394 EXPERIMENTAL INFECTIONS DURING LIFE 



To prepare the antigen for use, emulsify the whole of 

 the bacterial growth in 5 c.c. normal saline solution. 



Shake the emulsion in a test-tube with some sterilised 

 glass beads to ensure a homogenous emulsion, and ster- 

 ilise by heating to 60 C. in a water-bath for one hour. 



METHOD. 



1. Take five small test-tubes, and number them i to 5 

 with a grease pencil. 



2. Into tubes Nos. i, 3, 4 and 5 pipette o.i c.c. of 

 complement. 



3. Into tubes Nos. i and 2 pipette 0.2 c.c. of the 

 serum to be tested. 



4. Into tube No. 4 pipette 0.2 c.c. of control serum. 



5. Into tubes Nos. i, 2, 3 and 4 pipette i c.c. of the 

 bacterial emulsion which forms the antigen. 



6. Place the whole set of tubes in the incubator at 

 3 7 C. for a period of one hour. 



7. Remove the tubes from the incubator and pipette 

 i c.c. erythrocyte solution and 4 minimal hsemolytic 

 doses of the corresponding hsemolysin into each tube. 



8. Mix thoroughly and return the tubes to the incu- 

 bator at 37 C. for further period of one hour. 



9. At the expiration of that time transfer the tubes 

 to the ice chest, and allow them to stand for three 

 hours. 



10. Examine the tubes. 



Tubes 3, 4 and 5 should show complete haemolysis; 

 tube 2 should give no evidence whatever of haemolysis. 



These tubes form the controls to the first tube, which 

 contains the serum to be tested. 



In tube No. i the absence of haemolysis would indicate 

 the presence in the serum of the inoculated animal of a 

 specific antibody to the micro-organism used in the 

 inoculations; since it shows that the complement has 

 been bound by the immune body to the bacterial 

 antigen, and none has been left free to enter into the 



