296 THE ROLE OF AGGLUTINATION IN IMMUNITY, 



With the exception of perhaps Duclaux, all these authorities 

 experimented with the serum of animals that were naturally 

 immune or had been immunised with cultures of typhoid bacteria. 

 Although human serum was not employed, the results point to 

 the agglutinin of human serum being able to withstand in great 

 part or entirely an exposure to 60° for 15 minutes, and this would 

 destroy all opsonins. 



Technique. — In freeing the leucocytes from blood plasma, the 

 method of Wright and Douglas was followed. The blood was 

 drawn off into an equal volume of 1% citrate of soda contained in 

 bent capsules and centrifuged. The deposit was transferred into 

 ordinary centrifuge tubes containing about 20 volumes of normal 

 saline. The corpuscles were uniforml}^ distributed by blowing 

 air through the suspension, the capillary pipette being used for 

 the purpose. After centrifuging, the supernatant liquid was 

 removed and replaced by more normal saline. The corpuscles 

 were in this manner washed four times with normal saline. The 

 bacteria were grown upon nutrient agar for 24 hours at 37° and 

 suspended in normal saline. After being agglutinated, they were 

 washed twice with normal saline and distributed in as much 

 normal saline as sufficed to make an opalescent fluid. The clumps 

 were broken up and distributed by passing the suspensions in 

 and out of the capillary pipette. Equal volumes of the corpuscular 

 and bacterial suspensions were mixed upon a glass slide, and the 

 mixture was allowed to run into a capillar}^ tube and sealed, or it 

 was covered with a cover-glass and placed in a moist chamber. 

 The tests were incubated at 37° for 30 minutes. Films were 

 prepared and coloured by the Leishman stain. 



The Investigation. 

 Washed leucocytes and normal typhoid bacteria. — The films 

 showed a complete absence of phagocytosis. 



Washed leucocytes with agglutinated aud normal bacteria. — The 

 active serum agglutinated in ratios of 1:75 and under. 



Normal bacteria No phagocytosis. 



Agglutinated bacteria. 1:75 Phagocytosis 



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