spraying apparatus, such as is used for nasal medicaments.
3. Fill the reservoir of the instrument (previously sterilised) with the
fluid inoculum, and having attached the bellows, spray the inoculum into
the interior of the box.
4. On the completion of the spraying, open the box, spray the animal
thoroughly with a 10 per cent. solution of formaldehyde (to destroy any
of the virus that may be adhering to fur or feathers).
5. Transfer the animal to its cage.
6. Label, etc.
7. Thoroughly disinfect the inhalation chamber.
(b) _Fluid or Powdered Inoculum._--_Anaesthetic, A. C. E._
1. Anaesthetise the animal and secure it firmly to the operating table.
[Illustration: FIG. 190.--Gag for rabbits.]
2. Prop open the mouth by means of some form of gag; seize the tongue
with a pair of forceps and draw it forward.
The most convenient form of gag for the rabbit or cat is that shown in
Fig. 190. It is simply a strip of hard wood shaped at the middle and
provided with a square orifice through which a tracheal or oesophageal
tube can be passed.
3. Pass a previously sterilised glass tube (17 cm. long, 0.5 cm.
diameter, with its terminal 2 cm. slightly curved) down through the
larynx into the trachea.
4. Connect the straight portion of a ~Y~-shaped piece of tubing to the
upper end of the sterilised tube and couple one branch of the ~Y~ to a
separatory funnel containing the fluid inoculum, or insufflator
containing the powdered inoculum, and the other to a hand bellows.
5. Allow the fluid inoculum to run into the lungs by gravity, or blow in
the powdered inoculum by means of a rubber-ball bellows.
6. Remove the intratracheal tube; release the animal from the table.
7. Label, etc.
As an alternative method in the case of fairly large animals, such as
rabbits, etc., a sterile piece of glass tubing of suitable diameter may
be passed through the larynx down the trachea almost to its
bifurcation. Fluid cultivations may then be literally poured into the
lungs, or cultivations, dried and powdered, may be blown into the lung
by the aid of a small hand bellows or even a teat pipette.
~10. Intragastric Inoculation.~--_Fluid or semi-fluid inoculum.
(Anaesthetic none.)_
The method of performing the operation is varied slightly according to
the size of the experimental animal.
_A. Monkey, Rabbit, Guinea-pig._
1. Secure the animal to the operating table ventral surface uppermost.
2. Prop the mouth open with a gag; draw the tongue forward with forceps.
3. Sterilise a soft rubber catheter (No. 10 or 8 English scale, or No.
18 or 15 French) and lubricate it with sterile glycerine.
4. Pass it to the back of the pharynx, keeping the end in the middle
line.
5. Gently assist the progress of the catheter down the oesophagus
until it passes the cardiac orifice of the stomach. Do not use any
force.
6. Take up the required dose of inoculum into a sterilised pipette.
Insert the point of the pipette into the open end of the catheter and
allow the fluid to run down into the stomach. Remove the pipette and
drop it into a jar of lysol.
7. With another sterile pipette run one cubic centimetre of sterile
saline solution through the catheter to wash out the last traces of the
inoculum.
8. Withdraw the catheter.
9. Label, etc.
_B. Rats and Mice (Mark's Method)._
1. Secure the animal in the vertical position.
(a) _Rat._--Take a pair of catch sinus forceps about 22 cm. in length
and seize the animal by the loose skin of the head as far forward as
possible--fix the forceps, and holding the instrument vertically upward,
transfer to the left hand of an assistant who secures the animal's tail
between the fingers grasping the handle of the forceps. (See Fig. 191.)
[Illustration: FIG. 191.--Intragastric inoculation of rat.]
(b) _Mouse._--An assistant grasps the loose skin between the ears as far
forwards as possible between the forefinger and thumb of the left hand.
He now grasps the tail with the right hand, draws the mouse straight and
passes the tail between the fourth and little fingers of the left hand
and secures it there.
2. The assistant takes a closed pair of thin-bladed forceps in his right
hand, passes the ends into the animal's mouth, then allows the blades to
separate. This opens the animal's jaw and serves as a gag.
3. Moisten the sterilised oesophageal tube with sterile water. (This
tube is of silk rubber, 6.5 cm. in length, with the distal end rounded,
the proximal end mounted in a syringe needle head, which fits the
nozzles of the two sterile syringes to be used.)
4. Grasp the tube about its middle and pass it into the animal's mouth,
downwards and a little to one side or the other until its length is lost
in the digestive tract and mouth. Gentle guidance is alone necessary. Do
not use any force.
5. Take up the required dose of inoculum into the syringe; insert the
nozzle of the syringe into the needle-mount, and force the piston down.
