﻿STUDIES IN PLAGUE IMMUNITY. 311 



At the time of the beginning of the attenuation the strain "Pest 

 Avirulent Manila," when inoculated subcutaneously, killed guinea pigs 

 of from 200 to 300 grams weight in doses of about 2 oesen. At the time 

 the experiments were discontinued, the lethal dose for guinea pigs of 

 the same weight was about one agar slant culture, although the animal 

 sometimes even survived such a dose. 



The organism during the time of its attenuation was grown for five 

 different periods in the bouillon containing the different percentages 

 of alcohol. Series 59 (p. 312) gives the details of the change in virulence. 



At the time of the beginning of the experiments in the attenuation of 

 the strain "Pest Virulent" the organism possessed a virulence so great 

 that a portion of a spleen of a guinea pig which had died from infection 

 with it, when it was rubbed over the abdomen of a second guinea pig, 

 always caused the death of the animal from acute plague infection, and 

 5 oesen of a suspension (prepared by adding 5 cubic centimeters of 

 saline solution to a 24-hour agar slant culture of the organism) when 

 rubbed over a shaved and scarified area on the abdomen of a guinea pig, 

 also invariably caused it to succumb to plague. After the virulent strain 

 had been grown for nearly three months in the alcoholic bouillon, | oese 

 of a 24-hour agar culture of the organism when injected subcutaneously 

 into a guinea pig, still caused its death ; after the experiment had been 

 carried on for eleven months the virulence of the organism was found 

 to be such that 2 oesen of the 48-hour agar culture, when rubbed over 

 a shaved and scarified area on the abdomen of a guinea pig, or the same 

 amount injected subcutaneously, no longer caused the death of the inocu- 

 lated animals. However, \ agar slant after two months more of attenua- 

 tion, invariably caused fatal infection. At the time of the termination 

 of the experiments which lasted for about seventeen months, the virulence 

 was so reduced that \ agar slant culture of the organism, when injected 

 subcutaneously into guinea pigs, did not cause the death of more than 

 40 per cent of the animals so inoculated, the remaining 60 per cent 

 remaining alive and Avell. At the termination of the experiment the 

 same original strain (unattenuatecl) which had been kept in a sealed 

 tube and protected from light during the same period of time, was grown 

 for two successive transplants upon fresh agar and its virulence again 

 tested. It was found that 0.1 oese of this culture, when rubbed over the 

 scarified abdomen of a guinea pig, still caused the death of the animal, 

 showing that no practical decrease in the virulence of the organism had as 

 yet occurred. The details of the attenuation of the strain are recorded 

 in the series of experiments numbered 60. 



