380 TETANUS. 



serum or a deep tube of glucose agar is inoculated 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 gelatin, 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 com- 

 pares the colonies in gelatin plates to those of the B. subtilis. 

 They consist of a thick centre with shoots radiating out on all 

 sides. They liquefy the gelatin 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. Inocu- 

 lations 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 inoculation 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 tetanus spores which can develop in pure 

 culture. 



(3) Some method of anaerobically making plates, such as 

 that of Bulloch or Novy, may be employed. The isolation of 

 the tetanus bacillus is in many cases a difficult matter, and 

 various expedients require to be tried. 



Characters of Cultures. -Pure cultures having been obtained, 

 sub-cultures can be made in deep upright glucose gelatin or agar 

 tubes. On glucose gelatin in such a tube there commences, an 

 inch or so below the surface, a growth consisting of fine straight 

 threads, rather longer in the lower than in the upper parts of the 

 tube, radiating out from the needle track (Fig. 131). Slow lique- 



