74 LABORATORY COURSE IN SERUM STUDY 



4. 10 per cent salt solution. 



5. Copper sulphate solution 0.8 per cent. 



Prepare typhoid suspension (killed with formalin as in previous 

 lesson for the sake of safety) . Place in each of two centrifuge tubes, with 

 pointed tip, 2 c.c. of the suspension. To tube A add 2 c.c. of agglutinat- 

 ing serum diluted 1-50. To tube B add 2 c.c. of distilled water. Allow 

 the tubes to stand at 37 C. for 30 minutes. Centrifugalize the tubes at 

 high speed until the supernatant fluid is clear. Resuspend the sedi- 

 ments in 5 c.c. of distilled water and again centrifugalize. To the washed 

 sediments add 2 c.c. of distilled water and draw the mixture repeatedly 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% salt sol. 0.09 c.c. Dist. water 0.4 c.c. 



Copper sulphate sol. 0.8 % Distilled water 



3. Sediment A 0.5 c.c. 0.02 c.c. 0.5 c.c. 



4. Sediment B 0.5 c.c. 0.02 c.c. 0.5 c.c. 



5. Sediment B 0.5 c.c. 0.1 c.c. 0.5 c.c. 



6. Sediment B 0.5 c.c. 0.5 c.c. 



The tubes are placed in the water bath at 37 for one hour and then 

 observed. Tubes 2, 3 and 5 should show agglutination. 



This experiment shows that electrolytes are necessary for the 

 appearance of agglutination and that bacteria even though they 

 may have united with the specific serum agglutinins do not clump 

 in the absence of such salts. It is the mainstay of Bordet's view 

 of agglutination as a "two-phase" reaction in which one step is 

 the union with agglutinins, the other the actual clumping brought 

 about by the salts. 



Each student needs: 8 watch glasses, 2 centrifuge tubes, 6 f-inch test 

 tubes, glass tubing, test tube rack. 



Bacillus typhosus, 24-hr broth culture, 24-hr, agar slant. 



LESSON XI 

 ABSORPTION OF BACTERIAL AGGLUTININS 



THE serum of an animal immunized simultaneously with two 

 species of bacteria develops agglutinative power for each of these 

 species. The agglutinins for the two species may be shown to 

 be distinct by treating the serum with a heavy emulsion of one of 



