86 THE PRINCIPLES OF IMMUNOLOGY 



dilutions oi 1-128 to 1-4096. The fact that the other two organisms 

 are agglutinated is due to the phenomenon of " group reactions." 

 In the same way, if an animal were immunized to bacillus coli the 

 serum would agglutinate coli in high dilutions and typhosus in 

 lower dilutions. The principle is also well shown in a table taken 

 from Citron : 



Typhoid Cholera 



Agglutination of immune immune 



serum serum 



Against B. typhosus ....,,,,.,, 1-2,000 i-io 



Against B. paratyphosus i-ioo i-io 



Against B. coli 1-25 i-io 



Against V. cholerae i-io 1-3,000 



Absorption of Agglutinins. If specific sera for paratyphosus and 

 coli were interposed in the above diagram it would be seen that these 

 sera would clump the homologous bacteria in high dilution, and the 

 others of the group in only low dilutions. This indicates that in 

 each serum we may assume there are several agglutinins, one for 

 the homologous organism, a major agglutinin or main agglutinin, 

 and one for each of the other organisms of the group, minor agglu- 

 tinins or partial agglutinins. This statement may be accepted for 

 the present, although the conception will be somewhat altered in the 

 theoretical discussion. Castellani has shown that if the major 

 agglutinin is absorbed by its homologous organism the minor agglu- 

 tinins disappear also, but that if one or several of the minor agglu- 

 tinins be absorbed by other members of the group of organisms the 

 major agglutinin remains. In order to make this clear we shall first 

 illustrate the process of absorption and then apply it to the group 

 reaction. It is well known that an animal may be simultaneously 

 immunized to two or more types of organisms ; for example, bacillus 

 typhosus and bacillus coli. The resulting serum may agglutinate 

 typhosus in dilution of 1-4000 and coli in dilution of i-iooo. The 

 absorption of the agglutinins may be shown as follows : 



Prepare thick suspensions of bacillus typhosus and of bacillus coli com- 

 munis by suspending the twenty-four-hour surface growth of three slant agar 

 cultures in about 5 c.c. saline. This is done by placing 5 c.c. in the first tube, 

 making the suspension, then transferring to the second tube, suspending that 

 culture and repeating in the third tube. The typhoid emulsion is killed by 

 heat of 56 C. for one hour and the colon by heat at 60 C. for one hour. 

 Add to 1.5 c.c. serum an equal volume of thick suspension of dead bacillus 

 typhosus and in another tube place 1.5 c.c. serum with an equal volume of 

 thick suspension of dead colon bacilli. The tubes are marked A and B. After 

 mixing the emulsion of bacilli and serum the tubes are incubated at 37 C. 

 and placed in the ice-chest for twelve hours. The tubes are centrifuged and 

 the supernatant fluid pipetted off. The bacteria are resuspended and the sus- 

 pensions diluted with salt solution about 1-20 or more, in order that agglu- 

 tination may be easily observed. The supernatant fluid represents a 1-2 

 dilution of the original serum. Place 0.5 c.c. each into test tubes and add 4.5 c.c. 

 saline, making a dilution of 1-20, well under the titer of the serum. Of the 

 diluted fluid A which has been absorbed by typhosus place 0.5 c.c. in a series 

 of two tubes and add 0.5 c.c. thin emulsion of colon. After incubation the first 

 tube will show no agglutination, and the second tube containing colon, whose 

 agglutinin has not been absorbed, will show agglutination. Conversely place 

 0.5 c.c. diluted fluid B in a series of two tubes, and add in order 0.5 c.c. thin 



