Pasteur devoted the remainder of his life to the study of this subject.  He collected some saliva from the mouth of a child, on December 11, 1880, who had died at the Hospital Trousseau four hours before.  This saliva he diluted with distilled water, and this mixture he injected into rabbits, and they all died, and the saliva taken from these rabbits when injected into other rabbits caused their death with rabies.  He found also that saliva from rabid dogs almost always caused the disease.  The incubation period varied within wide limits, and very often the animals lived.  He then used the blood of rabid dogs for inoculation, but these blood inoculations always failed to produce the disease.  Pasteur was convinced after careful study of rabid animals during the many months necessary to complete his experiments, that rabies was a disease of the nervous system, and that the poison (virus) was transmitted from the wound to the brain by the way of the nerve trunks.  Then to prove his theory Pasteur removed a portion of the brain of a dog that had died of rabies.  A part of this was rubbed up in sterile water and used to inoculate other animals; and subcutaneous inoculations with this material almost always produced death.

After this Pasteur tried a new method and injected directly into the nervous system, either into the nerve trunk or directly into the brain, after trephining, and all such injections produced rabies in the injected animal and death.  He also found that rabbits inoculated in the brain always died in the same length of time.  When he injected into the nerve trunk the inoculation period was longer, depending upon the distance from the brain.  Two problems now remained for Pasteur to solve, and these were, how could he obtain the definite virulence and how could he reduce the virulence regularly and gradually, so that it could be used by inoculation safely as a vaccine to produce immunity to rabies in healthy animals, and also to prevent the development of rabies in animals bitten by rabid animals.  He first tried successive inoculations.  These inoculations were made, after trephining, directly to the brain, and he used a portion of the brain as a virus each time.  He inoculated rabbit number one with a portion of brain taken from a rabid dog, and this rabbit died on the fifteenth day.  He then inoculated rabbit number two with a portion of the brain of rabbit number one; from the brain of rabbit number two the virus was supplied for inoculating rabbit number three, and thus the brain of each inoculated rabbit was taken, after its death, for material to inoculate the next rabbit in the series.  This experimentation showed him that each rabbit in the series died a little sooner, showing that the virus was becoming more virulent, till no increase in activity of the poison was shown after the fiftieth successive inoculation.  “Rabbits inoculated with a brain suspension of rabbit number fifty all died in seven days.”  This caused Pasteur to name the virus of number fifty “virus fixe,” a virus of definite length.  He now had obtained a virus of definite strength and the next question was, how could the virulence be gradually and definitely reduced.