capsule III (1 c.c. of bouillon in capsule III contains 0.00001 loop),
and then from capsule III to capsule IV (1 c.c. of bouillon in capsule
IV contains 0.0000001 loop).
The dilutions thus prepared may be summarised in a table;
Capsule I = 1 loopful + 10 c.c. water [.'.] 1 c.c.=0.1 loop.
Capsule II = 0.1 c.c. capsule I + 9.9 c.c. water [.'.] 1 c.c.=0.001 loop.
Capsule III = 0.1 c.c. capsule II + 9.9 c.c. water [.'.] 1 c.c.=0.00001 loop.
Capsule IV = 0.1 c.c. capsule III + 9.9 c.c. water
[.'.] 1 c.c. = 0.0000001 loop.
6. With sterile graduated pipettes remove the necessary quantity of
bouillon corresponding to the various divisors of ten of the loop from
the respective capsules, and transfer each "dose" to a separate sterile
capsule and label; and to such doses as are small in bulk, add the
necessary quantity of sterile bouillon to make up to 1 c.c.
7. Multiples of the loop are prepared by emulsifying 1, 2, 5, or 10
loops each with 1 c.c. sterile bouillon in separate sterile capsules.
8. Inoculate a series of animals with these measured doses, filling the
syringe first from that capsule containing the smallest dose, then from
the capsule containing the next smallest, and so on. If care is taken,
it will not be found necessary to sterilise the syringe during the
series of inoculations.
9. Plant tubes of gelatine or agar, liquefied by heat, from each of the
higher dilutions, say from 0.0000001 loop to 0.01 loop; pour plates and
incubate. When growth is visible enumerate the number of organisms
present in each, average up and calculate the number of bacteria present
in one loopful of the inoculum.
10. The smallest dose which causes the infection and death of the
inoculated animal is noted as the minimal lethal dose.
_Toxins._--
Prepare flask cultivations of the organism under observation in glucose
formate broth, and incubate for fourteen days under optimum conditions.
(a) Intracellular or Insoluble Toxins:
1. Heat the fluid culture in a water-bath at 60 deg. C. for thirty
minutes. (The resulting sterile, turbid fluid is often spoken of as
"killed" culture,)
2. Inoculate a tube of sterile bouillon with a similar quantity, and
incubate under optimum conditions. This "control" then serves to
demonstrate the freedom of the toxin from living bacteria.
[Illustration: FIG. 160.--Apparatus arrange for toxin filtration.]
3. Inject intraveneously that amount of the cultivation corresponding to
1 per cent. of the body-weight of the selected animal, usually one of
the small rodents.
4. Observe during life or until the completion of twenty-eight days, and
in the event of death occurring during that period, make a complete
post-mortem examination.
5. Repeat the experiment at least once. In the event of a positive
result estimate the minimal lethal dose of "killed" culture for each of
the species of animals experimented upon.
(b) Extracellular or Soluble Toxins:
1. Filter the cultivation through a porcelain filter candle (Berkefeld)
into a sterile filter flask, arranging the apparatus as in the
accompanying figure (Fig. 160).
2. Inoculate mice, rats, guinea-pigs, and rabbits subcutaneously with
that quantity of toxin corresponding to 1 per cent. of the body-weight
of each respectively, and observe, if necessary, until the completion of
one month.
3. Inoculate a "control" tube of bouillon with a similar quantity and
incubate, to determine the freedom of the filtered toxin from living
bacteria.
4. In the event of a fatal termination make complete and careful
post-mortem examinations.
5. Repeat the experiments and, if the results are positive, ascertain
the minimal lethal dose of toxin for each of the susceptible animals.
The estimation of the _m. l. d._ of a toxin is carried out on lines
similar to those laid down for living bacteria (_vide_ page 316) merely
substituting 1 c.c. of toxin as the unit in place of the unit "loopful"
of living culture.
It frequently happens, during the course of casual investigations that a
bouillon-tube culture is available for a toxin test whilst a flask
cultivation is not. In such cases, Martin's small filter candle and tube
(Fig. 161) specially designed for the filtration of small quantities of
fluid, is invaluable. This consists of a narrow filter flask just large
enough to accommodate an ordinary 18 x 2 cm. test-tube. The mouth of the
tubular Chamberland candle 15 x 1.5 cm. is closed by a perforated rubber
cork into which fits the end of the stem of a thistle headed funnel,
whilst immediately below the butt of the funnel is situated a rubber
cork to close the mouth of the filter flask. When the apparatus is fixed
in position and connected to an exhaust pump, the cultivation is poured
into the head of the funnel and owing to the relatively large filtering
surface the germ free filtrate is rapidly drawn through into the
test-tube receiver.
