TOXIN 495 



Hueppe and Scholl grew the vibrio in the interior of fowls' eggs (p. 53, A.), the 

 object being to cultivate it under conditions similar to those obtaining in the human 

 intestine. After incubation the contents of the eggs were precipitated with alcohol 

 and the precipitate dissolved in sterile water ; this solution was highly toxic and 

 killed guinea-pigs in a few minutes. Gruber and Wiener however showed that this 

 result was in no way specific but can be produced by the sulphuretted hydrogen 

 developed in the cultures and by the alcohol used in the precipitation. 



II. Gamaleia maintains that there are several cholera toxins. This observer 

 grew the vibrio in calf's-foot broth for a fortnight at 37 C. and then left 

 the cultures standing for some days at the temperature of the laboratory to 

 allow the intra-cellular products to diffuse. This fluid is said to contain 

 two toxins : one thermolabile, producing diarrhoea in rabbits ; the other 

 thermostable, killing rabbits without any symptoms of diarrhoea. 



In Pfeiffer's view, the toxin is contained within the bodies of the vibrios 

 themselves (endotoxin) and only diffuses on the death and disintegration of 

 the organism : the toxins found in solution in cultures are according to this 

 observer merely a more or less modified product derived from the bodies of 

 the vibrios. 



Schmitz, Jurro, Blell, extracted a nucleo-protein from cholera vibrios 

 which was very toxic to guinea-pigs (p. 497). The procedure was similar to 

 that adopted by Lustig and Galeotti in the case of plague (p. 470). The 

 vibrios were dissolved in a 1 per cent, solution of potash and the solution 

 precipitated with acetic acid. 



Krawkoff by a different method extracted a similar nucleo-protein from 

 unfiltered broth cultures. 



III. Metchnikoff, Roux, and Taurelli-Salimbeni prepared a very powerful 

 soluble toxin which diffuses during the life of the organisms: Ransom 

 obtained similar results. 



Toxin of Roux, Metchnikoff and Taurelli-Salimbeni. 



Preparation of the toxin. 1. Select a highly toxigenic vibrio. 



It is best to isolate a vibrio direct from a patient suffering from cholera and one 

 which has not been passed through animals. Vibrios isolated from man are generally 

 very toxigenic, and to retain their virulence they should be sub-cultivated at room 

 temperature on agar but as seldom as possible. 



Vibrios which have been passed through the peritoneal cavities of guinea-pigs to 

 increase their virulence are generally only feebly toxigenic, but their toxin-producing 

 capacities can be augmented by passages in collodion sac cultures in guinea-pigs. 

 After filling a collodion sac with peptone water sow it with the vibrio (p. 175) and then 

 introduce it with aseptic precautions into the peritoneum of a guinea-pig ; leave 

 it for 48 hours and then inoculate the contents directly into the peritoneal cavity 

 of another guinea-pig : sow a second sac with the peritoneal exudate from this 

 second animal and repeat the operation several times, each intra- peritoneal 

 incubation in collodion sacs being alternated with direct inoculation into the peri- 

 toneal cavity. In this way the vibrio acquires such a degree of virulence that the 

 contents of the collodion sac rapidly kill a medium-sized guinea-pig when inoculated 

 intra- peritoneally in doses of 0'006 c.c. 



The virulence is maintained by making collodion sac cultures in the peritoneum 

 every third or fourth day. 



2. Sow the organism in the following medium : 



Martin's peptone solution, - 1000 grams. 



Gelatin, - 20 



Boil the solution to dissolve the gelatin, make absolutely neutral to litmus and 

 add 12 c.c. of normal soda solution. Heat at 115 C. for 20 minutes, filter through 

 Chardin paper, distribute in flasks and sterilize at 110-112C. for 20 minutes. 



When cool, add to the contents of each flask one-fourth its volume of horse serum. 

 The blood should have been collected 3 weeks previously and the serum should be 



