BIOLOGICAL PROPERTIES 467 



The Advisory Committee found that twenty-six passages from rat to rat 

 (M. rattus and M. decumanus), by sub-cutaneous inoculation without inter- 

 mediate culture, occupying in all 89 days, had no effect on the virulence 

 of the organism. Similarly its virulence was unaltered by passage through 

 rats by cutaneous inoculation without intermediate culture. These experi- 

 ments, however, demonstrated a varying susceptibility of the Bombay rat 

 to plague.] 



2. Bio-chemical reactions. 



[Media containing carbohydrates : The plague bacillus produces acid but 

 no gas in glucose, Isevulose, galactose, maltose, mannite and dextrin while 

 in lactose, saccharose, raffinose, sorbite, adonit, inulin, and dulcite no acid 

 is formed (MacConkey and others). 



[ The plague bacillus produces no change of colour in litmus milk. 



[In media containing sodium taurocholate the plague bacillus grows 

 well.] 



3. Toxin. 



Filtered cultures of the plague bacillus are only slightly toxic (Yersin ; 

 Calmette and Borrel). Markl showed that the toxin was adherent to the 

 bodies of the bacilli, so that in order to obtain it cultures several weeks old 

 [in which the toxin had diffused into the medium] had to be used. 



Roux prepared a very toxic product from a bacillus which had been 

 increased in virulence by growing it in collodion sacs in the peritoneal cavities 

 of guinea-pigs. This bacillus when grown in 0*5 per cent, gelatin-broth 

 yielded a toxin which killed mice in less than 12 hours when inoculated in 

 quantities of 0'014 c.c., but which had very little effect on rabbits and 

 guinea-pigs. Cultures grown in this way were macerated under toluol for 

 several weeks then filtered through paper and precipitated with ammonium 

 sulphate ; by these means a powder was obtained of which the fatal dose for 

 a mouse was O25 mg. 



Plague toxin is a very unstable substance. Its toxicity is diminished by 

 exposure to a temperature of 70 C. and is rapidly destroyed by light and 

 air. 



Besredka isolated a plague endotoxin by using a method similar to that adopted 

 by him for the preparation of typhoid endotoxin. The bacilli are dried and triturated 

 with salt, water is then added and the mixture allowed to macerate for 12 hours. 

 After centrifuging, the supernatant liquid contains the endotoxin. The latter is 

 thermolabile and is fatal to mice in doses of 0*006 c.c. 



[Rowland's toxin. Washing living plague bacilli with chloroform water 

 while killing the cell removes a certain amount of a nucleo-protein (substance 

 A) but only traces of the substances which are toxic and immunizing for rats. 

 Organisms which have been thus treated are toxic and possess immunizing 

 power for rats. By appropriate treatment (vide infra) a further nucleo- 

 protein (substance B) can be dissolved out which is more toxic and more 

 highly immunizing for rats than substance A and moreover bacilli from 

 which this substance has been removed are no longer toxic or immunizing 

 for these animals. 



[Preparation. The bacillus is grown on a neutral lemco-peptone-agar in Roux 

 flasks, incubated at 32 C. for 4 days and after being sterilized with chloroform 

 vapour the growth is emulsified in 10 c.c. dilute saline solution. The emulsion is 

 centrifuged and the deposit re-emulsified in salt solution, filtered through fine linen 

 and again centrifuged. This second deposit is pounded in a mortar with anhydrous 

 sulphate of soda until a dry powder is obtained. The powder is left in the ice-chest 

 overnight, then warmed to 37 C., well stirred and replaced in the ice-chest. The 

 freezing and thawing is repeated several times. Water is then added to make 



