210 Chapter VIII 



evident, then, that it is not an easy matter to set up an acquired 

 immunity in the Invertebrata. 



It was necessary, therefore, to go higher up the animal scale and 

 have recourse to cold-blooded vertebrates. The choice naturally 

 fell on the frog. I asked Dr Gheorghiewski 1 , who was working 

 in my laboratory, to try to vaccinate the Batrachians against pyo- 

 cyanic disease. I ought first to state that the bacillus of blue pus 

 is pathogenic for the frog, which it kills both at the ordinary laboratory 

 temperature, and at that of the incubator, 30 37 C. In the first 

 case the fatal dose is much greater than in the second, but it is 

 always easy to induce a fatal infection. In this respect, therefore, the 

 Bacillus pyocyaneus is much better adapted for study than the 

 anthrax bacillus or many other micro-organisms. Gheorghiewski 

 vaccinated green frogs (Rana esculenta), which had been accustomed 

 to the incubator temperature, 30 C., by injecting every 4 to 7 days 

 considerable doses of cultures of Bacillus pyocyaneus heated to 80 C. 

 in order to kill all the micro-organisms. Some (3 4) weeks after- 

 wards the prepared frogs became more resistant to the Bacillus 

 pyocyaneus than were the controls placed under the same conditions. 

 The frogs, inoculated with a fatal dose of the bacilli, clearly exhibited 

 a certain, though slight, degree of acquired immunity. They withstood 

 a dose that was always fatal to the controls or even a dose and a half, 

 but died when injected with double the fatal dose. The lymphatic 

 fluid of the vaccinated frogs feebly agglutinated (1 : 20 1 : 30) the 

 Bacillus pyocyaneus although it still formed an excellent culture 

 medium for this organism. Gheorghiewski satisfied himself that the 

 agglutination was insufficient to assure immunity to the frog. The 

 bacilli agglutinated into clumps were very virulent. 



A detailed examination of the phenomena observed in the im- 

 munised frogs revealed the following facts. During the earliest 

 stage the bacilli, injected into the dorsal lymphatic sac, were found 

 free in the fluid, retained their form and were not transformed into 

 granules. The bacilli carried by the lymphatic current spread rapidly 

 [222] throughout the body. Very shortly after inoculation, however, some 

 of the leucocytes began to ingest the bacilli which became trans- 

 formed into spherules within these cells. Later, the phagocytic 

 reaction increased and at the end of 15 to 20 hours all the bacilli 

 were found inside leucocytes. It was easy to demonstrate that these 



1 Ann. de Hnst. Pasteur, Paris, 1899, t xm, p. 314. 



