The Preparation of Hæmolytic Serum

Apparatus Required:

Small centrifuge, preferably electrically driven, with two receptacles
for tubes, and enclosed in a safety shield (Fig. 162).
Sterile centrifuge tubes (10 c.c. capacity), Fig. 163.
Sterile pipettes (10 c.c. graduated) in case.
Sterile glass capsules (in case).
Sterile test-tubes.
Sterile all glass syringe (5 c.c. or 10 c.c. capacity)
and needle.

Reagents Required:

Normal saline solution.
10 per cent. sodium citrate solution in normal saline.
Human blood (vide infra).

Method.—

1. Select a healthy full-grown rabbit of not less than 2500 grammes weight in accordance with the directions already given (page 322) and prepare it for intraperitoneal inoculation.

2. Measure out 2 c.c. citrated human blood (collected at a surgical operation or a venesection, or withdrawn by venipuncture from the median basilic or median cephalic vein of a normal adult) into a centrifuge tube and centrifugalise thoroughly.

3. Wash with three changes of normal saline (vide also page 388).

4. Transfer the washed cells to a sterile capsule by means of a sterile pipette. Add 5 c.c. of normal saline and mix thoroughly.

5. Take up the mixture of cells and saline in the all-glass syringe and inject into the peritoneal cavity of the rabbit.

6. Seven days later inject intraperitoneally the washed cells from 5 c.c. human blood mixed with 5 c.c. normal saline.

7. Seven days later inject the washed cells from 10 c.c. human blood mixed with 5 c.c. normal saline.

8. After a further interval of seven days repeat the injection of washed cells from 10 c.c. human blood mixed with 5 c.c. normal saline.

Note.—Better results are obtained if the second and subsequent injections are made intravenously, even when smaller quantities of washed red cells are employed. If, however, the intravenous route is selected exceeding great care must be exercised to avoid the introduction of air into the vein—an accident which is followed, within a few minutes, by the death of the rabbit from pulmonary embolism.

9. Allow five days to elapse, then collect a preliminary sample of blood, say about 2 c.c., from the rabbit's ear. Allow it to clot, separate off the serum and transfer to a sterile test-tube. Place the test-tube in a water-bath at 56° C. for fifteen minutes (to inactivate) and test the serum quantitatively for hæmolytic properties in the following manner: