hours--then centrifugalize thoroughly.
Pipette off the clear serum, and fill in quantities of 1 c.c., into
small glass ampoules or pipettes, and hermetically seal in the blowpipe
flame, care being taken to avoid scorching the serum.
Place the ampoules when filled with serum and sealed, in a water-bath at
56 deg. C. for 30 minutes. This destroys the complement, i. e.,
inactivates the serum, and at the same time, provided the various
operations have been carried out under aseptic precautions, ensures its
sterility. A longer exposure reduces the haemolytic power.
Place the ampoules in a closed metal box and store in the ice chest for
future use.
FOOTNOTES:
[10] The quantities here given are not absolutely correct. If exactitude
is essential the student must calculate the amount required by the aid
of the Percentage Formula, Appendix, page 496.
[11] See Percentage Formula, Appendix, page 496.
XVII. EXPERIMENTAL INOCULATION OF ANIMALS.
The use of living animals for inoculation experiments may become a
necessary procedure in the Bacteriological Laboratory for some one or
more of the following reasons:
A. ~Determination of Pathogenetic Properties of Bacteria already Isolated
in Pure Culture~ (see page 315).
The exact study of the conditions influencing the virulence (including
its maintenance, exaltation and attenuation) of an organism, and precise
observations upon the pathogenic effects produced by its entrance into,
and multiplication within the body tissues can obviously only be carried
out by means of experimental inoculation; whilst many points relating to
vitality, longevity, etc., can be most readily elucidated by such
experiments.
B. ~Isolation of Pathogenetic Bacteria.~
Certain highly parasitic bacteria (which grow with difficulty upon the
artificial media of the laboratory) can only be isolated with
considerable difficulty from associated saprophytic bacteria when
cultural methods alone are employed; but if the mixture of parasite and
saprophytes is injected into an animal susceptible to the action of the
former, the pathogenic organism can readily be isolated from the tissues
of the infected animal. The pneumococcus for example occurs in the
sputum of patients suffering from acute lobar pneumonia, but usually in
association with various saprophytes derived from the mouth and pharynx.
The optimum medium for the growth of the pneumococcus, blood agar, is
also an excellent pabulum for the saprophytes of the mouth, and plate
cultures are rapidly overgrown by them to the destruction of the more
delicate pneumococcus. But inoculate some of the sputum under the skin
of a mouse and three or four days later the pneumococcus will have
entered the blood stream (leaving the saprophytes at the seat of
inoculation) and killed the animal. Cultivations made at the post-mortem
(see page 398) from the mouse's heart blood will yield a pure growth of
the pneumococcus.
C. ~Identification of Pathogenetic Bacteria.~
The resemblances, morphological and cultural, existing between certain
pathogenetic bacteria are in some cases so great as to completely
overwhelm the differences; again the same bacterium may under varying
conditions assume appearances so different from those regarded as
typical or normal as to throw doubt on its identity. In each case a
simple inoculation experiment may decide the point at once. As a
concrete example may be instanced an autopsy on an animal dead from an
unknown infection. Cultivations from the heart blood gave a pure growth
of a typical (capsulated) pneumococcus. Cultivations from the liver gave
a pure growth of what appeared to be a typical (non-capsulated)
Streptococcus pyogenes longus. The latter inoculated into a rabbit
caused the death of the animal from pneumococcic septicaemia, and
cultures from the rabbit's blood gave a pure growth of a typical
(capsulated) pneumococcus.
~D. Study of the Problems of Immunity.~
It is only by a careful and elaborate study of the behaviour of the
animal cell and the body fluids vis-a-vis with the infecting bacterium
that it becomes possible to throw light upon the complex problem whereby
the cell opposes successful resistance to the diffusion of the invading
microbe, or succeeds in driving out the microbe subsequently to the
occurrence of that diffusion.
At the moment, however, our attention is directed to the first of these
broad headings, for it is by the application of the knowledge acquired
in its pursuit that we are able to deal with problems arising under any
of the remainder.
For whatever purpose the inoculation is performed, it is essential that
the experiment should be planned to secure the maximum amount of
information and the minimum of discomfort to the animal used. Every care
therefore must be taken to ensure that the virus is introduced into the
exact tissue or organ selected; and the operation itself must be carried
out with skill and expedition, and under strictly aseptic conditions.
