196 ACTIVE IMMUNIZATION 



bacilli per c.c. of bouillon. The first point can be reached by passing 

 the organism through a few guinea-pigs, while the second has to be 

 tried out. Whether either condition is really imperative may be 

 questioned. To my mind it is more important that the vaccine 

 should be polyvalent, i. e., that it should represent a mixture of 

 a number of different strains. As medium for growth ordinary 

 1 per cent, peptone broth is used, which should be as nearly neutral 

 as possible. Equal quantities of this, in suitable flasks, are inocu- 

 lated and kept at body temperature for twenty-four to forty-eight 

 hours, after which they are mixed, care being taken that the neck or 

 the upper portion of the mixing flask is not soiled by the emulsion, 

 as otherwise some of the organisms may escape destruction when 

 the mixture is sterilized, which is the next step in the operation. 

 This is carried out in a suitable water bath, at as low a temperature 

 as possible; 60 C. is the usual temperature for this purpose, though 

 some writers advocate 52 C. Care should be taken that the water 

 in the bath stands at least as high as the culture in the flask, and 

 that the temperature corresponds to the contents of the flask and 

 not to the surrounding water. To this end a second flask, filled with 

 water, is placed alongside the culture flask and a thermometer sus- 

 pended in it. As soon as the temperature reaches 60 C., the flame 

 of the water bath is turned off. The flask remains in the hot water 

 for ten to fifteen minutes longer, and is then removed. After this 

 the sterility of the contents is tested by plating out a given quantity 

 (1 to 10 c.c., according to the amount, of material) in agar or by 

 inoculation of broth. The contents of bacteria is next determined 

 as follows: 



Determination of the Number of Bacteria (according to Wright). 

 A capillary pipette provided with a rubber nipple (Plate III, Fig. d) 

 is marked with a glass pencil about three-quarters of an inch from 

 the end, and is then charged with one volume, of blood (obtained by 

 puncture of the thumb near the root of the nail), one of the bacterial 

 emulsion and three volumes of saline (0.9 per cent.), the individual 

 portions being separated from one another by little air-bubbles. 1 

 Blood and bacteria are thoroughly mixed by repeatedly blowing 

 the contents of the capillary upon the slide and drawing them up 



1 With other bacteria, where the content may be much smaller, one may take 

 two or three volumes of the emulsion, instead of saline, due allowance being 

 made in the calculation by dividing with two, three, or four, as the case may be. 



