ISOLATION OF THE BACILLUS. 381 



function as a saprophyte is unknown. From such sources 

 and from the pus of wounds in tetanus, occurring naturally 

 or experimentally produced, it has been isolated by means 

 of the methods appropriate for anaerobic bacteria. The 

 best methods for dealing with such pus are as follows : 



(1) The principle is to take advantage of the resistance 

 of the spores of the bacillus to heat. A sloped tube of 

 inspissated serum or a deep tube of glucose agar is inocu- 

 lated with the pus and incubated at 37 C. for forty-eight 

 hours, at the end of which time numerous spore-bearing 

 bacilli can often be observed microscopically. The culture 

 is then kept at 80 C. for from three-quarters to one hour, 

 with the view of killing all organisms except those which 

 have spored. A loopful is then added to glucose gelatine, 

 and roll-tube cultures are made in the usual way and kept 

 in an atmosphere of hydrogen at 22 C. ; after five days the 

 plates are ready for examination. Kitasato compares the 

 colonies in gelatine plates to those of the B. subtilis. They 

 consist of a thick centre with shoots radiating out on all 

 sides. They liquefy the gelatine more slowly than the B. 

 subtilis. This method of isolation is not always successful, 

 partly because along with the tetanus bacilli, both in its 

 natural habitats outside the body and in the pus of wounds, 

 other spore -forming obligatory and facultative anaerobes 

 occur, which grow faster than the tetanus bacillus, and thus 

 overgrow it. 



(2) If in any discharge the spore-bearing tetanus bacilli 

 be seen on microscopic examination, then a method of 

 isolation based on the same principle as the last may be 

 adopted. Inoculations with the suspected material are 

 made in half a dozen deep tubes of glucose agar, previously 

 melted and kept at a temperature of 100 C. After inocu- 

 lation they are again placed in boiling water and kept for 

 varying times, say for half a minute, for one, three, four, 

 five, and six minutes respectively. They are then plunged 

 in cold water till cool, and thereafter placed in the incubator 

 at 37 C., in the hope that in one or other of the tubes all 

 the organisms present will have been killed, except the 