In the course of inoculation studies many instances of natural immunity,
both racial and individual, will be met with; but it must be recollected
that natural immunity is relative only and never absolute, and care be
taken not to label an organism as _non-pathogenic_ until many different
methods of inoculation have been performed upon different species of
animals, combined when necessary with various procedures calculated to
overcome any apparent immunity, and have invariably given negative
results.
In some countries experiments upon animals are only permitted under
direct license from the Government, and then only within premises
specially licensed for the purpose. In England this license is in the
grant of the Home Secretary, and confers the permission to experiment
upon animals under general anaesthesia, provided that after the
experiment is completed the animal must be destroyed before regaining
consciousness. If it is intended to carry out simple hypodermic
inoculations and superficial venesections, Certificate A, granting this
specific permission and dispensing with the necessity for general
anaesthesia must be obtained _in addition to the license_; whilst if the
inoculation entails more extensive operative procedures, and it is
necessary to observe the subsequent course of the infection, should such
occur, the license must be _coupled with Certificate B_--since this
certificate removes the compulsion to destroy the animal whilst under
the anaesthetic. Further special certificates and combinations of
certificates are required if cats, dogs, horses, asses or cattle are to
be the subjects of experiment. Under every certificate it is expressly
stipulated that if the animal shows signs of pain it must be destroyed
immediately.
The animals generally employed in the study of the pathogenic properties
of the various micro-organisms are:
_Cold Blooded._ _Warm Blooded._ _Hot Blooded._
Frog. Mouse. Fowl.
Toad. Rat. Pigeon.
Lizard. Guinea pig.
Rabbit.
Monkey.
~Preparation.~--Before inoculation, the experimental animals should be
carefully examined, to avoid the risk of employing such as are already
diseased: since it must be remembered that in a state of nature, as well
as in captivity, the animals employed for laboratory inoculations are
subject to infection by various animal and vegetable parasites, and in
some instances such infection presents no symptoms which are obvious to
the casual examination; the sex should be noted, the weight recorded,
and the rectal temperature taken. The remaining items of importance are
the time of the inoculation, the material that is inoculated, and the
method of inoculation, and finally under what authority the experiment
is performed. In the author's laboratory these data are entered upon a
pink card which forms part of a card index system. The card further
provides space for notes on the course of the resulting infection, and
carries on the reverse the weight and temperature chart (Figs. 164 and
165).
[Illustration: Fig. 164.--Front of inoculation card.]
~Preliminary Inspection and Examination.~--The preliminary examination
should comprise observation of the animal at rest and in motion; the
appearance of the fur, feathers or scales, inspection of the eyes, and
of external orifices of the body; tactile examination of the body and
limbs, and palpation of the groins and abdomen; and in many cases the
microscopical examination of fresh and stained blood-films.
Some of the commoner forms of naturally acquired infection may be
briefly mentioned, without however touching upon the various fleas, lice
and ticks which at times infect the ordinary laboratory animals.
[Illustration: FIG. 165.--Back of inoculation card.]
~The Rabbit~, particularly in captivity, is subject to attacks of Psoric
Acari, and the infection is readily transmitted to rabbits in
neighbouring cages and also to guinea pigs, but not to rats and mice.
One species (_Sarcoptes minor_ var. _cuniculi_) gives rise to the
ordinary mange. The infection first shows itself as thick yellowish
scales and crusts around the nose, mouth and eyes, spreads to the bases
and outer surfaces of the ears (never to the inside of the concha), to
the fore and hind legs and into the groins and around the genitals. The
acari can be readily demonstrated microscopically in scrapings of the
skin, treated with liquor potassae. Another form of scabies (due to
Psoroptes _communis cuniculi_) commences at the bottom of the concha,
which is filled with whitish-yellow masses consisting of dried crusts,
scales, faeces, and dead acari. The base of the ear is hard and swollen,
and lifting the animal by the ears--as is usually done--gives rise to
considerable pain; indeed this symptom may be the one which first
attracts attention to an infection, which causes progressive wasting and
terminates in death. A mixed infection--sarcoptic plus psorotic
acariasis--is sometimes seen.
