496 THE CHOLERA VIBRIO 



left standing on the clot in the ice chest for a week before being decanted. In the 

 original method there was no serum in the culture medium, its addition being sug- 

 gested by the investigations of Brau and Denier (vide infra). 



Distribute the medium in Roux bottles 50 c.c. in each and heat the latter 

 with their contents at 60 C. for 3 hours to destroy the bactericidal substances in the 

 serum. 



Sow the medium liberally with 16 or 18-hour agar cultures and incubate 

 at 38 C. Growth takes place in the form of a thin pellicle and the contents 

 should be shaken daily for the first 4 days to increase the aeration. Filter 

 the culture on the seventh day (when the content of toxin is at its maximum) 

 first through paper then through a Chamberland filter. 



Brau and Denier state that a very constant yield of toxin can be obtained in the 

 following medium : 



Sterile Martin's gelatin-broth, - 45 c.c. 



Normal horse-serum (3 weeks old), - 45 



Defibrinated horse-blood (3 weeks old), - 10 



Mix and heat the mixture at 60 C. for 3 hours and distribute in Roux bottles. Sow 

 the medium freely and filter after incubating for 7 days, at 39 C. 



Properties of the toxin. The filtrate obtained is alkaline and has a charac- 

 teristic smell : it kills guinea-pigs in doses of Ol-0'3 c.c. per 100 grams of 

 body weight. The toxin is unchanged by heating at 100 C. for 20 minutes. 

 Its toxicity is diminished by exposure to air especially in presence of sun- 

 light but it retains its properties if stored in tubes which are exactly filled, 

 sealed in the blow-pipe flame and kept in the dark. Absolute alcohol and 

 ammonium sulphate precipitate the active principle from the solution. 



Action of the toxin on animals. Small or medium-sized guinea-pigs are 

 more susceptible to cholera toxin than any other animal : large guinea-pigs 

 are more immune. The toxin is equally virulent whether inoculated sub- 

 cutaneously or intra-peritoneally ; death takes place in 10-30 hours after 

 inoculation of medium-sized doses (1 c.c. sub-cutaneously or 0*3 c.c. intra- 

 peritoneally for guinea-pigs weighing 250 grams). By using a large quantity 

 of toxin a fatal result can be obtained in a few minutes, especially if it be 

 inoculated intra-peritoneally. 



The symptoms are similar to those which follow the inoculation of living cultures 

 of the organism, but the incubation period is shorter : immediately after inoculation 

 the temperature falls to below normal and continues low until death occurs, when it 

 may have fallen to 24 or 25 C. Post mortem, there is a little oedema at the site of 

 inoculation, a little fluid in the peritoneum, hyperaemia of the small intestine and 

 stomach and congestion of the abdominal viscera : the intestine is distended with 

 liquid diarrhceal matter. When the quantity of toxin inoculated is very small 

 there is a temporary rise of temperature followed by a fall to below normal and the 

 animal recovers. 



Rabbits are weight for weight more resistant than guinea-pigs : for a 

 rabbit the dose must be one-third greater than that required to kill a similar 

 weight of guinea-pig. A fatal result in adult rabbits can only be produced by 

 in tra- venous inoculation. 



Mice, rats, pigeons and fowls are almost immune. 



4. Vaccination. 



The ease with which laboratory animals can be vaccinated against the 

 cholera vibrio has for a long time stimulated attempts to vaccinate man. 

 Numerous methods have been described but only two, Ferran's and Haff- 

 kine's, have been applied on a large scale, and the evidence adduced as to 

 the value of these is based entirely upon statistics. 



I. Ferran's vaccine. Ferran demonstrated that guinea-pigs which have 