~Raising the Virulence of an Organism.~--If it is desired to raise or
"exalt" the virulence of a feebly pathogenic organism, special methods
of inoculation are necessary, carefully adjusted to the exigencies of
each individual case. Among the most important are the following:
1. _Passage of Virus._--The inoculation of pure cultivations of the
organism into highly susceptible animals, and passing it as rapidly as
possible from animal to animal, always selecting that method of
inoculation-e. g., intraperitoneal--which places the organism under
the most favorable conditions for its growth and multiplication.
[Illustration: FIG. 161--Martin's filtering apparatus for small
quantities of fluid.]
2. _Virus Plus Virulent Organisms._--The inoculation of pure
cultivations of the organism together with pure cultivations of some
other microbe which in itself is sufficiently virulent to ensure the
death of the experimental animal, either into the same situation or into
some other part of the body. By this association the organism of low
virulence will frequently acquire a higher degree of virulence, which
may be still further raised by means of "passages" (_vide supra_).
3. _Virus Plus Toxins._--The inoculation of pure cultivations of the
organism into some selected situation, together with the subcutaneous,
intraperitoneal, or intravenous injection of a toxin--e. g., one of
those elaborated by the proteus group--either simultaneously with,
before, or immediately after, the injection of the feeble virus. By
this means the natural resistance of the animal is lowered, and the
organism inoculated is enabled to multiply and produce its pathogenic
effect, its virulence being subsequently exalted by means of "passages."
~Attenuating the Virulence of an Organism.~--Attenuating or lowering the
virulence of a pathogenic microbe is usually attained with much less
difficulty than the exaltation of its virulence, and is generally
effected by varying the environment of the cultivations, as for example:
1. Cultivating in such media as are unsuitable by reason of their (a)
composition or (b) reaction.
2. Cultivating in suitable media, but at an unsuitable temperature.
3. Cultivating in suitable media, but in an unsuitable atmosphere.
4. Cultivation in suitable media, but under unfavorable conditions as to
light, motion, etc.
Attenuation of the virus can also be secured by
5. Passage through naturally resistant animals.
6. Exposure to desiccation.
7. Exposure to gaseous disinfectants.
8. By a combination of two or more of the above methods.
IMMUNISATION.
The further study of the pathogenetic powers of any particular bacterium
involves the active immunisation of one or more previously normal
animals. This end may be attained by various means; but it must be
remembered that immunisation is not carried out by any hard and fast
rule or by one method alone, but usually by a combination of methods
adapted to the exigencies of each particular case. The ordinary methods
include:
A. Active Immunisation.
I. By inoculation with dead bacteria (i. e., bacteria
killed by heat; the action of ultra-violet rays, of chemical
germicides, or by autolysis).
II. By the inoculation of attenuated strains of bacteria.
III. By the inoculation of living virulent bacteria (exalted
in virulence if necessary).
B. Combined Active and Passive Immunisation:
IV. By the inoculation of toxin-antitoxin mixtures.
ACTIVE IMMUNISATION.
The immunisation of the rabbit against the Diplococcus pneumoniae may be
instanced as an example of the general methods of immunisation of
laboratory animals.
1. Take a full grown rabbit weighing not less than 1200 to 1500 grammes
(large rabbits of 2000 grammes and over are the most suitable for
immunising experiments). Observe weight and temperature carefully during
the few days occupied in the following steps.
2. Inoculate a small rabbit intraperitoneally with one or two loopfuls
of a twenty-four-hour-old blood agar cultivation of a _virulent_ strain
of Diplococcus pneumoniae.
Death should follow within twenty-four hours, and in any case will not
be delayed beyond forty-eight hours.
3. Under aseptic precautions, at the post-mortem, transfer a loopful of
heart blood to an Erlenmeyer flask containing 50 c.c. sterile nutrient
broth. Incubate at 37 deg. C. for twenty-four hours.
4. Prepare also several blood agar cultures from the heart blood of the
rabbit, label them all O.C. (original culture). After twenty-four hours
incubation at 37 deg. C. place an india-rubber cap over the plugged mouth
of the tube of all but one of these cultures and paint the cap with Canada
balsam or shellac varnish, dry, and replace in the hot incubator.
This will prevent evaporation, and cultures thus sealed will remain
unaltered in virulence for a considerable time.
