440 Dr. G. Sims Woodhead [Feb. 8, 



shown.] Through this composite plug three tubes are passed into 

 the flask ; the two lateral tubes are bent at right angles, both inside 

 and outside the flask ; whilst the centre tube is fitted with a small 

 thistle-head, which may be plugged with cotton wadding, and then 

 closed with an indiarubber cap. The outlets of the lateral tubes are 

 also plugged with cotton wadding, and the whole apparatus is kept 

 for an hour or two in steam maintained at a temperature of 100° C. 

 (Flasks so treated may be preserved for years without any change, 

 beyond some slight evaporation, taking place in the broth.) A small 

 quantity of a pure broth culture of the virulent diphtheria bacillus 

 is now drawn into a long thin pipette, the indiarubber cap and the 

 cotton wadding plug are removed from the thistle-head, and the con- 

 tents of the pipette are introduced; the pipette is withdrawn, the 

 cotton wadding is replaced, the indiarubber cap is fitted in position, 

 and the flask is placed in an incubator which is maintained at the 

 temperature of the body (98° -4 F., or 38° -2 C), or better still a 

 degree or two below this. As soon as the growth is well started 

 (usually at the end of about 24 hours), a current of moist air is 

 made to pass continuously over the surface of this cultivating fluid, 

 the air being first warmed and saturated with moisture, in order as far 

 as possible to prevent evaporation. A fine flocculent deposit soon 

 makes its appearance on the bottom of the vessel, the supernatant 

 fluid remaining clear. This deposit increases in thickness, much 

 more luxuriant growth going on after the first ten days. Toxine is 

 formed by the diphtheria bacilli so long as they can grow freely — 

 that is, so long as they can obtain sufficient nutrient material from the 

 fluid and from the air that is continually passing over the surface. 

 At the end of three weeks, or even less, if all these precautions are 

 taken, the toxine should be of such a strength that T L of a c.c. (about 

 two or three drops) injected into a guinea-pig weighing 500 grammes 

 (over 17 ounces) will kill it within 48 hours. The strength of the 

 toxine or poison may be a little greater or a little less than this, but 

 it is a comparatively easy matter to measure the strength, and there- 

 fore to graduate the dose to be used in immunising the horse. This 

 only holds good, however, if the active diphtheria bacilli are removed 

 or destroyed ; these, if left in the fluid, would be a complicating and 

 inconstant factor in the equation. In order to kill these bacilli the 

 Germans recommend the addition of i per cent, of carbolic acid to 

 the culture ; the dead bacilli falling to the bottom, leave a perfectly 

 clear supernatant fluid. The French, on the other hand, recommend 

 the separation of the bacilli from the fluid by means of a Pasteur- 

 Chamberland filter. By this means a clear virulent poison which 

 does not contain any diphtheria bacilli is obtained. With this fluid, 

 a horse with a good constitution, and which has been proved to be 

 free from tubercle and glanders, is injected under the skin of the side 

 of the neck in front of the shoulder. Small doses are first injected, 

 either pure or with the addition of ^ of the volume of weak solution 

 of iodide of potassium. If the fluid is of full strength, only about 



