~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 s.p.a.ce in plasticine block.

[Ill.u.s.tration: FIG. 194.--Plasticine block with materials arranged for opsonin estimations.]

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).

[Ill.u.s.tration: FIG. 195. Opsonin pipette.]

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 gla.s.s 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 gla.s.s staining rack which can be lowered in turn into each of a series of gla.s.s 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.

[Ill.u.s.tration: FIG. 196. Gla.s.s staining trough for blood films.]

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_.

IMMUNE BODY.

Immune body or amboceptor is the name given to a substance present in the serum of an infected animal that has successfully resisted inoculation with some particular micro-organism, and which possesses the power of linking the complement normally present in the serum to bacteria of the species used as antigen in such a manner that the micro-organisms are rendered innocuous, and ultimately destroyed. The presence of the immune body in the serum can be demonstrated _in vitro_ by the reaction elaborated by Bordet and Gengou, known as the complement fixation test, the existence or the absence of the phenomenon of complement fixation being rendered obvious macroscopically by the absence or presence of haemolysis on the subsequent addition of "sensitised" red blood corpuscles, (e. g., a mixture of crythrocyte solution and the appropriate haemolysin--two of the three essentials in the haemolytic system, _vide_ page 326).

_Apparatus Required:_

Sterile pipettes 1 c.c., (graduated in tenths).

16 2 cm. test-tubes.

9 1 cm. test-tubes.

Test-tube racks for each size of test-tube.

_Reagents Required:_

Normal saline solution.

Erythrocyte solution (human red cells, page 329) = E.

Haemolytic serum (for human cells) = H.S.

Complement (fresh guinea-pig serum) = C.

Specific serum from inoculated animal, inactivated = S.S.

Control pooled serum from normal animals of same species, Inactivated = P.S.

_Antigen_ (cultivation upon solid medium of the organism (e. g., B. typhosus) which has already served as antigen in the inoculation of the experimental animal) = A.

To prepare the antigen for use, emulsify the whole of the bacterial growth in 5 c.c. normal saline solution.

Shake the emulsion in a test-tube with some sterilised gla.s.s beads to ensure a h.o.m.ogenous emulsion, and sterilise by heating to 60 C. in a water-bath for one hour.

METHOD.--

1. Take five small test-tubes, and number them 1 to 5 with a grease pencil.

2. Into tubes Nos. 1, 3, 4 and 5 pipette 0.1 c.c. of complement.

3. Into tubes Nos. 1 and 2 pipette 0.2 c.c. of the serum to be tested.

4. Into tube No. 4 pipette 0.2 c.c. of control serum.