 

 of the toxins prepared by Sanarelli and by Chantemesse ; but its action on 

 rabbits appears to be more constant. 



10. Endotoxin of Besredka. Dried typhoid bacilli killed by heating for 1 

 hour at 60 C. are ground up with sodium chloride until an impalpable powder 

 is obtained. This powder is diluted with water added drop by drop and the 

 mixture left over-night. Next morning it is warmed in a water bath to 

 60-62 C. for 2 hours, and then allowed to settle. The supernatant liquid 

 contains the endotoxin. The average lethal dose for white mice is 0'05 c.c. 

 intra-peritoneally. The endotoxin is destroyed only by temperatures above 

 127C. 



11. Typho-lysin. As early as the second day filtered cultures of the typhoid 

 bacillus show distinct powers of haemolysis. This property increases with 

 the age of the culture up to the fifteenth day when it is at its maximum 

 (E. and P. Levy). Macfadyen and Rowland demonstrated the presence 

 of typho-lysin in an eight-day culture grown on macerated spleen. This 

 haemolysin of the typhoid bacillus is not destroyed at 55 C. 



The red cells of the dog are very sensitive to typho-lysin, and dogs which have 

 been repeatedly treated with heated cultures yield an antitypholytic serum. 



