Microbic l)iseases Individually Considered. 287 



as gelatin and agar, are sometimes freed from air by- 

 boiling for half an hour; the mass is solidified by 

 rapid cooling, inoculated by deep puncture, and the 

 surface of the medium covered with a layer of ster- 

 ilized oil which prevents the absorption of the at- 

 mospheric oxygen. Deoxygenation can also be ac- 

 complished by the addition to the same media of sub- 

 stances which readily absorb oxygen. Kitasato ad- 

 vises the addition to gelatin or agar, of glucose two 

 per cent, sulpho-indigotate of soda 0*1 per cent, blue 

 turnsol five per cent. 



The most favorable temperature varies between 

 38° and 39°, but multiplication still occurs at 18° and 

 continues even at 43°. 



The natural inoculation substances at our disposal : 

 pus of wounds which have given rise to tetanus, earth, 

 forage, etc., are always contaminated with other germs 

 than those of this disease ; hence, impure cultures 

 are first obtained from which the bacillus of i^icolaier 

 must be isolated. With this aim we take advantage 

 of the great resistance of its spores, and heat the 

 sporulated cultures in a water bath at 80° to 90° for 

 three quarters of an hour to one hour ; traces of 

 these cultures are sowed in anaerobic tubes which are 

 then closed over the flame and the gelatin spread out 

 on their walls by a rotatory motion. 



The colonies which then develop are composed ex- 

 clusively of tetanus bacilli, as may be proved by in- 

 oculation. If some spores of the septic vibrio have 

 resisted the heat and produced a collateral vegetation, 

 we should then resort to the action of antiseptics ; 

 1 to 1000 sublimate solution kills the tetanus bacillus 



