MICHIGAN ACADEMY OF SCIENCE. 
127 
The salt solution containing the separated germs is transferred to an empty 
sterile test-tube and the neck of the tube sealed in the flame (see Fig. 25). 
When cool the tube is vigorously shaken, so as to thoroughly emulsify the 
bacteria and disintegrate any clumps that may have formed. After shaking, 
the tube is opened and a small portion (about one drop) taken out and re- 
served for the count. The tube is again sealed and placed in the water- 
bath, at 60° C., for such time as is required to kill all the bacteria contained 
in it — usually one-half hour is sufficient. 
The enumeration of the bacteria in the portion reserved for the count is 
done as follows: By means of an opsonizing pipette and nipple, take up one 
part of freshly drawn normal blood, one part of the vaccine, and two or three 
parts of physiological salt solution. These are blown out on to a slide and 
mixed in the usual way, and than a portion is spread out into a film. This 
film should be made very thin and uniform, the same as a film for a differen- 
Fig. 25. — The tube of 
bacterial suspension 
sealed ready to shake. 
Fig. 26. — Photomicrograph showing cross-hairs, bac- 
teria and red blood corpuscles. 
tial leucocyte count, and not like the smear described above for a phago- 
cytic count. The film is stained, and the number of bacteria and of red 
corpuscles in successive fields of the microscope are counted until at least 
200 corpuscles have been counted. As the number of red blood-corpuscles 
per cubic centimeter is five thousand million, the number of bacteria per 
cubic centimeter can be determined from the results of the counting by a 
simple arithmetical proportion (see Table II). If the bacterial vaccine is very 
dense, it is well to take one part of vaccine and a sufficient number of parts 
of normal blood, so that the number of bacteria does not differ greatly from 
the number of red blood-corpuscles. The vaccine can now be standardized 
by dilution to any required strength. The count of the concentrated vaccine 
divided by the count required in the dilute form will give the number of 
times it should be dilutee! (see Table II). The vaccine should be sterilized 