6. Steadying the needle-mount with the left hand, detach the syringe.
7. Draw up some sterile water in the second (sterile) syringe, and
inserting its nozzle into the needle-mount force a few drops of water
through the tube to wash it out.
8. With one quick upward movement remove the tube from the animal's
mouth.
9. Label, etc.
One other method of inoculation remains to be described, which does not
require operative interference.
~11. Feeding.~--
1. _Fluid Inoculum._--Small pieces of sterilised bread or sop
(sterilised in the steamer at 100 deg. C.) are soaked in the fluid
inoculum and offered to the animals in a sterile Petri dish or capsule.
2. _Solid Inoculum._--Small pieces of tissue are placed in sterile
vessels and offered to the animals.
FOOTNOTES:
[12] This table is made by Messrs. Down Bros., St. Thomas's Street,
London, S. E.
[13] This modification is made for the author by Messrs. Down Bros., St.
Thomas's Street, London, S. E.
[14] Manufactured by Messrs. Francis Lepper, 56, Great Marlborough
Street, London, W.
XVIII. THE STUDY OF EXPERIMENTAL INFECTIONS DURING LIFE.
The possession of pathogenetic properties by an organism under study is
indicated by the "infection" of the experimental animal--a term which is
employed to summarise the condition resulting from the successful
invasion of the tissues of the experimental animal by the
micro-organisms inoculated and by their multiplication therein.
Infection is considered to have taken place:
1. When the death of the animal is produced as a direct consequence of
the inoculation.
2. When without necessarily producing death the inoculation causes local
or general changes of a pathological character.
3. When either with or without death, or local or general changes
occurring, certain substances make their appearance in the body fluids,
which can be shown (_in vitro_ or _in vivo_) to exert some profound and
specific effect when brought into contact with subcultivations of the
organism originally inoculated.
The important factors in the production of infection are:
A. Seed. Virulence of organism.
Dose of organism.
B. Soil. Resistance offered by the cells of the experimental animal.
The first two factors, although variable, are to a certain extent under
the control of the experimenter. Thus by suitable means the virulence of
an organism can be exalted or attenuated, whilst the size of the dose
may be increased or diminished. The third factor also varies, not only
amongst different species of animals, but also amongst different
individuals of the same species. The essential causes of this variation
are not so obvious, so that beyond selecting the animals intended for
similar experiments with regard to such points as age, size or sex, but
little can be done to standardise cell resistance.
Immediately an animal has been inoculated a period of clinical
observation must be entered upon, which should only terminate with the
death of the animal. The general observations should at first and if the
infection is an acute one, be made daily--later, and if the animal
appears to be unaffected or if the infection is chronic, both general
and special observations should be carried out at weekly intervals. If
the animal appears to be still unaffected, it should be killed with
chloroform vapour at the end of two or three months and a complete
post-mortem carried out.
A. The ~general observations~ should take cognisance of:
1. _General appearance._ The experimental animal should be inspected
daily, not only with a view to detecting symptoms due to the
experimental infection, but also to prevent any intercurrent infection,
naturally acquired, from escaping notice (_vide_ page 337).
2. _The weight_ of the inoculated animal should be observed and recorded
each day during the course of an experimental infection at precisely the
same hour, preferably just before the morning feed.
3. _The temperature_ should similarly be recorded daily, if not more
frequently, during the whole period the animal is under observation, and
carefully charted--individual variations will at once become apparent.
It should be borne in mind that the temperature regarded as normal for
man (37.5 deg. C.) is not the normal average temperature of any of the
lower animals save the rat and mouse. The accompanying table of normal
averages for the animals usually employed in bacteriological research
may be of use in preventing the erroneous assumption that pyrexia is
present in an animal, which merely shows its own normal temperature.
NORMAL AVERAGES.
----------------------------------------------------
| Rectal | Pulse. | Respirations.
Animal. | Temp. |------------------------
| deg. C. | Rate per minute.
----------------------------------------------------
| | |
Frog | 8.9-17.2 | 80 | 12
Mouse | 37.4 | 120 | ...
Rat | 37.5 | ... | 210
Guinea pig | 38.6 | 150 | 80
Rabbit | 38.7 | 135 | 55
Cat | 38.7 | 130 | 24
Dog | 38.6 | 95 | 15
Goat | 40.0 | 75 | 16
Ox | 38.8 | 45 | ..
Horse | 37.9 | 38 | 11
Monkey (Rhesus) | 38.4 | 100 | 19
Pigeon | 40.9 | 136 | 30
Fowl | 41.6 | 140 | 12
| | |
----------------------------------------------------
B. ~Special observations~ comprise some or all of the following, according
to the method of inoculation and the character of the virus.
1. _The site of inoculation_ should be minutely examined at least at
weekly intervals, and the neighbouring lymphatic glands palpated.
