426 PRINCIPLES OF VETERINARY SURGERY 



ISOLATION OF THE MICRO-ORGANISM.— In or- 

 der to isolate the microbe of tetanus Kitasato implanted 

 tetanic pus in serum. At a temperature of 36 to 38 degrees C. 

 he obtained impure cultures at the end of forty-eight hours 

 which contained numerous bacilli and foreign micro-organ- 

 isms. He submitted them to a temperature of 80 degrees 

 C. .for three-quarters of an hour to one hour, and found 

 that Nicolaier's bacillus alone resisted. Cultures made in 

 vacuum or in an atmosphere of hydrog'en are the on'y ones 

 that developed to show the agent of tetanus in pure colo- 

 nies. These colonies cultivated in serum or agar-agar and 

 incubated in a vacuum or in hydrogen show growth consti- 

 tuted only of the tetanus microbe, which, inoculated into 

 mice, produced the characteristic tetanus. 



By this process it is possible to isolate Nicolaier's bacil- 

 lus because it g'ives spores more rapidly than the other 

 bacteria which accompany it. The latter are killed at the 

 temperature of 80 to 90 degrees, while the tetanus spores 

 resist. It is owing to the great resistance of the spores that 

 the isolation occurs. Vaillard and Vincent cultivated tet- 

 anus pus in beef tea at 38 to 39 degrees, sheltered from the 

 air, for five to six days, and obtained growths of different 

 varieties of micro-organisms. With a sand bath they then 

 heated the culture to 100 degrees for one to two minutes 

 in a closed vessel. The spores of Nicolaier's bacillus re- 

 sisted and the other microbes were killed, and when culti- 

 vated on gelatine or hot bouillon pure cultures were ob- 

 tained. Sometimes a single heating did not destroy every 

 impurity, in which event a second or thirc^ operation was 

 necessary. The vibrio septic and the hay bacillus can 

 resist this temperature and remain mixed with the tetanus 

 bacillus. In order to isolate the latter recourse must then 

 be had to Roux's process of cultivation on gelatine or of 

 Vignal's method of separating anaerobic micro-organisms. 



