90 THE PRINCIPLES OF IMMUNOLOGY f> 



whereas others do not disappear until 80 C. has been reached. 

 Wells states that " purified typhoid agglutinin may resist 80 to 90 

 degrees." Agglutinins cannot be reactivated by the addition of 

 fresh serum, even though the temperature may have been adjusted 

 so that the agglutinoid remains. 



A simple experiment for the demonstration of the influence of heat on 

 agglutinins is as follows: The typhoid immune serum, the production of 

 which has been described above, and also the killed typhoid suspension may 

 be used. In each of three tubes place 0.5 c.c. serum diluted i-io, and into a 

 fourth tube 0.5 c.c. salt solution. Tube I is heated in a water bath at 56 C. 

 for one-half hour, tube 2 heated at 70 to 75 C. for one-half hour, and 

 tubes 3 and 4 kept at room temperature. After cooling tubes i and 2, add 

 0.5 c.c. bacterial emulsion to each tube and incubate for one hour at 37 C. 

 Agglutination will not occur in tube 2, the serum having been heated to 70 to 

 75 C., nor in the control tube with saline. The unheated serum and the 

 serum heated to 56 C. will agglutinate powerfully. It will be found also that 

 the addition of o.i c.c. fresh guinea-pig serum (complement) to tube 2, and 

 subsequent incubation will fail to produce agglutination. 



It is of interest to note that the degree of concentration of serum 

 has some influence on the degree of heat necessary for destruction. 

 For example, Koeckert in this laboratory found that normal un- 

 diluted iso-hemagglutinins are destroyed at 65 to 66 C. for 

 thirty minutes, but that in high dilutions they are destroyed at 62 C. 

 for thirty minutes. 



The influence of electrolytes on the phenomenon of agglutination 

 is of considerable importance from the theoretical point of view 

 because of the resemblance to flocculation of colloidal suspensions. 

 Bordet, who discovered this fact, compared the reaction to the 

 throwing down of the alluvial matter in rivers as the fresh water 

 meets the salt water of the ocean. By previously dialyzing the salts 

 out of the bacterial suspension and the specific serum he showed 

 that agglutination would not occur, but that if the mixture was 

 salted in proper concentration the reaction would take place. It is 

 possible, however, to agglutinate bacteria by certain concentration 

 of salts, particularly of the heavy metals, but such concentration is 

 always much stronger than is necessary for salting, as described in 

 the Bordet experiment. 



The demonstration of the influence of salts may be seen in the following 

 experiment, taken from Zinsser, Hopkins and Ottenberg. For this experi- 

 ment the killed typhoid suspension and the anti-typhoid serum as employed 

 in previous experiments may be used. " Place in each of two centrifuge tubes 

 with pointed tip 2.0 c.c. of the suspension. To tube A add 2.0 c.c. of agglu- 

 tinating serum diluted 1-50. To tube B add 2.0 c.c. distilled water. Allow 

 the tubes to stand at 37 C. for thirty minutes. Centrifugalize the tubes at 

 high speed until the supernatant fluid is clear." Pipette off the fluid and " to 

 the washed sediments add 2.0 c.c. distilled water and draw the mixture re- 

 peatedly in and out of the capillary pipette in order to break up the clumps and 

 obtain an even suspension. Set up the following tests in agglutination tubes : 



1 Sediment A 0.5 c.c Distilled water 0.5 c.c. 



2 Sediment A 0.5 c.c. 10 per cent NaCl 0.09 c.c. Distilled water 0.5 c.c. 



3 Sediment A 0.5 c.c. 0.8 per cent. CuSO 4 0.02 c.c. Distilled water 0.5 c.c. 

 4 Sediment B 0.5 c.c. 0.8 per cent. CuSO 4 0.02 c.c. Distilled water 0.5 c.c. 



5 Sediment B 0.5 c.c. 0.8 per cent. CuSO 4 o.i c.c. Distilled water 0.5 c.c. 

 6 Sediment B 0.5 c.c Distilled water 0.5 c.c. 