2. Any _local reaction_ at the site of inoculation and any other readily
accessible lesion should be carefully investigated. Any suppurative
process which may occur, whether in the subcutaneous tissues or in
joints, should be explored and the pus carefully examined both
microscopically and culturally.
Fluid secretions and excretions, such as pus or serous exudates when
accessible are collected direct from the body in sterile capillary
pipettes (_vide_ Fig. 13a,) in the following manner:
1. Open the case containing the pipettes, grasp one by the plugged end,
remove it from the case, and replace the lid of the latter.
2. Attach a rubber teat (_vide_ page 10) to the plugged end of the
pipette and use the teat as the handle of the pipette.
3. Pass the entire length of the pipette twice or thrice through the
flame of the Bunsen burner.
4. Snap off the sealed end of the pipette with a pair of sterile
forceps.
5. Compress the india-rubber teat, thrust the point of the pipette into
the secretion; now relax the pressure on the teat and allow the pipette
to fill.
6. Remove the point of the pipette from the secretion, allow the fluid
to run a short distance up the capillary stem and seal the point of the
pipette in the flame. (If using a pipette with a constriction below the
plugged mouthpiece (Fig 13b), this portion of the pipette may also be
sealed in the flame.)
When ready to examine the morbid material snap off the sealed end of the
pipette with sterile forceps and eject the contents of the pipette into
a sterile capsule. The material can now be utilized for cover-slip
preparations, cultivations and inoculation experiment.
3. _The peripheral blood_ should be examined from time to time for from
this tissue is often obtained the fullest information as to the course
and progress of an infection.
a. The ~histological examination of the blood~ should be directed
chiefly to observations on the number and kind of white cells; and since
but few bacteriologists are at the same time expert comparative
haematologists, some notes on the normal characters of the blood of the
commoner laboratory animals, contrasted with those of man, are inserted
for reference. These have been very kindly compiled for me by my friend
and one time colleague Dr. Cecil Price Jones.
COMPARATIVE HAEMOCYTOLOGY OF LABORATORY ANIMALS.
--------------------------------------------------------------------
| Totals | Percentages
|------------------------------------------------------------
Animal | | | Hb, |Lympho-|Large |Poly- |Eosin-| Mast
|Red cells |White | per | cytes,|monos,|morph,| oph, |cells,
| | cells|cent.| per | per | per | per | per
| | | | cent. | cent.| cent.|cent. |cent.
--------------------------------------------------------------------
Frog | 490,000| 8,000| 58 | 40 | 10.0 | 22.0 |15 | 13
Mouse | 8,700,000| 8,000| 78 | 60 | 21.5 | 17.0 | 1.4 | 0.1
Rat | 9,000,000| 9,000| 85 | 54 | 7.0 | 37.5 | 1.3 | 0.2
Guinea-| | | | | | | |
pig | 5,700,000|10,000| 99 | 55 | 9.0 | 32.8 | 3.0 | 0.2
Rabbit | 6,000,000| 7,000| 70 | 50 | 2.0 | 46.0 | 0.6 | 1.4
Rhesus | 4,500,000|13,000| 77 | 43 | 5.0 | 50.0 | 1.3 | 0.7
Goat |14,600,000|15,000| 58 | 35 | 6.3 | 56.7 | 1.25 | 0.75
Fowl | 3,500,000|30,000| 100 | 49 | 3.0 | 42.0 | 1.0 | 5.0
Pigeon | 3,500,000|20,000| 101 | 43 | 9.0 | 43.0 | 3.0 | 2.0
--------------------------------------------------------------------
Man | | | | | | | |
(adult)| 5,000,000| 7,500| 100 | 25 | 5.5 | 65 | 4.0 | 0.5
Normal | (4.5-5) | (7-9)|(95- |(20-30)| (4-8)|(55- |(3-5) |(0.5-2)
limits.| millions.| thou-| 101)| | | 68) | |
| |sands.| | | | | |
--------------------------------------------------------------------
The above table represents in each case the average of a large number of
counts.
REMARKS.
_Frog._--The _red cells_ are large oval nucleated (20-25 mu by 12-15 mu)
discs, the nucleus relatively small and irregularly elongated or oval,
about 10 mu in length. Many primitive and developing forms are usually
observed--also free nuclei and many cells in various stages of
degeneration. Haemoglobin estimation is difficult owing to turbidity of
the blood after dilution with water. The _polymorphonuclear_ leucocytes
are large cells, about 20 mu; no definite granules can be observed. The
_eosinophile_ cells contain large deeply staining coccal-shaped
granules.
_Mouse._--The granules of the _polymorphonuclear_ leucocytes are usually
not stained, or only very faintly so. The nucleus of the _eosinophile
cell_ is ring-shaped or much divided, and the granules are coccal and
stain oxyphile. The remarkable character of the blood is the high
percentage of large _mononuclear_ cells.
