IMMUNIZATION OF GUINEA PIGS. 247 



animals, and in 3 cases of living animals twenty-five days after 

 inoculation in pure culture. On account of the lack of virulence 

 of this strain of plague, it w^as impossible to identify it by guinea 

 pig inoculations; but the morphology, staining, and character- 

 istic growth on various media, including the growth in broth 

 covered with sterile vaseline, served to identify the organism 

 satisfactorily. The fact that several tests showed the same 

 plague-like organism in pure culture helped to confirm the iden- 

 tification. One animal inoculated with 3 cubic centimeters of 

 agar with avirulent plague bacilli died from some unknown cause 

 twenty-nine days after inoculation. In it the avirulent plague 

 bacillus in apparently pure culture was found at the point of 

 inoculation, while no growth of any sort was obtained from the 

 spleen, liver, lungs, or peritoneal cavity. There was still a small 

 mass of agar remaining. This case is the more remarkable 

 since there, had been some necrosis of the skin over the inoculated 

 agar. This had entirely healed at the time of the death of the 

 animal. 



It was demonstrated then that, following a small initial dose 

 of avirulent pest bacilli in agar, pest colonies form in the agar, 

 a portion of agar remains as long as twenty-nine days, and that 

 pest bacilli may be recovered in pure culture after that inter- 

 val. Gradual dosage over a long period of time may, therefore, 

 be attained by this method. 



To test for immunity, the animals inoculated with the agar- 

 avirulent-pest were subjected to infection with virulent plague 

 bacilli. In the first group, included under Table I, relatively 

 small amounts of pest bacilli were mixed with the agar and 

 various quantities of agar were inoculated. The test dose in all 

 animals included in Table I was 0.5 cubic centimeter of a suspen- 

 sion of a 24-hour culture made directly from an infected guinea 

 pig. This strain had been kept at a high degree of virulence 

 by long passage through guinea pigs and was regularly fatal to 

 guinea pigs, inoculated cutaneously, in three to five days. The 

 number of bacteria in the test dose was estimated by means of 

 the Thoma Zeiss counting chamber at 750,000, counting each 

 element of a chain as one. By plating dilutions, the test dose 

 gave 390,000 colonies, a lower number than that obtained by 

 counting, since a chain or united pair could give but one colony. 

 The test dose was given subcutaneously. 