5. Make a fresh subcultivation on blood agar from the uncapped O.C.
cultivation and after twenty-four hours incubation at 37 deg. C.
determine the minimal lethal dose of this strain upon a series of mice
(see page 316).
6. Suspend the flask containing the twenty-four-hour-old broth culture
(step 3) in the water-bath at 60 deg. C. for one hour. Cool the flask
rapidly under a stream of cold water.
7. Determine the sterility of this (?) killed cultivation by
transferring one cubic centimetre to each of several tubes of nutrient
broth, and incubate at 37 deg. C. for twenty-four hours. If growth of
Diplococcus pneumoniae occurs, again heat culture in water-bath at 60
deg. C. for one hour and again test for sterility.
8. Inject the selected rabbit intravenously (see page 363) with 2 c.c.
of the killed cultivation, and inject a further 10 c.c. into the
peritoneal cavity.
During the next few days the animal will lose some weight and perhaps
show a certain amount of pyrexia.
9. When the temperature and weight have again returned to
normal--generally about seven days after the inoculation--again inject
killed cultivation, this time giving a dose of 5 c.c. intravenously and
20 c.c. intraperitoneally. A temperature and weight reaction similar to,
but less marked than that following the first injection will probably be
observed, but after about a week's interval the animal will be ready for
the next injection.
10. When ready to give the third injection prepare a fresh blood agar
subculture from another O.C. tube and after twenty-four hours incubation
prepare a minimal lethal dose (as determined in 5) and inject it
subcutaneously into the rabbit's abdominal wall.
A slight local reaction will probably be observed as well as the weight
and temperature reactions.
11. A week to ten days later inject a similar minimal lethal dose into
the peritoneal cavity.
12. Observe the weight and temperature of the rabbit very carefully, and
regulating the dates of inoculation by the animal's general condition,
continue to inject living cultivations of the pneumococcus into the
peritoneal cavity, gradually increasing the dose by multiples of ten.
13. At intervals of two months samples of blood may be collected from
the posterior auricular vein and the serum tested for specific
antibodies.
14. Under favourable conditions it will be found after some six months
steady work that the rabbit may be injected intraperitoneally with an
entire blood agar cultivation without any ill effects being apparent;
and this characteristic--resistance to the lethal effects of large doses
of the virus--is the sole criterion of _immunity_. Further, the serum
separated from blood withdrawn from the animal about a week after an
injection, if used in doses of .01 c.c., will protect a mouse against
the lethal effects of at least ten minimal lethal doses of living
pneumococci.
In the foregoing illustration it has been assumed that complete acquired
active immunity has been conferred upon the experimental rabbit in
consequence of the formation of antibody, specific to the diplococcus
pneumoniac, sufficient in amount to ensure the destruction of enormous
doses of the living cocci--the _antigen_ (that is the substance injected
in response to which _antibody_ has been elaborated) in this particular
case being the bacterial protoplasm of the pneumococcus with its
endo-toxins.
But provided death does not immediately follow the injection of the
antigen, specific antibody is always formed in greater or lesser amount;
and in experimental work a sufficient amount of any required antibody
can often be obtained without carrying the process of immunisation to
its logical termination.
For instance, if the immunisation of a rabbit toward Bacillus typhosus
is commenced on the lines already set out it will often be found, after
a few injections of "killed" cultivation that the blood serum of the
animal (even when diluted with several hundred times its volume of
normal saline) contains specific agglutinin for B. typhosus--and if the
sole object of the experiment has been the preparation of agglutinin the
inoculations may well be stopped at this point, although the animal is
not yet immune in the strict meaning of the word.
Again, antibodies may be formed in response to antigens other than
infective particles--thus the injection into suitable animals of foreign
proteins such as egg albumin, heterologous blood sera or red blood discs
from a different species of animal, will result in the formation of
specific antibodies possessing definite affinities for their respective
antigens.
The most important antibody of this latter type is Haemolysin, a
substance that makes its appearance in the blood serum of an animal
previously injected with washed blood cells from an animal of a
different species. The serum from such an animal possesses the power of
disintegrating red blood discs of the variety employed as antigen and
causing the discharge of their contained haemoglobin, and is specific in
its action to the extent of failing to exert any injurious effect upon
the red blood cells of any other species of animal.
The action of this serum is due to the presence of two distinct bodies,
complement and haemolysin.
_Complement_ (or alexine) is a thermo-labile readily oxidised body
present in variable but unalterable amount in the normal serum of every
animal. It is a substance which exerts a lytic effect upon all foreign
matter introduced into the blood or tissues; but by itself is a
comparatively inert body, and is only capable of exerting its maximum
lytic effect in the presence of and in combination with a specific
antibody, or immune body.
