180 LABORATORY COURSE IN SERUM STUDY 



(c) Other Methods 



A number of other methods have been devised for standardization 

 of vaccines. 



One method (Hopkins) is to centrifugalize at high speed in a special 

 tube with graduated tip until the supernatant fluid is clear. The num- 

 ber of organisms for a number of species in such a closely packed 

 sediment has been determined, and is as follows : 



Staphylococcus aureus 0.01 c.c. equals 10 billion 



Streptococcus haemolyticus 0.01 c.c. equals 8 billion 



Gonococcus 0.01 c.c. equals 8 billion 



Pneumococcus (capsulated) 0.01 c.c. equals 2.5 billion 



B. typhosus 0.01 c.c. equals 8 billion 



B. coli 0.01 c.c. equals 4 billion 



Another method of standardization is by the turbidity of the emul- 

 sion. Standard tubes of carefully counted suspensions are prepared, 

 and the vaccine to be standardized is then diluted until the turbidity 

 is equal to that in the standard tube. 



III. STERILIZATION 



The most reliable means of sterilization is by means of heat. For 

 staphylococci and streptococci 59 to 60 C. for half an hour is satis- 

 factory ; for typhoid bacilli 55 to 56 for an hour is usually used. In 

 heating, the bacteria should be placed in a sealed tube and the entire 

 tube immersed in the water bath. After exposure to heat test the steril- 

 ity of the vaccine by transferring two or three loopfuls of the suspen- 

 sion to an agar slant. 



IV. DILUTION 



The stock suspension prepared is as a rule too concentrated to allow 

 the accurate measurement of the dose desired, which may vary accord- 

 ing to the organism from 20 to 500 millions or even more. After 

 the vaccine has been sterilized by heat, sufficient 5 per cent phenol 

 or an equivalent quantity of other aromatic disinfectant should be 

 added to bring the content of phenol in the mixture up to 0.5 per cent. 



Each student will prepare a suspension from the cultures 

 given out, standardize this suspension by methods (a) and (ft), 

 heat at 59 for half an hour, test for sterility, and prepare two 

 dilutions, one containing 200 million and one containing 1000 

 million per c.c. 



