11 
TECHNIQUE EMPLOYED IN COUNTING BACTERIA IN VACCINE VIRUS. 
The virus was suspended in a measured quantity of sterile bouillon 
and agitated so that all the clumps were broken up and a uniform 
suspension obtained as nearly as possible. The dry points were first 
softened in a small quantity of bouillon and then rubbed clean, always 
using’ the usual bacteriological precautions to prevent contamination 
from the outside. The glycerinated virus was mixed with a small 
quantity of bouillon, and the capillary tube washed out by drawing 
the liquid in and out of the tube a number of times. 
The mixing was done in test glasses of appropriate size and the 
mixture thoroughly agitated. It is very important to agitate the sus- 
pension thoroughly in order to break up the clumps. This may best 
be done b}^ drawing the fluid into pipettes and blowing it out vigor- 
ously, repeating the operation a number of times. 
This suspension was now planted in agar and plated on Petri dishes. 
No less than three plates were made of each point or capillary tube; 
one or two drops of the suspension being planted in the first plate, 
five or ten drops in the second, and the total quantity remaining in 
the third plate. In this way the figures gave an accurate count of all 
the colonies that grew^ from each vaccine examined, excepting in 
those instances where the vaccine contained excessive numbers of bac- 
teria. In such cases it was necessary to estimate the total number from 
the first or second plate. 
The plates were grown in the incubator at 37° C., and the counts 
made when the maximum growth appeared, usually upon the third day. 
It is practicall}" impossible to determine the absolute number of 
organisms in a given virus, for it consists of an inflammatory product 
which is very variable in its physical characteristics. Upon dry 
points it coagulates into a hard, insoluble film; and mixed with 
glycerin it always contains little masses and agglutinated flakes and 
particles which hold enmeshed the micro-organisms. It is practically 
impossible to ultimately break up these masses. Therefore, the sus- 
pensions are not uniform and the counts we make are only approxi- 
mations. Micro-organisms have a well-known tendency to group or 
cling together, so that every colony upon an agar plate does not rep- 
resent the growth from one single microbe. The figures as given, 
therefore, are misleading only in that they give an underestimate of 
the number of organisms contaminating vaccine virus, and therefore 
some of the results, as bad as they are, do not fully represent the 
actual conditions. 
The capillary tubes containing glycerinated virus vary considerably 
in capacity; some hold three and four times as much as others. This 
fact partly explains the discordance in some of the figures, but it is 
not sufficient to justify the marked discrepancy which we have found 
existing between tubes from the same package and bearing the same 
laboratory number as given by the manufacturer. 
