INOCULATION WITH DILUTED VIRUS. 
107 
other diseases. With fowl cholera a cultivation of the virus was 
made in sterilized broth, the flask being arranged to admit filtered 
air. This was set aside for an indefinite period, varying from 
five to eight months, during which time the attenuation occurred. 
A second cultivation, made from this first one after this indefinite 
period, was the vaccinal virus. During this time the activity of 
the germs is gradually lessened, but the same degree of attenu¬ 
ation does not always occur in the same length of time ; and 
when preparing fresh vaccine the degree of attenuation must be 
determined by inoculation experiments. 
When the vaccine is once obtained it may be preserved for .a 
considerable time by frequently renewing the cultivations of it, 
but this cannot be done indefinitely, as Pasteur at first announced. 
This vaccine gradually loses its strength, no matter what pre¬ 
cautions are observed ; consequently it must from time to time 
be renewed, the setting aside for from live to eight months, and 
the testing of the activity, which involves much trouble and ex¬ 
pense, to say nothing of the skill required, all must be gone over 
again as at first. What makes this still worse, there must be two 
vaccines, one stronger than the other, because the weaker vaccine 
is too inactive to grant complete immunity. 
With charbon virus, the attenuation occurs more rapidly, be¬ 
ing complete in less than eight days for the weaker vaccine; but 
here there is also an obstacle to its preparation. The virus must 
be kept at a constant temperature of 42° to 43° C. to prevent the 
formation of spores. If spores are formed there is no attenu¬ 
ation, and the vaccine obtained will produce fatal effects. Koch 
and Klein have each attempted to show that virus could not be 
attenuated in this way; but they undoubtedly failed by allowing 
spores to form either by variation of the temperature, by the 
liquids being too shallow, or from neglecting to supply some 
other requiijed conditions. These experiments, while they can¬ 
not overthrow Pasteur’s general conclusions, are sufficient to 
demonstrate the difficulties and the uncertainties of the method 
when attempted by others less skilful than the originator. 
With this method of attenuation by dilution, as I have de¬ 
veloped it, most of these difficulties are avoided. The prepara- 
