PREPARATION OF VACCINES. 133 



we have a handle with which to draw out the test-tube when heated 

 about i inch from the mouth in a blowpipe flame. Drawing this out, 

 we let it cool, and then filing the constricted portion we break it off 

 and seal it in the flame. By shaking up and down vigorously for 5 to 

 15 minutes, the bacteria are distributed evenly in the salt solution. 

 The sealed test-tube is then placed in a water-bath at 60 C. and heated 

 at this temperature for i hour. Again shake. The constricted sealed 

 end is again filed off and a few drops shaken out in a watch glass for 

 standardization, and at the same time a few drops are deposited on 

 an agar slant as a test for sterility. (Incubation for 24 hours should 

 not show growth). 



Wright found that by taking a definite quantity of blood and a 

 similar quantity of bacterial emulsion, mixing the blood and bacterial 

 emulsion, then making a smear and staining, it was possible to de- 

 termine the ratio of bacteria to red cells, and from this the number 

 of bacteria per cubic centimeter could be determined. For example, 

 if we find 3 bacteria to each red cell we should have 15,000,000 

 bacteria to i cubic millimeter. (There being 5,000,000 red cells to 

 the cubic millimeter.) As one cubic centimeter is 1,000 times greater 

 than i cubic millimeter, there would be 15,000,000,000 bacteria in 

 each cubic centimeter of such an emulsion, or vaccine, as it is 

 termed. The standardization may be made with a haemacytometer.* 



Having determined the strength of the stock vaccine, we should pre- 

 pare a dilute vaccine for injection. This is most conveniently carried 

 out by filling vials with 50 c.c. of salt solution, plugging with cotton, 

 then sterilizing in the autoclave. A sterile rubber cap is now drawn 

 over the mouth of the vial. Sterility is insured by plunging the rubber 

 cap and neck in boiling water. If the stock vaccine showed 5,000,000,- 

 ooo bacteria per c.c. and we desired to have a vaccine containing 200,- 

 000,000 bacteria per c.c., it would only be necessary to draw out 2 c.c. 

 of the salt solution by means of a sterile syringe needle inserted 

 through the rubber cap and replace it with 2 c.c. of the bacterial emul- 

 sion. Example : In introducing 2 c.c. of a vaccine containing 5 billion 



*This is best done by drawing up the vaccine to .5 with either the red or white 

 pipette, according to concentration, and then sucking up i to 10 dilute carbol 

 fuchsin to 1 1 or 101. Allow the bacteria to settle on the shelf for 10 minutes before 

 counting. Count as in making a red count. 



