~The Macroscopical Reaction:~

Sterile graduated capillary pipettes to contain 90 c. mm.

Eighteen to twenty-four-hours-old bouillon cultivation of the organism to be tested.

Three test-tubes containing the 10, 1, and 0.1 per cent.

solutions of specific serum (about 90 c. mm. remaining in each).

Tube containing 50 per cent. solution of pooled serum.

Sedimentation pipettes (_vide_ page 17) or teat pipettes.

METHOD.

1. Pipette 90 c. mm. of the bouillon culture into each of the tubes containing the diluted serum; and the same quant.i.ty into the tube containing the pooled serum.

2. Fill a sedimentation tube (by aspirating) or a teat pipette from the contents of each tube. Seal off the lower ends of the sedimentation tubes in the Bunsen flame.

3. Label each tube with the dilution of serum that it contains--viz., 5, 0.5, and 0.05 per cent.

4. Place the pipettes in a vertical position, in a beaker, in the incubator at 37C., for one or two hours.

5. Observe the granular precipitate which is thrown down when the reaction is positive, and the uniform turbidity of the negative reaction as compared with the appearances in the control pooled serum.

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 gla.s.s box or tray to hold pipettes.

Incubator regulated at 37C.

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 ma.s.s 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 ma.s.s 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 pneumoniae) 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 ma.s.ses 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 ma.s.ses. If unsatisfactory prepare another emulsion, this time sc.r.a.ping up the surface growth with a platinum spatula, transferring it to an agate mortar and grinding it up with successive small quant.i.ties of normal saline. If satisfactory insert the tube in the plasticine block next to that containing the washed cells.