Complement is obtained (unmixed with antibody) by collecting fresh blood
serum from any healthy normal (that is uninoculated) animal.
Guinea-pigs' serum is that most frequently employed for experimental
work.
_Haemolysin_ (immune body, copula, sensitising body, amboceptor) is a
_thermostable_ antibody formed in response to the injection of red cells
which although in itself inert is capable of linking up complement
present in the normal serum to the red cells of the variety used as
antigen--a combination resulting in haemolysis.
Haemolysin is obtained by collecting fresh blood serum from a suitably
inoculated animal and exposing it to a temperature of 56 deg. C. (to
destroy the thermo-labile complement) for 15 to 30 minutes before use.
It is then referred to as _inactivated_, and is _reactivated_ by the
addition of fresh normal serum--that is serum containing complement.
Haemolysin is of importance academically owing to the fact that many of
the problems of immunity have been elucidated by its aid; but its
present practical importance lies in the application of the _haemolytic
system_ (that is haemolysin, corresponding erythrocyte solution and
complement) to certain laboratory methods having for their object either
the identification of the infective entity or the diagnosis of the
existence of infection.
For use in these laboratory methods of diagnosis it is most convenient
to prepare haemolytic serum specific for human blood--whether the
laboratory is isolated or attached to a large hospital. Ox blood, sheep
blood or goat blood if readily obtainable, may however be used instead,
and although the following method is directed to the preparation of
human haemolysin the same procedure serves in all cases.
THE PREPARATION OF HAEMOLYTIC SERUM.
_Apparatus Required:_
Small centrifuge, preferably electrically driven, with two
receptacles for tubes, and enclosed in a safety shield (Fig. 162).
Sterile centrifuge tubes (10 c.c. capacity), Fig. 163.
Sterile pipettes (10 c.c. graduated) in case.
Sterile glass capsules (in case).
Sterile test-tubes.
Sterile all glass syringe (5 c.c. or 10 c.c. capacity)
and needle.
[Illustration: FIG. 162.--Small electrical centrifuge.]
[Illustration: FIG. 163.--Centrifuge tube.]
_Reagents Required:_
Normal saline solution.
10 per cent. sodium citrate solution in normal saline.
Human blood (_vide infra_).
METHOD.--
1. Select a healthy full-grown rabbit of not less than 2500 grammes
weight in accordance with the directions already given (page 322) and
prepare it for intraperitoneal inoculation.
2. Measure out 2 c.c. citrated human blood (collected at a surgical
operation or a venesection, or withdrawn by venipuncture from the median
basilic or median cephalic vein of a normal adult) into a centrifuge
tube and centrifugalise thoroughly.
3. Wash with three changes of normal saline (_vide_ also page 388).
4. Transfer the washed cells to a sterile capsule by means of a sterile
pipette. Add 5 c.c. of normal saline and mix thoroughly.
5. Take up the mixture of cells and saline in the all-glass syringe and
inject into the peritoneal cavity of the rabbit.
6. Seven days later inject intraperitoneally the washed cells from 5
c.c. human blood mixed with 5 c.c. normal saline.
7. Seven days later inject the washed cells from 10 c.c. human blood
mixed with 5 c.c. normal saline.
8. After a further interval of seven days repeat the injection of washed
cells from 10 c.c. human blood mixed with 5 c.c. normal saline.
NOTE.--Better results are obtained if the second and
subsequent injections are made intravenously, even when
smaller quantities of washed red cells are employed. If,
however, the intravenous route is selected exceeding great
care must be exercised to avoid the introduction of air into
the vein--an accident which is followed, within a few
minutes, by the death of the rabbit from pulmonary embolism.
9. Allow five days to elapse, then collect a preliminary sample of
blood, say about 2 c.c., from the rabbit's ear. Allow it to clot,
separate off the serum and transfer to a sterile test-tube. Place the
test-tube in a water-bath at 56 deg. C. for fifteen minutes (to
inactivate) and test the serum quantitatively for haemolytic properties
in the following manner:
THE TITRATION OF HAEMOLYTIC SERUM.
_Apparatus Required:_
Electrical centrifuge.
Sterile centrifuge tubes.
Water-bath regulated at 56 deg. C.
Sterilised pipettes 10 c.c. graduated in tenths.
Sterilised pipettes 1 c.c. graduated in tenths.
Sterile test-tubes, 16 x 2 cm.
Small sterile test-tubes, 9 x 1 cm.
