48 CLINICAL BACTERIOLOGY AND HEMATOLOGY 



mixed with some boiled water (previously cooled) and then 

 sucked up into the pipette ; the end of the latter is then to be 

 sealed in the flame, care being taken that the material itself is 

 not heated. 



Having filled and sealed the pipette, heat some water in a 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. 



At the end of this time the pipette will contain no living object 

 other than spores. Break off its point and insert it gently into 

 the glucose 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 inoculated 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. These do not 

 appear in the upper portion 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 culture is pure 

 — and this is not likely to be the case, as these wounds are 

 usually very highly contaminated — no gas will be produced by the 

 growth, whereas the bulk of the other organisms which are 

 likely to occur produce gas. A bubble or two in the course 

 of the growth, therefore, indicates that the culture is not pure 

 tetanus bacillus, but that organism may nevertheless be present. 

 If the tube shows such a growth, it should be submitted to a 

 microscopic examination. 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. 



