In the blood of animals, as in the cultures at the beginning, the splenic fever microbe appears at first in transparent filaments, more or less divided into segments. Up to that point, the resemblance between the microbe of splenic fever and the microbe of cholera is complete. But this blood, or the cultures exposed to the free contact of the air, instead of continuing this first mode of generation, frequently exhibit, even in the course of twenty-four hours, spores distributed more or less regularly along the length of the filaments. All around these corpuscles the matter of the filaments is absorbed, in the manner formerly illustrated by Pasteur in the diagrams of his work on the diseases of silkworms, when treating of the bacilli of putrefaction. Little by little, all cohesion between the spores disappears, and the whole collection soon forms nothing more than a dust of germs. But—and here lies the great difficulty which experimenters encountered in applying to splenic fever the method of gradual attenuation which was practised with the microbe of cholera—these germs of splenic fever may be exposed for years to the air without losing their virulence, always ready to reproduce themselves without any appreciable change, and to manifest their effects in the bodies of animals. How can it be hoped to discover a vaccine of splenic fever by the method used with the contagium of fowl cholera, since the splenic fever virulence, at the end of twenty-four hours, is concentrated in a spore? Before the oxygen of the air has had time to attenuate the contagium, the virulence of the parasite would be encased in these spores. Yet this objection did not appear insuperable to Pasteur. Since (said he to himself), under its filamentous form, the microbe of splenic fever is quite analogous to the microbe of fowl cholera, may not the problem of exposing the splenic microbe to the air be reduced to the following one: to determine the conditions which would prevent the production of spores? The difficulty would thus be surmounted; for, once we have got rid of the spores, the splenic filaments might be maintained in contact with air for any length of time, and we might then no doubt fall back upon the conditions which had produced the attenuation of the cholera microbe.
Pasteur and his two assistants gave themselves up to this research. Days passed and experiments were multiplied. Pasteur became more and more engrossed: he had, what his daughter called, 'the face of an approaching discovery.'
'Ah! what a grand thing it would be,' he was heard from time to time to murmur to himself with a suppressed voice, 'if one could arrive at that—if the fact that the attenuation of the microbe of fowl cholera proved not to be an isolated one!' But if anyone ventured to ask him a timid question as to the phase his experiments were going through, he would reply, 'No, I can tell you nothing. I dare not express aloud what I hope.'
At last one day he came up from his laboratory with a triumphant face. His joy was such that tears stood in his eyes. I have never seen a more radiant expression of the highest and most generous emotions than emanated from his countenance.
'I should never console myself,' he said while embracing us, 'if a discovery such as my assistants and I have just made were not a French discovery.'
And with the clearness which is the charm of this powerful mind, he related to us the most recent discoveries of his laboratory.
In neutralised chicken infusion the splenic microbe can no longer be cultivated at a temperature of 44 or 45 degrees. Its cultivation, on the contrary, is easy at 42 or 43 degrees; and in these conditions the microbe produces no spores. At this latter temperature, therefore, and in contact with pure air, we can maintain a culture of filamentous parasites of splenic fever, deprived of all germs. In some weeks the crop dies—that is to say, when this culture is sown in fresh broth the sterility of the broth remains complete. But during the preceding days life exists in the cultivating liquid. If after two, four, six, or eight days of exposure to the air and to heat, the contagium is tried upon animals, its virulence is found to be continually changing with the time of its exposure to the air, and, consequently, it represents a series of attenuated contagia. From the moment when the formation of the spores of the splenic fever bacillus is prevented, all becomes substantially the same as in the case of the microbe of fowl cholera. Moreover, as in the cholera microbe, each of these states of attenuated virulence can be reproduced by cultivation. Finally, splenic fever not being recurrent, each of these splenic fever microbes constitutes a vaccine for the more virulent microbe.
In order to apportion the virulence of the vaccine to the species it is desired to vaccinate, it must be tried on a certain number of individuals of the same species. If some vaccinated animals are inoculated with the virulent virus, and none of them perish, the vaccine is good. Among individuals of the same species, however, the difference of receptivity is in general great enough to make it prudent, and even necessary, to have recourse to two vaccines, one weak and the other stronger, with an interval of from 12 to 15 days between the two inoculations.