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 mass 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
glass cutting knife and snapping the tube at that point, the blood-serum
becomes readily accessible for testing purposes.
If larger quantities of blood are required, the animal, after puncturing
the vein, should be inverted, an assistant 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 deg. 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 masses, 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 x 0.5 cm.
Normal saline solution in flask or test-tube.
Pipette of specific serum.
Glass cutting knife, or three-square file.
Glass 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 glass 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 glass 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.
3. Fit a rubber teat to the barrel of each of the pipettes.
4. Make a mark with the grease pencil on the stem of one of the pipettes
about 2 or 3 cm. from the open extremity.
[Illustration: FIG. 193.--Filling the capillary teat pipette.]
5. Compress the teat between the finger and thumb (Fig. 193) to such an
extent as to drive out the greater part of the contained air.
6. Maintaining the pressure on the teat pass the stem of the pipette
into the capsule holding the saline solution, until the open end of the
pipette is below the level of the fluid.
7. Now cautiously relax the pressure on the teat and let the fluid enter
the pipette and rise in the stem until it reaches the level of the
grease pencil mark. As soon as this point is reached, check the movement
of the column of fluid by maintaining the pressure on the teat, neither
relaxing nor increasing it.
8. Withdraw the point of the pipette clear of the fluid, and again relax
the pressure on the teat very slightly. The column of saline solution
rises higher in the stem, and a column of air will now enter the pipette
and serve as an index to separate the first volume of fluid drawn into
the stem from the next succeeding one.
9. Again introduce the end of the pipette into the fluid and draw up a
second volume of saline to the level of the grease pencil mark, and
follow this with a second air index.
10. In like manner take up seven more equal volumes of saline solution
and their following air bubbles. There are now nine equal volumes of
normal saline in the pipette.
11. Now pass the point of the pipette into the blood tube and dip the
open end below the surface of the serum. Proceeding as before, aspirate
a volume of serum into the capillary stem up to the level of the pencil
mark.
12. Eject the contents of the pipette into the small tube marked 10 per
cent. by compressing the rubber teat between thumb and finger.
13. Mix the one volume of serum with the nine volumes of saline solution
very thoroughly by repeatedly drawing up the whole of the fluid into the
pipette and driving it out again into the test-tube.
14. Now take a clean pipette and proceed precisely as before, 4 to 10.
15. Having aspirated nine equal volumes of saline into this second
pipette, now take up one similar volume of the fluid in the "10 per
cent. tube."
16. Eject the contents of this pipette into the second tube marked 1 per
cent. and mix thoroughly as before.
17. In similar fashion make the 0.1 per cent. solution and transfer to
the third tube.
18. Further dilutions in multiples of ten can be prepared in the same
way, and by varying the number of volumes of diluting fluid or serum any
required dilution can be made (see Appendix, Dilution Tables).
NOTE.--The saline diluting fluid _must always_ be taken into
the pipette first, otherwise if the serum contains a very
large amount of agglutinin the traces of this serum added to
the saline solution may be sufficient to entirely vitiate
the subsequent observations--whilst if more than one sample
of serum is diluted from the same saline solution serious
errors may be introduced into the experiments.
~The Microscopical Reaction:~
_Apparatus Required:_
Five hanging-drop slides (or preferably two slide), with two
cells mounted side by side on each (Fig. 62, a), and one
slide with one cell only.
Vaseline.
Cover-slips.
Platinum loop.
Grease pencil.
Eighteen to twenty-four-hour-old bouillon cultivation of the
organism to be tested (e. g., Bacillus typhi abdominalis)
Pipette end with the remainder of the specific serum
labelled ~s.s.~
Tubes containing the three solutions of the specific serum,
10, 1, and 0.1 per cent. respectively.
Pipette end with pooled normal serum labelled ~p.s.~
METHOD.--
1. Make five hanging-drop preparations, thus:
(a) One loopful of bouillon cultivation + one loopful pooled serum;
label "Control."
(b) One loopful culture + one loopful undiluted specific serum; label
50 per cent.
Mount these two cover-slips on a double-celled slide.
(c) One loopful bouillon culture + one loopful 10 per cent. serum;
label 5 per cent.
Mount this on single-cell slide.