If it is decided to try and save animals suffering from infection by
these parasites, they must be segregated, the scabs carefully cleaned
from the infected areas and the denuded surfaces washed with 5 per cent.
solution of Potassium persulphate (a few drops being allowed to run into
the concha), or with a preparation containing equal parts of soft
paraffin and vaseline with a few drops of lysol. This treatment should
be repeated daily until the acarus is destroyed and the animal has
regained its normal condition. The cages should be disinfected and all
neighbouring animals carefully examined, and any which show signs of
infection should be treated in a similar manner. Favus also attacks the
rabbit, and the typical spots are first noted around the base of the
ear.
Infection by _Coccidium oviforme_ is very common, without however
presenting any symptoms by which the infection may be recognised.
Usually the condition is only noted post-mortem, when the liver is found
to be studded with numerous cascating tubercles, which on examination
prove to be cystic areas crowded with coccidia. Sometimes too the liver
of a rabbit dead from some intentional or accidental bacterial infection
is found at the post-mortem to be marked by fine yellowish streaks and
small tubercles due to the embryos of _Taenia serrata_, while the cystic
form (_Cysticercus pisiformis_) is often noted free in the peritoneal
cavity, or invading the mesentery.
Abscess formation from infection with ordinary pyogenic bacteria occurs
naturally in the rabbit, and frequently the animal house of a laboratory
is decimated by an infective septicaemia due to _B. cuniculicida_.
The ~Mouse~ and ~Rat~ suffer from septicaemia, and from the cysticercus
form of _Taenia murina_; the cystic form (_Cysticercus fasciolaris_) of
_T. crassicollis_ has its habitat in their livers. These small rodents are
frequently infected with scabies, but if freely provided with clean
straw will clean themselves by rubbing through it. The mouse is also
attacked by favus, and the rat is often infected with _Trypanosoma
Lewisi_.
The ~Guinea pig~, like the rabbit, suffers from scabies and coccidiosis.
In addition it is often naturally infected with _B. tuberculosis_, and
it is a wise precaution to test animals as soon as they reach the
laboratory by injecting Koch's Old Tuberculin--0.5 c.c. causing death in
the tuberculous cavy within 48 hours.
The ~Monkey~ is naturally prone to tuberculosis, and should be injected
with 1 c.c. Old Tuberculin on arrival in the laboratory. The tissues of
the monkey also serve as the habitat for a Nematode worm parasitic in
cattle (_Oesophagostoma inflatum_) resembling the Anchylostomum, and
this parasite frequently bores through the intestinal wall, and
provokes the formation of small cysts in the immediately adjacent
mesentery. The presence of these cysts may give rise to considerable
speculation at the post-mortem.
The ~Pigeon~ may be infected by _Haemosporidia_, and its blood show the
presence of halteridia. This bird may also be the subject of a bacterial
infection known as pigeon diphtheria; while the fowl may be subject to
scabies and ringworm, or suffer from fowl cholera or fowl
septicaemia--infections due to members of the haemorrhagic septicaemia
group.
~Weighing.~--The larger animals are most conveniently weighed in a decimal
scale provided with a metal cage for their reception instead of the
ordinary pan (Fig. 166). Mice and rats are weighed in a modification of
the letter balance, weighing to 250 grammes, which has a conical wire
cage, (carefully counterpoised) substituted for its original pan (Fig.
167).
[Illustration: FIG. 166.--Rabbit scales.]
~Temperature.~--To take the rectal temperature of any of the laboratory
animals, the animal should be carefully and firmly held by an assistant.
Introduce the bulb of an ordinary clinical thermometer, well greased
with vaseline, just within the sphincter ani. Allow it to remain in this
position for a few seconds, and then push it on gently and steadily
until the entire bulb and part of the stem, as far as the constriction,
have passed into the rectum. Three to five minutes later, the time
varying of course with the sensibility of the thermometer used, withdraw
the instrument and take the reading. The thermometers employed for
recording temperature should be verified from time to time by comparison
with a standard Kew certified Thermometer kept in the laboratory for
that purpose.
[Illustration: FIG. 167.--Mouse scales]
~Cages.~--During the period which elapses between inoculation and death,
or complete recovery, the experimental animals must be kept in suitable
receptacles which can easily be kept clean and readily disinfected.
