Sterilise by boiling

2. Prepare the animal as for intravenous inoculation (see page 363) and introduce the syringe needle into the lumen of the selected vein.

3. Slowly withdraw the piston of the syringe. When sufficient blood has been collected direct the assistant to release the proximal compression of the vein; and withdraw the needle.

4. Remove the needle from the nozzle of the syringe and deliver the citrated blood into a small Ehlenmeyer flask containing about 250 c.c. of nutrient broth.

5. Label, incubate and examine daily until growth occurs or until the expiration of ten days.

c. The serological examination of the blood is directed to the demonstration of the presence of certain specific antibodies in the sera of experimentally infected animals, and within certain limits to an estimation of their amounts.

The chief of these bodies are:

Antitoxin.
Agglutinin.
Precipitin.
Opsonin.
Immune body or Bacteriolysin.

None of these substances are capable of isolation in a state of purity apart from the blood serum, consequently special methods have been elaborated to permit of their recognition. In every instance the behaviour of serum from the experimental animal, which may be termed "specific" serum, is studied in comparison with that of serum from an uninoculated animal of the same species, and which is termed "normal" serum. In view of minor differences in constitution exhibited by the serum of various individuals of the same series, it is usual to employ a mixture of sera obtained from several different normal animals of the same species as the inoculated animal, under the term "pooled serum." The method of collecting blood (e. g., from the rabbit) for serological tests is as follows:

Collection of Serum.

Apparatus required:

Razor.
Liquid soap.
Cotton-wool.
Lysol 2 per cent. solution, in drop bottle.
Ether in drop bottle.
Flat Hagedorn needles.
Blood pipettes (Fig. 16, page 12).
Centrifugal machine.
Centrifuge tubes.
Glass cutting knife.
Bunsen flame.
Writing diamond or grease pencil.

Method.

1. Shave the dorsal surface of the ear over the course of the posterior auricular vein (see Fig. 192).

2. Sterilise the skin by washing with lysol.

The lysol should be applied with sterile cotton-wool and the ear vigourously rubbed, not only to remove superficial scales of epithelium, but also to render the ear hyperæmic and the vein prominent.

3. Remove the lysol with ether dropped from a drop bottle, and allow the ether to evaporate.

4. Puncture the vein with a sterile Hagedorn needle.

5. Take a small blood-collecting pipette (Fig. 161) and hold it at an angle to the ear, one end touching the issuing drop of blood, the other depressed.

The blood will now enter the pipette at first by capillarity; afterward gravity will also come into play and the pipette can be two-thirds filled without difficulty.

6. Hold the tube by the end containing the blood, the clean end pointing obliquely upward—warm this end at the bunsen flame to expel some of the contained air; then seal the clean point in the flame.

7. Place the pipette down on a cool surface (e. g., a glass slide). The rapid cooling of the air in the clean end of the pipette creates a negative pressure, and the blood is sucked back into the pipette, leaving the soiled end free from blood. Seal this end in the bunsen flame.

8. Mark the distinctive title of the specimen (e. g., animal's number) upon the pipette with a writing diamond or grease pencil.

9. When the sealed ends are cold and the blood has clotted, place the pipette on the centrifuge, clean end downward; counterpoise and centrifugalise thoroughly. On removing the pipette from the centrifuge, the red cells will be collected in a firm mass at one end, and above them will appear the clear serum.

10. By marking the blood pipette above the level of the serum with the glass cutting knife and snapping the tube at that point, the blood-serum becomes readily accessible for testing purposes.

If larger quantities of blood are required, the animal, after puncturing the vein, should be inverted, an assistant holding it up by the legs. Blood to the volume of several cubic centimetres will now drop from the punctured vein, and should be caught in a tapering centrifuge tube, the tube transferred to the incubator at 37° C. for two hours, then placed in the centrifugal machine, counterpoised and centrifugalised thoroughly. The three most important of the antibodies referred to which can be demonstrated with a certain amount of facility are agglutinin, opsonin and bacteriolysin; and the methods of testing for these bodies will now be considered.