BACTERIAL INOCULATION 219 



theria and tetanus, are more appropriately treated by 

 immune sera or antitoxins. 



Preparation of Bacterins. — The earliest, simplest 

 and commonest method of bacterin preparation con- 

 sists in washing off the appropriate media twenty- 

 four-hour — with certain bacteria the time is much 

 longer — pure cultures of the bacterium, using a plati- 

 num spatula. The growth is suspended in normal 

 saline solution, containing, if desired, 0.25 to 0.5 per 

 cent, phenol. After shaking for one-half hour in a 

 mechanical shaker (Fig. 20), the suspension is stand- 

 ardized to contain from 100,000,000 to 1,000,000,- 

 000 bacteria per cubic centimetre. This is best 

 accomplished by the hsemocytometer method, first 

 employed by one of the authors (Thomas) in 1906. 

 The bacterial suspension is drawn up in an ordinary 

 red blood-cell counting pipette to the mark 0.5, fol- 

 lowed by freshly filtered carbol-thionin, Leishman's 

 or Jenner's stain, sufficient to stain the bacteria. After 

 mild agitation for two or three minutes the bulb is 

 filled to the 101 mark, and after thorough dissemina- 

 tion of the bacteria a drop of the pipette content is 

 placed on the hsemocytometer stage and the micro- 

 organisms are comited precisely as are red blood-cells, 

 determining the number per cubic centimetre. It is 

 advisable to use a cover-slip not over 0.13 mm. thick; 

 otherwise there may be interference with the oil-im- 



