566 THE BACILLUS OF MALIGNANT (EDEMA 



After inoculation the vibrio multiplies and rapidly invades all the tissues of the 

 body : it is not therefore to be expected that its toxin will be as powerful as that of 

 micro-organisms which like the tetanus bacillus and the diphtheria bacillus only 

 grow at the site of inoculation. While the toxins of these latter organisms are 

 fatal to small laboratory animals in almost infinitesimal doses, filtered cultures of 

 the bacillus of malignant oedema have to be inoculated in doses of several c.c. to 

 produce a similar result. 



Roux and Chamberland by filtering through a porcelain bougie an emulsion 

 made with the muscles of guinea-pigs and rabbits which had died of the 

 disease obtained a liquid which proved fatal when inoculated in quantities 

 of 40 c.c. into the peritoneal cavity of a guinea-pig. 



(ii) Besson re-investigated the toxin of the bacillus. Cultures and the 

 exudate from animals which had just died of the acute experimental disease 

 were used after being filtered through a Chamberland bougie. 



(a) For cultures, it was necessary to select a medium favouring the forma- 

 tion of the largest quantity possible of toxin. Cultures in ordinary broth 

 were not suitable. Better results were obtained with cultures grown on 

 meat pulp. 



Besson's method for the preparation of the toxin. To 500 grams of minced beef 

 in a flask of 1500 c.c. capacity and plugged with wool add a few c.c. of a 1 per cent, 

 solution of soda and heat at 115 C. in the autoclave for 20 minutes. When cool, sow 

 the medium with a little of the exudate obtained from a guinea-pig which has died 

 as the result of the inoculation of a bacillus the virulence of which has been increased 

 by passage through guinea-pigs. For the cotton-wool plug substitute a sterile 

 india-rubber cork carrying two tubes, one of which dips into the contents of the flask 

 and outside is bent at an acute angle and terminates in a fine point : this tube serves 

 for the decantation of the toxin after incubation. The other tube only passes just 

 through the cork and outside is bent at a right angle, plugged with wool, and con- 

 stricted near the end. Attach the latter tube to the water pump and when a vacuum 

 is created seal the tube in the blow-pipe at the constricted part. Incubate the 

 flask at 37 C. After incubating about 20 hours numerous bubbles of gas burst 

 on the surface of the doughy mixture in the flask, the meat has a characteristic bright 

 pink colour and tends to divide into two layers, i.e. the culture consists of a semi- 

 solid irregular broken mass bathed in a reddish turbid fluid. A considerable amount 

 of gas is formed during incubation and if allowed to accumulate will check the growth 

 of the organism and prevent the formation of toxin ;. it is therefore advisable after 

 about 48 hours to break off the sealed end of the tube, when the gas, which has a 

 most offensive odour, will escape under considerable pressure. The tube need not 

 be sealed again because the amount of gas formed is sufficient to keep the culture 

 under anaerobic conditions. 



Experience has shown that the toxin content is greatest about the sixth day of 

 incubation and afterwards rapidly diminishes, so that the flask must then be taken 

 out of the incubator. Decant the liquid and press the solid portion in the meat 

 press. Add the juice obtained from the latter to the liquid and filter the mixture 

 through a Chamberland bougie. 



This toxin is more active than E-oux and Chamberland's. A dose of 

 3-5 c.c. injected into the peritoneal cavities of guinea-pigs weighing 450-600 

 grams produces symptoms similar to those seen in the last stages of the 

 septicsemic form of the disease but from which the animals rapidly recover. 

 Similar or larger doses inoculated into the sub-cutaneous tissues have much 

 less effect on the general condition and hardly any effect at all on the tem- 

 perature, but give rise to a local oedema or slough. 



Intra-peritoneal inoculation of doses of 5-10 c.c. is rapidly fatal to guinea- 

 pigs weighing 300-400 grams and the symptoms are not preceded by any 

 incubation period. 



Guinea-pigs and rabbits inoculated frequently with small doses of toxin 

 suffer as a rule from a chronic intoxication and ultimately die. 



