tte 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 deg. C. for a
period of one hour.

7. Remove the tubes from the incubator and pipette 1 c.c. erythrocyte
solution and 4 minimal haemolytic doses of the corresponding haemolysin
into each tube.

8. Mix thoroughly and return the tubes to the incubator at 37 deg. 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. 1 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 haemolytic system; on the
other hand the presence of haemolysis 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 haemolytic serum, and was ready to combine with those reagents to
complete the haemolytic 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 deg. C. for one hour.
--------------------------------------------------------------------------

1 c.c. E. 1. c.c. E. 1 c.c. E. 1 c.c. E. 1 c.c. E.

H.S.^{4} H.S.^{4} H.S.^{4} H.S.^{4} H.S.^{4}
--------------------------------------------------------------------------
Incubate at 37 deg. C. for one