CLINICAL BACTERIOLOGY AND H/EMATOLOGY 



small flask or large test-tube until it reaches 80 C, as measured 

 by the thermometer; insert the sealed end of the pipette in the 

 water, and maintain the temperature for ten minutes. The 

 thermometer is to be kept in the water the whole of the time, 

 and the flame is to be taken away when the temperature rises 

 above 80 C., and reapplied when it falls below that point. 

 The exact temperature does not matter, and they are not killed 

 even if they are boiled for a minute or two. 



At the end of this time the pipette will con- 

 tain no living object other than spores. Break 

 off its point and insert it gently into the glu- 

 cose agar, taking care to keep exactly in the 

 axis of the tube, until the tip of the pipette 

 reaches almost to the bottom of the test-tube. 

 Withdraw the pipette gradually, blowing out 

 its contents as you do so. The spores of the 

 tetanus bacillus (if present) will now be inocu- 

 lated deep down in the medium, far away from 

 the air. To reduce the supply of oxygen still 

 further it is a good plan to melt some paraffin 

 (a hard candle answers perfectly) and pour a 

 layer an inch thick over the surface of the 

 medium; or vaseline which has been heated to 

 the boiling-point of water (to sterilize it) may 

 be used. 



The cultures thus made are to be incubated 

 for a few days at the body temperature. After 

 about forty-eight hours the growth begins to 

 appear in the deeper portions of the tube as a 

 series of delicate wavy outgrowths from the 

 central stab. This is very much better seen in 

 cultures ig gelatin, which is very slowly lique- 

 fied. These do not appear in the upper por- 

 tion of the medium, where the oxygen (which 

 diffuses down from the air, unless the tube has been sealed 

 with paraffin) hinders their growth. If the tube shows such 

 a growth, it should be submitted to a microscopic examina- 

 tion. It is a good plan to break the tube and to split up the 

 cylinder of medium with a knife; films are made from the 

 growth and stained as above. Spores are formed after about 

 thirty-six hours. 



FIG. 15. BUCH- 

 NER'S TUBE. 



