﻿STUDIES IN PLAGUE IMMUNITY. 243 



cultivated at 30° C. there was almost no precipitation of the bacteria, 

 the fluid still being cloudy. My experience has led me to conclude 

 that the time limit of the agglutinative reaction with the plague bacillus 

 should be placed at certainly not over one or two hours at 37° C. 

 at the most, since after this time the results may be very inaccurate 

 and confusing owing to the tendency of the bacteria to become spon- 

 taneously precipitated. 



The testing of serum supposed to contain relatively small amounts of 

 plague agglutinins has been very unsatisfactory where it was neces- 

 sary to employ concentrated mixtures of the serum, for example in 

 dilutions of 0.5 or 0.25. As already mentioned, the density and viscosity 

 of the serum in these dilutions evidently somewhat retards the aggluti- 

 nation of the pest organism, as was evident from the fact that the reaction 

 occurred in some of the experiments in the higher but not in the lower 

 dilutions. This phenomenon of retardation can not be explained on the 

 assumption of the presence of agglutinoid, since it was noted in several 

 instances with perfectly fresh sera. It may sometimes be observed in 

 fresh, specific immune sera with other bacteria and must be ascribed to 

 physical causes, the discussion of the nature of which will not be entered 

 into here. 



I have conchided from a large number of experiments that the agglu- 

 tinins are formed slowly and only in very small amounts in animals 

 which are being immunized against pest infection and that they only 

 occur in demonstrable quantities in those which have been very highly im- 

 munized. At most, only very minute traces of these substances are encoun- 

 tered after single inoculations of either the killed or the living organisms, 

 no matter how large the dose which is employed. I am convinced that 

 in my earlier experiments with plague agglutination I sometimes mistook 

 pseudo-agglutinations of the pest bacillus for true ones, and from a 

 study of the literature it seems to me very likely that other observers 

 have also erred in this respect. A study which I have made of the blood 

 sera of guinea pigs which have been vaccinated against pest infection and 

 which have later shown themselves to be immune to lethal and multiple 

 lethal doses of the pest bacillus has demonstrated that practically no traces 

 of agglutinins exist in such sera. The same may be said of the sera of 

 other animals immunized in a similar manner. Monkeys which have first 

 been vaccinated with attenuated pest cultures or inoculated with killed 

 cultures, and which have afterwards been shown to be immune to pest 

 infection by the injection in increasing closes of from \ oese to nearly 

 1 entire agar slant culture of the living virulent strain, still have 

 developed in their sera practically no traces of agglutinins. Indeed it is 

 very difficult to immunize monkeys to such a high degree that the 

 blood of the animals shows the presence of plague agglutinins. In a 

 series comprising twelve monkeys (numbers 1232, 1233, 1239, 1240, 



