The Elements of Bacteriological Technique Part 89

_Apparatus Required:_

Small centrifuge, preferably electrically driven, with two receptacles for tubes, and enclosed in a safety s.h.i.+eld (Fig. 162).

Sterile centrifuge tubes (10 c.c. capacity), Fig. 163.

Sterile pipettes (10 c.c. graduated) in case.

Sterile gla.s.s capsules (in case).

Sterile test-tubes.

Sterile all gla.s.s syringe (5 c.c. or 10 c.c. capacity) and needle.

[Ill.u.s.tration: FIG. 162.--Small electrical centrifuge.]

[Ill.u.s.tration: FIG. 163.--Centrifuge tube.]

_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 Measure out 2 c.c. citrated human blood (collected at a surgical operation or a venesection, or withdrawn by venipuncture from the median basilic or median cephalic vein of a normal adult) into a centrifuge tube and centrifugalise thoroughly.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-gla.s.s 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 quant.i.ties 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 quant.i.tatively for haemolytic properties in the following manner:

THE t.i.tRATION OF HaeMOLYTIC SERUM.

_Apparatus Required:_

Electrical centrifuge.

Sterile centrifuge tubes.

Water-bath regulated at 56C.

Sterilised pipettes 10 c.c. graduated in tenths.

Sterilised pipettes 1 c.c. graduated in tenths.

Sterile test-tubes, 16 2 cm.

Small sterile test-tubes, 9 1 cm.

Small test-tube rack, or roll of plasticine.

Capillary teat pipettes.

Stout rubber band or length of small rubber tubing.

_Reagents Required and Method of Preparation:_

1. Normal saline solution.

2. Haemolytic serum inactivated by preliminary heating to 56 C. for 15 minutes (_vide supra_) in test-tube labelled H. S.

3. Complement. Fresh guinea-pig serum in test-tube labelled C.

Kill a normal guinea-pig with chloroform vapour.

Open the thorax with all aseptic precautions, and collect as much blood as possible from the heart with a sterile Pasteur pipette.

Transfer it to a sterile centrifuge tube and place the tube in the incubator at 37 C. Two hours later separate the clot from the sides of the tube, and centrifugalise thoroughly.

Pipette off the clear serum to a clean sterilised test-tube.

4. Erythrocyte solution, in test-tube labelled E.

Collect and wash human red blood cells (see page 388, 1-8).

Measure the volume of red cells available and prepare a 2 per cent. suspension in normal saline solution.

METHOD.--

1. Take two test-tubes and number them 1 and 2, and pipette into each 9 c.c. of normal saline solution.

2. Add 1 c.c. of haemolytic rabbit serum to tube No. 1 and mix thoroughly: take up 1 c.c. of the mixture and add it to tube No. 2; mix thoroughly.

3. Set up ten small test-tubes in test-tube rack or in roll of plasticine, and number 1 to 10.

4. Pipette into tube No. 1 0.5 c.c. = 0.5 c.c.} haemolytic serum } From tube Pipette into tube No. 2 0.1 c.c. = 0.1 c.c. } H. S.

haemolytic serum }

Pipette into tube No. 3 0.5 c.c. = 0.05 c.c. } haemolytic serum } Pipette into tube No. 4 0.3 c.c. = 0.03 c.c. } haemolytic serum } From Pipette into tube No. 5 0.2 c.c. = 0.02 c.c. } tube 1.

haemolytic serum } pipette into tube No. 6 0.1 c.c. = 0.01 c.c. } haemolytic serum }

Pipette into tube No. 7 0.5 c.c. = 0.005 c.c. } haemolytic serum } Pipette into tube No. 8 0.3 c.c. = 0.003 c.c. } haemolytic serum } From Pipette into tube No. 9 0.2 c.c. = 0.002 c.c. } tube 2.

haemolytic serum } Pipette into tube No. 10 0.1 c.c. = 0.001 c.c. } haemolytic serum }

5. To each tube add 1 c.c. of erythrocyte solution.

6. When necessary (that is to say in tubes 2, 4, 5, 6, 8, 9 and 10) add normal saline solution to the mixture in the test-tubes till the column of fluid in each reaches to the same level.

7. Shake each tube in turn, so as to thoroughly mix its contents. Plug the mouth of each tube with cotton wool, and place entire set in the incubator at 37C. for one hour.