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 sterilise by heating to 60° C. in a water-bath for one hour. METHOD.-- 1. Take five small test-tubes, and number them 1 to 5 with a grease pencil. 2. Into tubes Nos. 1, 3, 4 and 5 pipette 0.1 c.c. of complement. 3. Into tubes Nos. 1 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. 1, 2, 3 and 4 pipette 1 c.c. of the bacterial emulsion which forms the antigen. 6. Place the whole set of tubes in the incubator at 37° C. for a period of one hour. 7. Remove the tubes from the incubator and pipette 1 c.c. erythrocyte solution and 4 minimal hæmolytic doses of the corresponding hæmolysin into each tube. 8. Mix thoroughly and return the tubes to the incubator 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 hæmolysis; tube 2 should give no evidence whatever of hæmolysis. These tubes form the controls to the first tube, which contains the serum to be tested. In tube No. 1 the absence of hæmolysis 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 hæmolytic system; on the other hand the presence of hæmolysis would show that no appreciable amount of antibody has yet been formed in response to the inoculations. In other words, there is an absence of infection, since the complement remained unfixed at the time of the addition of the erythrocyte solution and hæmolytic serum, and was ready to combine with those reagents to complete the hæmolytic system. The method may be shown diagramatically as under using the symbols already indicated Test-tubes. 1 2 3 4 5 0.1 c.c. C. ........ 0.1 c.c. C. 0.1 c.c. C. 0.1 c.c. C. 0.2 c.c. S.S. 0.2 c.c. S.S. ......... 0.2 c.c. P.S. ........ A. A. A. A. ........ -------------------------------------------------------------------------- Incubate at 37° C. for one hour. -------------------------------------------------------------------------- 1 c.c. E. 1. c.c. E. 1 c.c. E. 1 c.c. E. 1 c.c. E.