1 go ' PRACTICAL METHODS IN IMMUNITY 



Wright prefers Koch's T. R. or Neu Tuberculin in doses of from J 

 to J^oo rng. Some prefer Koch's more recent bazillen emulsion. In 

 case of other infections, however, and preferably with gonorrhceal in- 

 fections, the causative organism should be isolated from pus, sputum, 

 urine, blood, or other material from patient (autogenous vaccine). 



In the making of vaccines all media and apparatus should be sterilized with 

 scrupulous care to avoid the danger of tetanus infection. Having isolated the organ- 

 ism, it is inoculated upon one or more agar slants, and after a growth of from five 

 to seven hours with streptococci and pneumococci, or with eighteen hours for staphy- 

 lococci and colon, the growth on these inoculated slants is taken up with salt solu- 

 tion, thoroughly shaken up in the diluting solution and standardized. (Esmarch 

 roll of nutrient agar may be inoculated for growing cultures for vaccines.) 



The most practical way is to gently rub off the growth on the agar in about i or 



2 c.c. of salt solution with a platinum loop or sterile cotton swab. Then pour the 

 bacterial emulsion into a sterile test-tube and repeat the process with three to five 

 agar slants, until we have from 6 to 10 c.c. of the emulsion in the sterile test-tube. 

 By heating to melting-point in the flame a piece of glass tubing and attaching it to 

 the rim of the test-tube (also melted), 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 five to fifteen minutes, or 

 preferably in a mechanical shaker the bacteria are distributed evenly in the salt 

 solution. A piece of platinum wire, twisted into corkscrew shape, and fused in the 

 drawn-out end of the containing test-tube helps in breaking up the bacterial emulsion 

 and is a great aid in the preparation of streptococci or diphtheroid vaccines. 



The sealed test-tube is then placed in a water-bath at 6oC. and heated at this 

 temperature for one 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 twenty-four to forty-eight 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 three bacteria to each red cell we should have 15,000,000 

 bacteria to i c.mm. (There being 5,000,000 red cells to the cubic milli- 

 meter.) As i c.c. is 1000 times greater than i c. mm., there would 

 be 15,000,000,000 bacteria in each c.c. of such an emulsion, or vaccine, 

 as it is termed. 



The standardization made with a haemacytometer is best done by drawing up 

 the vaccine to 0.5 with either the red or white pipette, according to concentration, 



