330 MICROBIOLOGY 



bacteria. For this reason it is difficult to isolate. Kitasato 

 recommended the following method for isolating it : 



Inoculate slightly alkaline nutrient agar or glucose bouil- 

 lon with the tetanus-bearing material (pus or tissue from the 

 inoculation wound), keeping the culture under anaerobic 

 conditions for twenty-four to forty-eight hours at a tempera- 

 ture of 37 C., and, after the tetanus spores have formed, heat- 

 ing it for one-half hour at 80 C., to destroy the associated 

 bacteria. The spores are able to survive this exposure, so that 

 when anaerobic cultures are then made in the usual way the 

 tetanus colonies develop. When the tetanus bacilli are the 

 only spore-bearing bacteria present, pure cultures are readily 

 obtained ; when other spore-bearing anaerobes are present, the 

 isolation of a pure culture may be a difficult matter. It can 

 frequently be obtained in pure culture from the local lesion of 

 a guinea pig dead of tetanus after inoculation. 



Agar. In stick cultures, a growth occurs along the entire 

 needle tract, resembling in its ramifications somewhat a minia- 

 ture pine tree. On the surface, colonies present the appearance 

 of light fleecy clouds. Under magnification, they seem to be 

 a tangle of fine threads. 



Gelatin. Along the needle track the growth exhibits the 

 appearance of a cloudy linear mass, with prolongations radi- 

 ating into the gelatin on all sides (arborescent growth) . Lique- 

 faction takes place slowly and generally with the production 

 of gas. The colonies develop slowly and have a dense opaque 

 center surrounded by fine diverging rays. 



Potato. Growth is delicate and hardly visible. 



Serum. Its growth appears on serum very much as it does 

 on agar. 



Bouillon. This medium becomes cloudy within twenty- 

 four to thirty-six hours. Anaerobic conditions may be ob- 

 tained by removing the air by boiling and covering the surface 

 with a layer of sterile oil. It may be cultivated in ordinary 

 bottles by filling them full and tightly corking. It ferments 

 dextrose, producing acid and gas. The gas consists largely 

 of C0 2 but there are present volatile substances that give rise 



