The Elements of Bacteriological Technique Part 99

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.

Gla.s.s 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 hyperaemic 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.

[Ill.u.s.tration: FIG. 192.--Collecting blood from rabbit.]

7. Place the pipette down on a cool surface (e. g., a gla.s.s 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 t.i.tle 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 ma.s.s 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 gla.s.s cutting knife and snapping the tube at that point, the blood-serum becomes readily accessible for testing purposes.

If larger quant.i.ties of blood are required, the animal, after puncturing the vein, should be inverted, an a.s.sistant 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.

AGGLUTININ.

Agglutinin is the name given to a substance present in the blood-serum of an animal that has successfully resisted inoculation with a certain micro-organism. This substance possesses the power of collecting together in clumps and ma.s.ses, or agglutinating watery suspensions of that particular microbe.

~Dilution of the Specific Serum~:

_Apparatus required_:

Sterile graduated capillary pipettes to contain 10 c. mm. (Fig. 17).

Sterile graduated capillary pipettes to contain 90 c. mm. (Fig. 17).

Small sterile test-tubes 5 0.5 cm.

Normal saline solution in flask or test-tube.

Pipette of specific serum.

Gla.s.s cutting knife, or three-square file.

Gla.s.s capsule, nearly full of dry silver sand, or roll of plasticine.

Grease pencil.

METHOD.--

1. Take three sterile test-tubes and number them 1, 2 and 3.

2. Pipette 0.9 c.c. sterile normal saline solution into each tube, and stand tubes upright in the sand in the capsule, or in the plasticine block.

3. Make a scratch with the gla.s.s cutting knife on the blood pipette above the upper level of the clear serum, and snap off and discard the empty portion of the tube.

4. Remove 0.1 c.c. of the serum from the blood pipette tube, and mix it thoroughly with the fluid in tube No. 1; and label ~s.s.~, (specific serum), 10 per cent.

5. Remove 0.1 c.c. of the solution from tube No. 1 by means of a fresh pipette, and mix it with the contents of tube No. 2; and label ~s.s.~, 1 per cent.

6. Remove 0.1 c.c. of the solution from tube No. 2 by means of a fresh pipette, and mix it with the contents of tube No. 3; and label ~s.s.~, 0.1 per cent.

When the yield of serum from the specimen of blood which has been collected, or is available, is small, the above method of diluting is not practicable, and the dilution should be carried out by Wright's method in a capillary teat pipette.

~Dilution of Serum by Means of a Teat Pipette.~

_Materials required:_

Blood pipette containing sample of specific serum after centrifugalisation.

Capsule of diluting fluid--normal saline solution.

Supply of Pasteur pipettes (Fig. 13a).

India-rubber teats.

Small test-tubes.

A block of plasticine to act as a test-tube stand.

Grease pencil.

METHOD:

1. Mark three small test-tubes 10 per cent., 1 per cent. and 0.1 per cent. respectively, and stand them upright in the plasticine block.

2. Take a Pasteur pipette, nick the capillary stem just above the sealed end with a gla.s.s cutting knife, and snap off the sealed end with a quick movement so that the fracture is clean cut and at right angles to the long axis of the capillary stem--cut "square", in fact. Prepare several, say a dozen, in this manner.