ANTITOXINS 911 



3. Prepare two emulsions: one to contain about 500 million bacilli in each cubic 

 centimeter (first dose), and the second 1000 million per cubic centimeter (second and 

 third doses). 



EXPERIMENT 43. PREPARATION OF STAPHYLOCOCCUS VACCINE 



1. Prepare two to six agar slant cultures of Staphylococcus aureus and grow for 

 twenty-four hours at 37 C. If a patient with furunculosis is available, make cultures 

 of pus and secure Staphylococcus, of which a vaccine is prepared. 



2. Proceed in the preparation of the vaccine as given in the text, placing each 

 dose in separate ampules, and so diluting that each dose is of one cubic centimeter 

 and contains 1000 million of cocci. Count by the method of Wright and by the 

 counting chamber method. 



EXERCISE 16. ANTITOXINS 

 EXPERIMENT 44. STANDARDIZING DIPHTHERIA ANTITOXIN 



1. Prepare a strong diphtheria toxin with the Park-Williams Bacillus No. 8, after 

 the technic given in the text. 



2. Secure some of the dried Standard Antitoxin and dilute so that 1 ex. is equal 

 to one immunity unit. 



3. With this antitoxin determine the L+ dose of the toxin prepared according to 

 the technic given, using six guinea-pigs and the following doses of toxin: 0.1 c.c., 

 0.12 c.c., 0.15 c.c., 0.18 c.c., 0.2 c.c., 0.25 c.c. 



4. Secure a sample of diphtheria antitoxin in the open market containing about 

 4 c.c. serum and 2000 units of antitoxin. If the titration given on the label is still 

 correct, one would expect about 500 units of antitoxin per cubic centimeter of serum. 

 Carefully remove 1 c.c. of serum and dilute with 19 c.c. salt solution (1:20). From 

 this stock dilution prepare the following dilutions (taken from Bulletin No. 21, 

 Hygienic Laboratory, M. J. Rosenau): 



1 c.c.+14 c.c. NaCl solution 1 c.c. = .00333 or ^ = 300 units per c.c. 



1 c.c. +16 c.c. NaCl solution 1 c.c. = .00294 or ^ = 340 units per c.c. 



1 c.c. + 18 c.c. NaCl solution 1 c.c. = .00263 or ^ = 380 units per c.c. 



1 c.c. +20 c.c. NaCl solution 1 c.c. = .00238 or ^ = 420 units per c.c. 



1 c.c. +22 c.c. NaCl solution 1 c.c. = .00217 or ^ = 460 units per c.c. 



1 c.c. +24 c.c. NaCl solution 1 c.c. = .002 or -gfa = 500 units per c.c. 



5. Mix 1 c.c. of these various dilutions with the L+dose of toxin; stand aside 

 for an hour and inject subcutaneously in median abdominal line of 250- to 300-gram 

 guinea-pig as per the technic already given. 



6. Carefully observe all animals for a period of four days at least. Autopsy 

 those that succumb, paying particular attention to the condition of the abdominal 

 wall and suprarenal glands. If the serum should contain less than 300 units of anti- 

 toxin per cubic centimeter of serum, the test should be repeated with lower dilutions. 



7. Inject 1 c.c. of the serum subcutaneously into a white mouse to test for excess 

 of preservative. It requires 1 c.c. of a 0.5 per cent, solution of tricresol or 0.5 c.c. of 

 a 0.5 per cent, phenol solution to kill a medium-sized mouse. If the mouse shows 

 trembling, it would indicate that the serum contains nearly this percentage of tricresol 

 (Bulletin No. 21, Hygienic Laboratory). 



8. Inoculate 1 c.c. of the serum in a flask containing 100 c.c. sterile neutral 

 bouillon. Incubate at 37 C. format least four days. This will test the sterility of the 

 product. 