The _mouse_ is usually stored in a glass jar (Fig. 168) 11 cm. high and
11 cm. in diameter, closed by a wire gauze cover which is weighted with
lead or fastened to the mouth of the jar by a bayonet catch. A small
oblong label, 5 cm. by 2.5 cm., sand-blasted on the side of the
cylinder, is a very convenient device as notes made upon this with an
ordinary lead pencil show up well and only require the use of a damp
cloth to remove them (Fig. 168).
The _rat_ is kept under observation in a glass jar similar, but larger,
to that used for the mouse.
[Illustration: FIG. 168.--Mouse jar.]
[Illustration: FIG. 169.--Tripod.]
A layer of sawdust at the bottom of the jar absorbs any moisture and
cotton-wool or paper shavings should be provided for bedding. The food
should consist of bran and oats with an occasional feed of
bread-and-milk sop.
The use of a metal tripod, on the platform of which are soldered two
small cups for the reception of the food, inside the cage, prevents
waste of food or its contamination with excreta (Fig. 169).
After use the jars and tripods are sterilised either by chemical
reagents or by autoclaving.
The _rabbit_ and the _guinea-pig_ are confined in cages of suitable
size, made entirely of metal (Fig. 170). The sides and top and bottom
are of woven wire work; beneath the cage is a movable metal tray filled
with sawdust, for the reception of the excreta. The cage as a whole is
raised from the ground on short legs. The sides, etc., are generally
hinged so that the cage packs up flat, for convenience of storing and
also of sterilising.
The ordinary rat cage, a rectangular wire-work box, 30 cm. from front to
back, 20 cm. wide, and 14 cm. high, makes an excellent cage for
guinea-pigs if fitted with a shallow zinc tray, 35 cm. by 24 cm., for it
to stand upon.
[Illustration: FIG. 170.--Metal rabbit rage.]
A plentiful supply of straw should be provided for bedding and the food
should consist of fresh vegetables, cabbage leaves, carrot and turnip
tops and the like for the morning meal and broken animal biscuits for
the evening meal. Occasionally a little water may be placed in the cage
in an earthenware dish.
The tray which receives the dejecta should be cleaned out and supplied
with fresh sawdust each day, and the soiled sawdust, remains of food,
etc., should be cremated.
These cages are sterilised after use either by autoclaving or spraying
with formalin.
As ~animal inoculation~ is purely a surgical operation, the necessary
instruments will be similar to those employed by the surgeon, and, like
them, must be sterile. In the performance of the inoculation strict
attention must be paid to asepsis, and suitable precautions adopted to
guard against accidental contamination of the material to be introduced
into the animal. In addition, the hands of the operator should be
carefully disinfected.
The list of apparatus used in animal inoculations given below comprises
practically everything needed for any inoculation. Needless to remark,
all the apparatus will never be required for any one inoculation.
[Illustration: FIG. 171.--Hypodermic syringe with finger rests.]
Apparatus Required for Animal Inoculation:
1. Water steriliser (_vide_ page 33). It is also convenient
to have a second water steriliser, similar but smaller (23
by 7 by 5 cm.), for the sterilisation of the syringes.
2. Injection syringe. The best form is one of the ordinary
hypodermic pattern, 1 c.c. capacity graduated in twentieths
of a cubic centimeter (0.05 c.c.), fitted with finger rests,
but with the leather washers and the packing of the piston
replaced by those made of asbestos (Fig. 171). The
instrument must be easily taken to pieces, and spare parts
should be kept on hand to replace accidental breakage or
loss. Other useful syringes are those of 2 c.c., 5 c.c., 10
c.c., and 20 c.c. capacity. A good supply of needles must be
kept on hand, both sharp-pointed and with blunt ends. To
sterilise the syringe, fill it with water, loosen the
packing of the piston and all the screw joints, place it in
the steriliser and boil for at least five minutes. Disinfect
the syringe _after use_, in a similar manner. The needles,
which are exceedingly apt to rust after being boiled, should
be stored in a pot of absolute alcohol when not in use.
3. Operating table.
4. Surgical instruments. Sterilise these before use by
boiling, and disinfect them _after use_ by the same means.
