172 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS 



more successful. It is a safe general rule to state that an autogenous 

 vaccine is desirable. 



The preparation of vaccine is carried out as follows: 



1. Obtain pure cultures of the organisms from the lesion or what- 

 ever material is available. The details of culture vary with the type 

 of organism that is expected. 



2. Inoculation of the pure culture, or cultures in the event of mul- 

 tiple infection, in suitable media to furnish the desired amount of 

 growth. 



3. Removal of the growth, with sterile precautions, to a sterile 

 container, such as a test-tube containing sterile glass beads. This 

 is accomplished by washing the growth from the medium into sterile 

 saline solution : 5 to 1 c.c. of salt solution are required for an ordinary 

 agar slant culture. When enough growth is accumulated it is trans- 

 ferred to the sterile test-tube, being careful that no organisms con- 

 taminate the upper part, else they may escape sterilization. 



4. Sterilization: Heat the bacterial suspension in a water-bath. 

 Usually one hour at 60 to 65 C. suffices. Care must be taken that 

 the level of water in the water-bath is well above that of the level 

 of the suspension in the test-tube. 



5. Test sterility of the suspension. Inoculate suitable media and 

 observe the absence of growth. In skin infections it is sometimes 

 desirable to exclude the presence of the tetanus bacillus. 



6. Shake the suspension vigorously to distribute the organisms 

 uniformly in it. 



7. Standardize: Determine the number of bacteria in a cubic 

 centimeter. This is very simply accomplished by thoroughly mixing 

 equal volumes of freshly drawn blood and bacterial emulsion in a 

 pipette, spreading the mixture on a microscope slide, drying and 

 staining it with Wright's or Jenner's stain. Determine by actual 

 counting in a number of fields the proportion of bacteria to red cells. 

 Knowing the number of red blood cells in a cubic centimeter of blood 

 (5,000,000,000) and the proportion of bacteria to red blood cells, it is a 

 simple matter to determine the number of bacteria in the suspension. 



A more accurate procedure is to draw up one volume of vaccine in 

 the erythrocyte pipette of a hemocytometer, dilute to the 101 mark 

 with a dilute solution of fuchsin or other suitable stain, mix and 

 transfer to the counting chamber. An enumeration of the bacteria 

 is made in precisely the same manner that a blood count is made. 



