required bacterium (e. g.,
Diplococcus pneumoniae) grown upon sloped blood agar at 37 deg. 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.

[Illustration: 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).

[Illustration: 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 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