A method was therefore found by which rabies was contracted surely and swiftly. Trephinings were again performed on chloroformed animals—Pasteur had a great horror of useless sufferings, and always insisted on anæsthesia. In every case, characteristic hydrophobia occurred after inoculation on the brain. The main lines of this complicated question were beginning to be traceable; but other obstacles were in the way. Pasteur could not apply the method he had hitherto used, i.e. to isolate, and then to cultivate in an artificial medium, the microbe of hydrophobia, for he failed in detecting this microbe. Yet its existence admitted of no doubt; perhaps it was beyond the limits of human sight. “Since this unknown being is living,” thought Pasteur, “we must cultivate it; failing an artificial medium, let us try the brain of living rabbits; it would indeed be an experimental feat!”
As soon as a trephined and inoculated rabbit died paralyzed, a little of his rabic medulla was inoculated to another; each inoculation succeeded another, and the time of incubation became shorter and shorter, until, after a hundred uninterrupted inoculations, it came to be reduced to seven days. But the virus, having reached this degree, the virulence of which was found to be greater than that of the virus of dogs made rabid by an accidental bite, now became fixed; Pasteur had mastered it. He could now predict the exact time when death should occur in each of the inoculated animals; his predictions were verified with surprising accuracy.
Pasteur was not yet satisfied with the immense progress marked by infallible inoculation and the shortened incubation; he now wished to decrease the degrees of virulence—when the attenuation of the virus was once conquered, it might be hoped that dogs could be made refractory to rabies. Pasteur abstracted a fragment of the medulla from a rabbit which had just died of rabies after an inoculation of the fixed virus; this fragment was suspended by a thread in a sterilized phial, the air in which was kept dry by some pieces of caustic potash lying at the bottom of the vessel and which was closed by a cotton-wool plug to prevent the entrance of atmospheric dusts. The temperature of the room where this desiccation took place was maintained at 23° C. As the medulla gradually became dry, its virulence decreased, until, at the end of fourteen days, it had become absolutely extinguished. This now inactive medulla was crushed and mixed with pure water, and injected under the skin of some dogs. The next day they were inoculated with medulla which had been desiccating for thirteen days, and so on, using increased virulence until the medulla was used of a rabbit dead the same day. These dogs might now be bitten by rabid dogs given them as companions for a few minutes, or submitted to the intracranial inoculations of the deadly virus: they resisted both.
Having at last obtained this refractory condition, Pasteur was anxious that his results should be verified by a Commission. The Minister of Public Instruction acceded to this desire, and a Commission was constituted in May, 1884, composed of Messrs. Béclard, Dean of the Faculty of Medicine, Paul Bert, Bouley, Villemin, Vulpian, and Tisserand, Director of the Agriculture Office. The Commission immediately set to work; a rabid dog having succumbed at Alfort on June 1, its carcase was brought to the laboratory of the Ecole Normale, and a fragment of the medulla oblongata was mixed with some sterilized broth. Two dogs, declared by Pasteur to be refractory to rabies, were trephined, and a few drops of the liquid injected into their brains; two other dogs and two rabbits received inoculations at the same time, with the same liquid and in precisely the same manner.
Bouley was taking notes for a report to be presented to the Minister:
“M. Pasteur tells us that, considering the nature of the rabic virus used, the rabbits and the two new dogs will develop rabies within twelve or fifteen days, and that the two refractory dogs will not develop it at all, however long they may be detained under observation.”
On May 29, Mme. Pasteur wrote to her children:
“The Commission on rabies met to-day and elected M. Bouley as chairman. Nothing is settled as to commencing experiments. Your father is absorbed in his thoughts, talks little, sleeps little, rises at dawn, and, in one word, continues the life I began with him this day thirty-five years ago.”
On June 3, Bourrel sent word that he had a rabid dog in the kennels of the Rue Fontaine-au-Roi; a refractory dog and a new dog were immediately submitted to numerous bites; the latter was violently bitten on the head in several places. The rabid dog, still living the next day and still able to bite, was given two more dogs, one of which was refractory; this dog, and the refractory dog bitten on the 3rd, were allowed to receive the first bites, the Commission having thought that perhaps the saliva might then be more abundant and more dangerous.
On June 6, the rabid dog having died, the Commission proceeded to inoculate the medulla of the animal into six more dogs, by means of trephining. Three of those dogs were refractory, the three others were fresh from the kennels; there were also two rabbits.