Wipe perfectly dry immediately after the disinfection is
completed.
Scissors, probe and sharp-pointed.
Dissecting forceps of various patterns.
Pressure forceps.
Retractors (small self retaining Fig. 172).
Aneurism needles, sharp and blunt.
Scalpels, } Keratomes, } with metal handles. Trephines, }
Michel's steel clips and special forceps for applying the
same. These small steel clips enable the operator to easily
and rapidly close skin incisions and are most satisfactory
for animal operations.
Surgical needles.
Needle holder.
Soft rubber catheters, various sizes.
Gum elastic oesophageal bougies with connection to fit
syringe.
[Illustration: FIG. 172. Small self retaining retractors.]
5. Anaesthetic.
(a) General: The safest general anaesthetic for animals is an A. C. E.
mixture, freshly prepared, containing by volume alcohol 1 part,
chloroform 2 parts, ether 6 parts, and should be administered on a
"cone" formed by twisting up one corner of a towel and placing a wad of
cotton-wool inside it, or from a saturated cotton-wool pad packed into
the bottom of a small beaker.
(b) Local:
1. Cocaine hydrochloride, 2 per cent. in adrenalin 1 per mille
solution.
2. Beta-eucaine, 2 per cent. in adrenalin, 1 per mille solution.
3. Ethyl chloride jet.
6. Sterile glass capsules of various sizes.
7. Cases of sterile pipettes { 10 c.c. (in tenths of a cubic centimetre).
{ 1 c.c. (in hundredths of a cubic
centimetre).
8. Flasks (75 c.c.) containing sterilised normal saline solution (or
sterile bouillon).
9. Sterilised cotton-wool. Cotton-wool (absorbent) is packed loosely in
a copper cylinder similar to that used for storing capsules, and
sterilised in the hot-air oven.
10. Sterilised gauze. Gauze is sterilised in the same way as
cotton-wool.
11. Sterilised silk and catgut for sutures. These are sterilised, as
required, by boiling for some ten minutes in the water steriliser.
12. Flexible collodion (or compound tincture of benzoin).
13. Grease pencil.
14. Tie-on celluloid labels, to affix to the cages.
15. Razor.
16. Small pot of warm water.
17. Liquid soap. Liquid soap is prepared as follows: Measure out 100
grammes of soft soap and add to 500 c.c. of 2 per cent. lysol solution
in a large glass beaker; dissolve by heating in a water-bath at about
90 deg. C. Bottle and label "Liquid Soap."
18. In place of the liquid soap and razor it is sometimes convenient to
use a Depilatory powder.
Barium sulphide 1 part
Rice starch 3 parts
Dust the powder thickly over the area to be denuded of hair, sprinkle
with water and mix into a thin paste _in situ_; allow the paste to act
for three minutes, then scrape off with a bone spatula--the hair comes
away with the paste and leaves a perfectly bare patch. This process is
preferably carried out, the day previous to the operation.
~Material Utilised for Inoculation.~--The material inoculated may be
either--
1. Cultures of bacteria--grown in fluid media, or on solid media.
2. Metabolic products of bacterial activity--e. g., toxins in
solution.
3. Pathological products (fluid secretions and excretions, solid
tissues).
~The Preparation of the Inoculum.~--
(a) _Cultivations in Fluid Media._--
1. Flame the plug of the culture tube.
2. Remove the plug and flame the mouth of the tube.
3. Slightly raise the lid of a sterile capsule, insert the mouth of the
culture tube into the aperture and pour some of the cultivation into the
capsule.
4. Remove the mouth of the culture tube from the capsule, replace the
lid of the latter, flame the mouth of the tube, and replug.
5. Remove the syringe from the steriliser, squirt out the water from its
interior, and allow to cool.
6. Raise the lid of the capsule sufficiently to admit the needle of the
syringe and draw the required amount of the cultivation into the barrel
of the syringe.
(Or, remove a definite measured quantity of the cultivation directly
from the tube or flask by means of a sterile graduated pipette,
discharge the measured amount into a sterile capsule, and fill into the
syringe; or take up the required quantity of the cultivation directly
into the graduated syringe from the tube or flask.)
[Illustration: FIG. 173.--Conical separatory funnel, fitted for
injection of fluid cultivations.]