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 haemolytic doses of the corresponding haemolysin 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 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 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 C. for one hour.

-------------------------------------------------------------------------- (?) No haemolysis. |__________________________________|

Haemolysis.

NOTE.--It is sometimes more convenient to _sensitise_ the erythrocytes just before they are needed. This is done forty-five minutes after the experiment has been started (page 394, step 6), that is to say, before the completion of the first period of incubation, thus:

1. Measure out into a sterile test-tube (or flask) five c.c.

of erythrocyte solution.

2. Measure out twenty minimal haemolytic doses of haemolysin, add to the erythrocyte solution on the test-tube.

3. Allow the erythrocyte and haemolysin to remain in contact for fifteen minutes at room temperature. The red cells are then sensitised and ready for use.

4. When the tubes are removed from the incubator at the end of the first hour (i. e., step 7) add 1 c.c. sensitised red cells to each tube by means of a graduated pipette.

5. Mix thoroughly, return the tubes to the incubator at 37C. and complete the experiment as previously described (steps 8 onward).

XIX. POST-MORTEM EXAMINATIONS OF EXPERIMENTAL ANIMALS.

The post-mortem examination should be carried out as soon as possible after the death of the animal, for it must be remembered that even in cold weather the tissues are rapidly invaded by numerous bacteria derived from the alimentary tract or the cavities of the body, and from external sources.

The following outlines refer to a complete and exhaustive necropsy, and in routine work the examination will rarely need to be carried out in its entirety.

NOTE.--Throughout the autopsy the searing irons must be freely employed, and it must be recollected that one instrument is only to be employed to seize or cut one structure. This done, it must be regarded as contaminated and a fresh instrument taken for the next step.

~Apparatus Required~:

Water steriliser.

{ Scalpels.

Surgical instruments: { Scissors.

{ Forceps.

{ Bone forceps.

Spear-headed platinum spatula (Fig. 199).

Searing irons (Fig. 198).

Tubes of media--bouillon and sloped agar.

Surface plates in petri dishes (of agar or one of its derivatives).

Platinum loop.

Aluminium "spreader."

Grease pencil.

Sterile capillary pipettes (Fig. 13, a).

Sterile gla.s.s capsules, large and small.

Cover-slips or slides.

Bottles of fixing fluid (_vide_ page 114) for pieces of tissue intended for sectioning.

1. Place the various instruments, forceps, scissors, scalpels, etc., needed for the autopsy inside the steriliser and sterilise by boiling for ten minutes; then open the steriliser, raise the tray from the interior and rest it crosswise on the edges.

2. Heat the searing irons to redness in a separate gas stove.