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After at first having tried a rather different technique partly 

 in orientating experiments, and partly in the first part of the main 

 series of experiments, I finally fixed upon the following method. 



For the simple agglutination experiment and also for the absorp- 

 tion of agglutinins fresh suspensions of 36—42 hours' cultures on 

 haemoglobin or Fildes agar in a solution of 0.3 o/ NaCl and 0.3o/q 

 phenol in distilled water, were always used. The bacterial suspen- 

 sion was prepared in a tube of about 15 mm. internal diameter 

 and was made of such a density for the agglutination test that one 

 could just read through the tube, with certainty, rather heavy 

 letters, 3—4 mm. in height * on white paper held immediately behind 

 the tube. The light — preferably clear daylight from a window 

 — must fall perpendicular to the surface of the paper; the in- 

 vestigator therefore stands with his back to the window. Dilutions 

 of the serum are made with the carbolised salt solution in a series 

 of small tubes, so as to get the concentrations 1:10, 1:20, 1:40, 

 1 : 80 etc in a volume of 0.1 c.c. To each tube is added 0.3 c.c. 

 of culture, making the final dilutions of the serum 1 : 25, 1 : 50, 1 : 100, 

 1 : 200 etc. When it is found necessary, a tube containing only 

 carbolised salt solution and the bacterial suspension, is put up as a 

 control of spontaneous agglutination. The tubes are then placed 

 in a water-bath at 50° for 4 hours and then read macroscopically. 

 Typical complete agglutination consists of large clumps like those 



seen in typhoid agglutination. This is denoted by T while fine 



clumps are denoted by +> and partial agglutination by (-|-). Very 

 slight agglutination is disregarded. 



The following details concerning the technique may be given. 

 The relatively long cultivation period was chosen to diminish the 

 tendency to spontaneous sedimentation. In comparative tests it was 

 found that young cultures had this tendency in a greater degree 

 than old. For the same reason the salt concentration is made as 

 low as 0.3o/o. In this way spontaneous sedimentation of most of 

 the strains was completely avoided, or else it was reduced fo such 

 an extent that it did not interfere with the estimation of the agglu- 

 tination. There w r ere some strains however in which spontaneous 

 sedimentation or agglutination was so marked that the investigation 

 of the specific agglutination could not be undertaken. But these 

 strains were usually so divergent in their morphology, — macro- 

 scopic and microscopic, — that they would scarcely have reacted 

 with the sera, even if it had been possible to obtain the necessary 

 experimental conditions. The addition of phenol was required when 

 the test was done at 37°, as otherwise a growth of foreign bacteria 

 took place, and it seemed advantageous to make use of it also 

 when the test was done at 50°. Formalin (1% ) was also tried. 



* It naturally does not matter if the letters are a bit larger or 

 smaller. 



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