30 VACCINE AND SERUM THERAPY. 



suspensions of tubercle bacilli is attended by numerous difficul- 

 ties, because this organism when grown on artificial media forms 

 conglomerated masses. According to Wright and Douglas the 

 living tubercle bacilli are heated to 100 C. before breaking up 

 the clumps. Later Wright modified this techinque by heating 

 the bacilli to 100 C. on three successive days. The clumps after 

 this are ground up in an agate mortar or in a watch glass, two or 

 three drops of . 1 per cent salt solution being added at a time until 

 two or three c. c. have been added. One-tenth per cent salt 

 solution is used in making this suspension because with greater 

 concentration the bacilli are again clumped. Later Wright added 

 1 . 5 per cent salt solution in making up the emulsion of tubercle 

 bacilli because he found that this concentration is necessary to 

 prevent spontaneous phagocytosis. When the heated bacilli are 

 * thoroughly rubbed and suspended in 1 . 5 per cent salt solution 

 a homogeneous mixture, containing but few clumps and many 

 isloated bacteria, results. This mixture is then centrifuged at 

 high speed for about ten minutes. After this the supernatent 

 fluid is drawn ofT, and enough salt solution added to get the right 

 concentration of the emulsion. 



While this method gives a fairly homogenous suspension of 

 tubercle bacilli, still in an emulsion made in this way many of 

 the tubercle bacilli are broken up. In determining the opsonic 

 index it is necessary to count the number of bacilli taken up by 

 the leucocytes, and when there is fragmentation of bacilli it is 

 necessary either to count each fragment as one bacillus, or else to 

 determine the fractional part of a bacillus. Either of these 

 methods is most unsatisfactory. 



Sellard and Jeans have emulsified the living tubercle bacilli 

 in the same manner that has been used for the emulsification of 

 other bacteria. After this they have killed the bacilli by exposing 

 the emulsion to sunlight for a number of hours ten hours being 

 sufficient to kill all tubercle bacilli present. In such emulsions 

 they have gotten no spontaneous clumping, no fragmentation, nor 

 spontaneous phagocytosis. 



Walker has recommended a method, according to which Dor- 

 sett's egg medium is heavily inoculated with an actively growing 

 culture of the bacillus of tuberculosis. After fourteen to eighteen 

 hours of incubation at 37 C., salt solution is squirted over the 



