Methods of Making the Vaccine 275 



determination of its value can be estimated. This is done by cal- 

 culating the number of contained bacteria per cubic centimeter 

 of the fluid, and then either diluting or concentrating by means of 

 centrifugation until an appropriate result is reached. As the con- 

 centration by centrifugation is more difficult. than dilution it is best 

 to take care at the very beginning of the process not to add too much 

 fluid to the culture bottles for the purpose of washing off the culture. 

 Whatever dilution of the final product may be necessary is made by 

 the use of the 0.5 per cent, phenol solution. 



The most ready method of calculating the number of bacteria 

 in the fluid is that of A. E. Wright which will be found in the chapter 

 upon the "Calculation of the Opsonic Index." 



After having determined the number of bacterial cells per cubic 

 centimeter of fluid, dilution with the phenol solution is made until 

 single doses are contained in quantities easily injected into the 

 patient. As the doses vary with 'the particular organism to be 

 injected, the operator must calculate from the number of bacteria 

 in the fluid, how much solution must be added to constitute a dose. 

 Several doses of each desired size should be prepared. Quantities 

 of the dilutions containing single doses or a number of doses as may 

 be preferred are now transferred, by means of a sterile pipet, into 

 previously sterilized, appropriate sized "ampules" or glass bulbs 

 made for the purpose, and the necks sealed in a flame. 



The bacteria are, however, still alive, and though many of them 

 no doubt undergo autolysis in the phenol salt solution, it is necessary 

 to make certain that none remains ahve to infect the patient. 



The destruction of the vitality of the micro-organisms which is 

 the final step in the process of vaccine preparation is effected by ex- 

 posure to the lowest temperature that is known to be positively 

 destructive. As spore-producing micro-organisms may maintain 

 this vitality at temperatures beyond ioo°C., at, which the micro- 

 organismal substance as well as their products are altered by coagula- 

 tion and other destructive transformation, they are inappropriate 

 organisms to employ for purposes of vaccines, unless, through some 

 such ingenious means as was devised by Pasteur for the anthrax 

 bacillus, the production of spores can be prevented. 



With very few exceptions non-sporogenous bacteria are destroyed 

 by exposure for sixty minutes to a temperature of 60° C. Should any 

 escape destruction, they are probably so injured as to be incapable of 

 further injurious effect upon the human body. 



The destruction of the bacteria is, then, effected by heat: 



The ampules of vaccine are placed in some sufficiently commodious receptacle 

 filled with water, the heat Ijeing supplied by a flame below, and the temperature 

 determined by a thermometer whose bulb is at the center of the bath. When 

 small quantities of the vaccine are to be made for special cases, a large beaker 

 supported upon an asbestos plate upon a chemical tripod and heated by a 

 Bunsen's flame answers very well. The burner is allowed to heat the bath until 

 the proper temperature is reached, when it is removed. As soon as the tern- 



