Opsonin

Opsonin is the term applied by Wright to a substance, present in the serum of an inoculated animal, which is able to act upon or sensitise bacteria of the species originally injected, so as to render them an easy prey to the phagocytic activity of polymorphonuclear leucocytes. In the method for demonstrating opsonin about to be described, a comparison is made between the opsonic "power" of the pooled serum and the specific serum.

Apparatus:

Small centrifuge and tubes for same (made from the barrels of broken capillary pipettes by sealing the conical ends in the bunsen flame).

Capillary Pasteur pipettes.

India-rubber teats.

Grease pencil.

Bunsen burner with peep flame.

Electrical signal clock (see page 39) stop watch, or watch.

Rectangular glass box or tray to hold pipettes.

Incubator regulated at 37°C.

3 × 1 slides.

Piece of light rubber tubing.

Rectangular block of plasticine.

Flask of normal saline solution.

Flask of sodium citrate (1.5 per cent.) in normal saline solution.

Materials required, and their preparation:

Small tube of "washed cells" (red blood discs and leucocytes); human cells are used in estimating the opsonising power of the serum of experimental animals.

Small tube of emulsion of bacteria of the species responsible for the infection of the experimental animal.

Blood pipette containing specific serum.

Blood pipette containing "pooled" serum.

Washed Cells.—

1. Take a small centrifuge tube and half fill it with sodium citrate solution. Mark with the grease pencil the upper limit of the fluid.

2. Cleanse the skin of the distal phalanx of the second finger of the left hand above the root of the nail with lint and ether. Wind the rubber tubing tightly round the second phalanx; puncture with a sterile Hagedorn needle through the cleansed area of skin.

3. Take up a sufficiency of the issuing blood (more or less according to the number of tests to be performed) with a teat pipette, transfer it to the tube of citrate solution and mix thoroughly. Make a second mark on the tube at the upper level of the mixed citrate solution and blood.

4. Place the tube in the centrifuge, counterpoise accurately and centrifugalise until the blood cells are thrown down in a compact mass occupying approximately the same volume as is included between the two pencil marks.

The column of fluid in the tube now shows clear supernatant fluid (citrate solution and blood plasma) separated from the sharp cut upper surface of the red deposit of corpuscles by a narrow greyish layer of leucocytes.

5. Remove the supernatant column of citrate solution by means of a teat pipette, fill normal saline solution into the tube up to the upper pencil mark, and distribute the blood cells throughout the saline by means of the teat pipette. Centrifugalise as before.

6. Again remove the supernatant fluid and fill in a fresh supply of saline solution and centrifugalise once more.

7. Remove the supernatant saline solution as nearly down to the level of the leucocytes as can be safely done without removing any of the leucocytes.

8. Next distribute the leucocytes evenly throughout the mass of red cells by rotating the tube between the palms of the hands—just as is done with a tube of liquefied medium prior to pouring a plate.

9. Set the tube upright in the plasticine block near to one end.

Bacterial Emulsion.—

1. Take an 18- to 24-hour culture of the required bacterium (e. g., Diplococcus pneumoniæ) grown upon sloped blood agar at 37° C. Pour over the surface of the medium some 5 c.c. of normal saline solution.

2. With a platinum loop emulsify the growth from the surface of the medium as evenly as possible in the saline solution.

3. Allow the tube to stand for a few minutes so that the large masses of growth may settle down; transfer the upper portion of the saline suspension to a centrifuge tube and centrifugalise thoroughly.

4. Examine a drop of the supernatant opalescent emulsion microscopically to determine its freedom from clumps and masses. If unsatisfactory prepare another emulsion, this time scraping up the surface growth with a platinum spatula, transferring it to an agate mortar and grinding it up with successive small quantities of normal saline. If satisfactory insert the tube in the plasticine block next to that containing the washed cells.

Specific Serum.—

Pooled Serum.—

These sera are collected and treated as already described (see page 379), and the portions of the blood pipettes containing them are arranged in the remaining space in plasticine block.

The plasticine block now presents the appearances shown in Fig. 194.

Method for Determining the Opsonic Index.—

1. Take a capillary pipette fitted with a teat, cut the distal end square and make a pencil mark about 2 cm. from the end.

2. Aspirate into the pipette one volume of washed cells, air index, one volume of bacterial emulsion, air index, and one volume of specific serum (see Fig. 195).

3. Mix thoroughly on a 3 by 1 slide by compressing the teat and ejecting the contents of the pipette on to the surface of the slide, relaxing the pressure and so drawing the fluid up into the pipette again. These two processes should be repeated several times; finally take up the mixture in an unbroken column to the central portion of the capillary stem.

4. Seal the point of the pipette in the peep flame of the bunsen burner and remove teat.

5. Mark the pipette (with the grease pencil) with the distinctive number of the serum and place it in the glass box or tray.

6. Take another similarly prepared pipette and aspirate into it equal volumes of washed cells, bacterial emulsion and pooled serum. Treat precisely as in 3 and 4, label it "control" or "N.S." (normal serum) and place in the box by the side of the specific serum preparation.

7. Place the box with the pipettes in the incubator and set the signal clock to ring at 15 minutes (or start the stop watch).

8. At the expiration of the incubation time remove the pipettes from the incubator.

9. Cut off the sealed end of the specific serum preparation. Mix its contents thoroughly as in step 3, and then divide the mixture between two 3 by 1 slips and carefully spread a blood film (vide page 376) on each in such a way that only one-half of the surface of each slide is covered with blood—the free edge of the blood film approximating to the longitudinal axis of the slide.

Allow films to dry and label the slides with writing diamond.

10. Treat the contents of the control pipette in similar fashion.

11. Select the better film from each pair for fixing and staining.

12. Fixing and staining must be carried out under strictly comparable conditions, and to this end the slides are best handled by placing in a glass staining rack which can be lowered in turn into each of a series of glass troughs containing the various reagents (Fig. 196). Place the rack in the first trough which contains the alcoholic solution of Leishman's stain for two minutes to fix.

Transfer to the second trough containing the diluted stain for ten minutes.

Transfer to the third trough containing distilled water, and holding the trough over a sink, run in a stream of distilled water until washing is complete. Remove slides from the rack and dry.

Leishman's stain is the best for routine work for all bacteria other than B. tuberculosis. Films containing tubercle bacilli must of course be stained by the Ziehl Neelsen method.

13. Examine specific serum slide microscopically with 1/12 inch oil immersion. Find the edge of the blood film—along this the bulk of the leucocytes will be collected. Starting at one end of the film move the slide slowly across the microscope stage and as each leucocyte comes into view count and record the number of ingested bacteria. The sum of the contents of the first 50 consecutive polymorphonuclears that are encountered is marked down. (The average number of bacilli ingested per leucocyte = the "phagocytic index.")

14. In precisely similar manner enumerate the bacteria present in the first 50 cells of the control preparation. This number is recorded as the denominator of a vulgar fraction of which the numerator is the number recorded for the specific serum. This fraction, expressed as a percentage of unity = the opsonic index.