AGGLUTINATED TYPHOID BACILLI. 147 



obtained by Jules Rehns 1 by the injection of agglutinated typhoid 

 bacilli. He found that it was immaterial, so far as effect was con- 

 cerned, whether he injected the typhoid bacilli agglutinated or not 

 agglutinated. An entirely similar experiment has also been pub- 

 lished by Nicolle and Trenell. 2 



Having previously and independently of Rehns busied ourselves 

 with this question, and having seen that it is attended with con- 

 siderable experimental difficulties, we again took up the problem 

 on the publication of Rehns' article, especially because of the theo- 

 retical importance of the subject. Furthermore, our previous experi- 

 ences had given us the impression that Rehns' results were not gen- 

 erally applicable. 



The technique of our experiments was as follows: The typhoid 

 culture employed was an old laboratory culture especially adapted 

 to agglutination experiments. Of this, one-day agar cultures, sus- 

 pended in physiological salt solution and killed by exposure for one 

 hour to 60-70 C., were used for the injections. 



The preparation of the agglutinated typhoid bacilli was most 

 carefully attended to, it being deemed especially important to fully 

 satisfy the bacilli with the agglutinin. The agglutinin was a highly 

 active typhoid agglutinin derived from a horse, and agglutinated 

 even in dilutions of 1 : 50,000 ; only in the last experiments was a 

 weaker serum used. The agglutinin was added to the bacteria in 

 such amounts that about 500-1000 times the amount calculated 

 to be necessary was used. In order to effect as firm a union as pos- 

 sible between bacilli and agglutinin the latter was allowed to act 

 on the bacilli for one hour at 42-44 C., during which time the tubes 

 were shaken every ten minutes (at times with glass beads) in order 

 to loosen the larger clumps and secure the penetration of the agglutinin 

 to the central portions of the clumps. And in order to be on the 

 safe side, we centrifuged the bacteria from the first mixture and 

 repeated the saturating process in the same manner. After the 

 second saturation the mixture was again centrifuged, filled up with 

 salt solution, again centrifuged, and then washed several times. 

 The various decantations were saved and tested for the presence 

 of agglutinin; the last washings had to be free from agglutinin. 



Concerning the amount of injected bacilli in conformity to our 



1 J. Rehns, Compt. rend, de la Soc. de Biol., 1900, page 1058, 



2 Nicolle et Trenell, Compt. rend, de la Soc. de Biol., 1900, page 1088. 



