200 N. S. FERRY AND ARLYLE NOBLE 



these were planted on plain agar in quart whiskey flasks — except in the 

 case of Bad. influenzae for which whole-blood agar was used. It has 

 been found advantageous to use agar in whiskey flasks, as this method 

 not only gives a larger amount of suspension for less labor, but it also 

 gives a far heavier and healthier growth than when tubes are used, 

 probably because of the greater supply of media, moisture and air. 

 An abundant growth of Bact. pertussis can be obtained on plain agar 

 in flasks when it will grow only slightly or not at all on plain agar in 

 tubes. And a young, vigorous growth is necessary to the production of, 

 homogeneous suspensions. Also in using agar without either ascitic fluid 

 or blood, all possibility of clumping from this source is avoided. In the 

 case of the influenza bacillus it is necessary to use blood agar to obtain 

 any growth. Just enough whole blood is added to agar to insure growth 

 and it is used before any hemolysis takes place, in order that the sus- 

 pension may contain as little blood as possible. 



The flasks were incubated for from eighteen to twenty-four hours and 

 the growth washed off with 0.5 per cent formalin in physiologic salt 

 solution. The suspensions were then shaken for a few hours and later, 

 after being tested for sterility, were filtered through paper and stand- 

 ardized to about 2000 million per cubic centimeter. 



With this technique, homogeneous suspensions of all the organisms 

 used, were produced. 



Aggluiination tests. In carrying out the tests, the serum was diluted 

 with physiologic solution and each tube contained 0.5 cc. suspension 

 plus 0.5 cc. diluted serum. The tests were all macroscopic and were 

 incubated at 37°C. for twenty-four hours. (+ + + ) represents com- 

 plete agglutination with fluid clear; (+ + ) partial agglutination with 

 marked clumping, but fluid not entirely cleared up; (+) slight agglu- 

 tination, but still with positive clumping; and ( — ) no clumping, no 

 clearing. 



Specially graduated 1 cc. pipettes were used for making the serum 

 dilutions and a different pipette was used for each dilution. All glass- 

 ware used in connection with the tests was clean and sterile. 



AGGLUTINATION REACTIONS WITH SERUM FROM DISTEMPER RABBITS 



AND DOGS 



In testing apparently' normal rabbits for agglutinins, before 

 beginning inoculation for the production of antisera, we found 

 that if a serum agglutinated B. bronchisepticus in a dilution higher 



