160 PRACTICAL' METHODS IN IMMUNITY 



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

 to seven hours with streptococci and pneumococti, 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. 



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. Then pour the bacterial emulsion into 

 a sterile test-tube and repeat the process with three to five agar slants, until we have 

 from six 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 streptococcic or diphtheroid vaccines. 



The sealed test-tube is then placed in a water-bath at 60 C. 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 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 cubic millimeter. (There being 5,000,000 red cells to the 

 cubic millimeter.) As i cubic centimeter is 1000 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 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, 

 and then sucking up i to 10 dilute carbol fuchsin to n or 101. Allow the bacteria 

 to settle on the shelf for ten minutes before counting. Count as in making a red 

 count. 



A more satisfactory diluting fluid is that recommended by Callison. It is: 

 Hydrochloric acid 2 c.c., Bichloride of mercury (1-500 aq. sol.) 100 c.c., and sufficient 

 i% aqueous solution of acid fuchsin to color the diluting mixture a deep cherry red. 

 The diluting fluid should then 'be filtered. The bichloride forms an albuminate on 

 the surface of the bacteria which promotes rapid sedimentation and the fuchsin 

 stains the bacteria. 