Small test-tube rack, or roll of plasticine.
Capillary teat pipettes.
Stout rubber band or length of small rubber tubing.
_Reagents Required and Method of Preparation:_
1. Normal saline solution.
2. Haemolytic serum inactivated by preliminary heating to 56 deg.
C. for 15 minutes (_vide supra_) in test-tube labelled H. S.
3. Complement. Fresh guinea-pig serum in test-tube labelled
C.
Kill a normal guinea-pig with chloroform vapour.
Open the thorax with all aseptic precautions, and collect as
much blood as possible from the heart with a sterile Pasteur
pipette.
Transfer it to a sterile centrifuge tube and place the tube
in the incubator at 37 deg. C. Two hours later separate the clot
from the sides of the tube, and centrifugalise thoroughly.
Pipette off the clear serum to a clean sterilised test-tube.
4. Erythrocyte solution, in test-tube labelled E.
Collect and wash human red blood cells (see page 388, 1-8).
Measure the volume of red cells available and prepare a 2
per cent. suspension in normal saline solution.
METHOD.--
1. Take two test-tubes and number them 1 and 2, and pipette into each 9
c.c. of normal saline solution.
2. Add 1 c.c. of haemolytic rabbit serum to tube No. 1 and mix
thoroughly: take up 1 c.c. of the mixture and add it to tube No. 2; mix
thoroughly.
3. Set up ten small test-tubes in test-tube rack or in roll of
plasticine, and number 1 to 10.
4. Pipette into tube No. 1 0.5 c.c. = 0.5 c.c.}
haemolytic serum } From tube
Pipette into tube No. 2 0.1 c.c. = 0.1 c.c. } H. S.
haemolytic serum }
Pipette into tube No. 3 0.5 c.c. = 0.05 c.c. }
haemolytic serum }
Pipette into tube No. 4 0.3 c.c. = 0.03 c.c. }
haemolytic serum } From
Pipette into tube No. 5 0.2 c.c. = 0.02 c.c. } tube 1.
haemolytic serum }
pipette into tube No. 6 0.1 c.c. = 0.01 c.c. }
haemolytic serum }
Pipette into tube No. 7 0.5 c.c. = 0.005 c.c. }
haemolytic serum }
Pipette into tube No. 8 0.3 c.c. = 0.003 c.c. }
haemolytic serum } From
Pipette into tube No. 9 0.2 c.c. = 0.002 c.c. } tube 2.
haemolytic serum }
Pipette into tube No. 10 0.1 c.c. = 0.001 c.c. }
haemolytic serum }
5. To each tube add 1 c.c. of erythrocyte solution.
6. When necessary (that is to say in tubes 2, 4, 5, 6, 8, 9 and 10) add
normal saline solution to the mixture in the test-tubes till the column
of fluid in each reaches to the same level.
7. Shake each tube in turn, so as to thoroughly mix its contents. Plug
the mouth of each tube with cotton wool, and place entire set in the
incubator at 37 deg. C. for one hour.
8. Remove the tubes from the incubator and into each tube pipette 0.1
c.c. complement (guinea-pig's serum) and replace tubes in incubator at
37 deg. C. for further period of one hour.
9. Remove the tubes from the incubator, and if complete haemolysis has
not taken place in every tube, stand on one side, preferably in the ice
chest, for an hour.
10. Then examine the tubes.
Complete haemolysis is indicated by a clear red solution,
with no deposit of red cells at the bottom of the test-tube.
Absence of haemolysis is indicated by a clear or turbid
colourless fluid, with a deposit of red cells at the bottom
of the test-tubes.
The smallest amount of haemolytic serum that has caused complete
haemolysis is known as the minimal haemolytic dose (_M. H. D._) and if
haemolysis has occurred in all the tubes down to No. 7--the m. h. d. of
this particular serum is .005 c.c. = 200 minimal haemolytic doses per
cubic centimetre. Such a serum is strong enough for experimental work;
indeed, for many purposes, complete haemolysis down to tube 6 will
indicate a serum sufficiently strong(= 100 m. h. d. per cubic
centimetre). If, however, only the first one or two tubes are completely
haemolysed, this is an indication that the rabbit should receive further
injections in order to raise the haemolytic power to a sufficiently high
level.
STORAGE OF HAEMOLYSIN.
If, and when the haemolysin content of the rabbit's serum is found to be
sufficient, destroy the animal by chloroform vapour.
Remove as much of its blood as possible from the heart under aseptic
precautions into sterilized centrifuge tubes.