(d) One loopful bouillon culture + one loopful 1 per cent. serum;
label 0.5 per cent.
(e) One loopful bouillon culture + one loopful 0.1 per cent. serum;
label 0.05 per cent.
Mount these two cover-slips on a double-celled slide.
2. Note the time: Examine the control to determine that the bacilli are
motile and uniformly scattered over the field--not collected into
masses.
3. Next examine the 50 per cent. serum preparation.
If agglutinin is present and the test is giving a positive reaction, the
bacilli _will_ be collected in large clumps.
If the test is giving a negative reaction, the bacilli _may_ be
collected in large clumps owing to the viscosity of the concentrated
serum.
4. Observe the 5 per cent. preparation microscopically.
If the bacilli are aggregated into clumps, positive reaction.
If the bacilli are _not_ aggregated into clumps, observe until thirty
minutes from the time of preparation before recording a negative
reaction.
5. Examine the 0.5 and 0.05 per cent. preparations.
These may or may not show agglutination when the result of the
examination of the 5 per cent. preparation is positive, according to the
potency of the specific serum; and by the examination of a series of
dilutions a quantitative comparison of the valency of specific sera from
different sources, or of serum from the same animal at different periods
during the course of active immunisation may be obtained.
NOTE.--The graduated pipettes supplied with Thoma's
haematocytometer (intended for the collection of the specimen
of blood required for the enumeration of leucocytes), giving
a dilution of 1 in 10--i. e., 10 per cent.--may be
substituted for the graduated capillary pipettes referred to
above, if the vessel in which the serum has been separated
is of sufficiently large diameter to permit of their use.
~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 quantity 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 37 deg. C., 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 glass box or tray to hold pipettes.
Incubator regulated at 37 deg. C.
3 x 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 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 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.
[Illustration: FIG. 196. Glass 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 x 2 cm. test-tubes.
9 x 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 glass beads to
ensure a homogenous emulsion, and sterilise by heating to 60 deg. 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.
5. Into tubes Nos. 1, 2, 3 and 4 pipette 1 c.c. of the bacterial
emulsion which forms the antigen.
6. Place the whole set of tubes in the incubator at 37 deg. C. for a
period of one hour.
7. Remove the tubes from the incubator and pipette 1 c.c. erythrocyte
solution and 4 minimal haemolytic doses of the corresponding haemolysin
into each tube.
8. Mix thoroughly and return the tubes to the incubator at 37 deg. C.
for further period of one hour.
9. At the expiration of that time transfer the tubes to the ice chest,
and allow them to stand for three hours.
10. Examine the tubes.
Tubes 3, 4 and 5 should show complete haemolysis; tube 2 should give no
evidence whatever of haemolysis.
These tubes form the controls to the first tube, which contains the
serum to be tested.
In tube No. 1 the absence of haemolysis would indicate the presence in
the serum of the inoculated animal of a specific antibody to the
micro-organism used in the inoculations; since it shows that the
complement has been bound by the immune body to the bacterial antigen,
and none has been left free to enter into the haemolytic system; on the
other hand the presence of haemolysis would show that no appreciable
amount of antibody has yet been formed in response to the inoculations.
In other words, there is an absence of infection, since the complement
remained unfixed at the time of the addition of the erythrocyte solution
and haemolytic serum, and was ready to combine with those reagents to
complete the haemolytic system.
The method may be shown diagramatically as under using the symbols
already indicated
Test-tubes.
1 2 3 4 5
0.1 c.c. C. ........ 0.1 c.c. C. 0.1 c.c. C. 0.1 c.c. C.
0.2 c.c. S.S. 0.2 c.c. S.S. ......... 0.2 c.c. P.S. ........
A. A. A. A. ........
--------------------------------------------------------------------------
Incubate at 37 deg. C. for one hour.
--------------------------------------------------------------------------
1 c.c. E. 1. c.c. E. 1 c.c. E. 1 c.c. E. 1 c.c. E.
H.S.^{4} H.S.^{4} H.S.^{4} H.S.^{4} H.S.^{4}
--------------------------------------------------------------------------
Incubate at 37 deg. C. for one hour.
--------------------------------------------------------------------------
(?) No haemolysis. |__________________________________|
Haemolysis.