190 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS 
vaccines. So little is actually known of what vaccines may accom- 
plish in the body that it is impossible to answer this question definitely. 
It is desirable, however, to retain in the vaccine all possible antigenic 
properties which were possessed by the organism in the host. It is a 
well-known fact that certain kinds of organisms rapidly lose their 
ability to produce disease when they are grown for any length of time 
outside the body. Others retain their virulence for some time. This 
would appear to indicate that stock vaccines of the former would be 
unsatisfactory, while stock vaccines of the latter might be more success- 
ful. 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 10 cc. 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, ^^accines killed by small amounts of 
formaldehyde are said to be superior to heat killed vaccines. 
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 l)acteria 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 1 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 