_Rat._--The fine rod-shaped granules of the _polymorphonuclear_
leucocytes are usually very faintly stained. The granules of
_eosinophile_ cells are well stained and coccal-shaped, the nucleus is
often ring shaped. The _basophile_ granular cells are few--but the
granules are large, and stain deeply basophile.
_Guinea-pig._--Polychromasia and punctate basophilia of _red cells_ are
very commonly observed--nucleated red cells are also frequent. The large
_mononuclear_ cells often contain vacuoles--"Kurlow cells"--possibly of
a parasitic nature.
_Rabbit._--It is not uncommon to find nucleated _red cells_ in films
from quite healthy animals. The granules of the _polymorphonuclear_
leucocytes stain oxyphile. The coarse granules of the _eosinophile_
cells appear to stain less deeply oxyphile, probably owing to the
basophile staining of the cytoplasm.
_Rhesus monkey._--The blood cells resemble those met with in human
blood. The minute neutrophile granules of the _polymorphonuclear_
leucocytes are often very scanty, and sometimes apparently absent. The
_eosinophile_ cells are not so densely packed with coarse oxpyhile
granules as in the human eosinophile, and the nuclei of these cells are
usually much divided, or polymorphous.
_Goat._--The _red cells_ are small, nonnucleated discs, only about 4.5
mu diameter, not much more than half that of the human red cell. The
_polymorphonuclear_ leucocytes have only a few very minute
coccal-shaped oxyphile granules, the nucleus is polymorphous. The
_eosinophile_ cells are large cells up to 20 mu, the cytoplasm is
basophile and contains coarse coccal-shaped oxyphile granules, and the
nucleus is often much divided.
_Fowl._--The _red cells_ are oval nucleated discs about 12 mu by 6 mu, the
nucleus being relatively small (about 4 mu long), irregularly elongated or
oval; round, more deeply stained cells with round or diffuse nuclei,
also free nuclei and degenerated forms of red cells are often present.
The granules of the cells corresponding to the _polymorphonuclear_
leucocytes are rod-shaped, often beaded or with clubbed ends. The
nucleus is not polymorphous, but usually divided into two, though it may
be single. The cells probably corresponding to _eosinophile_ leucocytes
have fine coccal-shaped granules, faintly staining eosinophile or
neutrophile. The basophile granules of the "mast" cells are
coccal-shaped, of various size--often quite powdery.
_Pigeon._--_Red cells_ resemble those of the fowl, and similar varieties
of appearance may be noted. The granules of those cells which correspond
to _polymorphonuclear_ leucocytes are rod-shaped, but smaller and finer
than in the fowl, and do not show clubbed appearances. The nucleus is
not polymorphous, and only occasionally divided. The coccal-shaped
granules of the _eosinophile_ cells are stained more deeply oxyphile
than those of the corresponding cells of the fowl.
_The preparation of dried films_ for this histological examination of
the blood is carried out as follows:
1. Small samples of blood for the preparation of blood films are most
conveniently obtained from the veins of the ear in most of the ordinary
laboratory animals, viz., monkey, goat, dog, cat, rabbit, guinea-pig; in
the pigeon and fowl the axillary vein should be punctured; in the rat
and mouse either a vein in the ear or preferably by wounding the tip of
the tail; in the frog, the web of the foot should be selected.
2. Puncture the selected vein with a sharp needle. A flat Hagedorn
needle (size No. 8) with a cutting edge is the most useful for this
purpose. If the vein cannot be distended by proximal compression,
vigourous friction with a piece of dry lint may have the desired
effect--or a test-tube full of water at about 40 deg. C. may be placed
close to the vein. Failing these methods, a drop or two of xylol may be
dropped on the skin just over the vein, left on for a few seconds and
then wiped off with a piece of dry lint.
3. One of the short ends of a 3 by 1 glass slip is brought into contact
with the exuding drop of blood, so that it picks up a small drop.
4. The slide is then lowered transversely on to the surface of a second
3 by 1 slip, which rests on the bench near to one end at an angle of
about 45 deg., and retained in this position for a few seconds, while the
drop of blood spreads along the whole of the line of contact (see also
Fig. 69).
5. Draw the first slide firmly and evenly along the entire length of the
lower slide, leaving a thin regular film which will probably show the
blood cells only one layer thick.
6. Allow the film to dry in the air.
7. Stain with one of the polychrome blood stains (see page 97).
8. Examine microscopically.
b. The ~bacteriological examination of the blood~ is directed solely to
the demonstration of the presence in the circulating blood of the
organisms previously injected into the animal. For this purpose several
cubic centimetres of blood should be taken in an all-glass syringe from
an accessible vein corresponding to one of those suggested as the site
of intravenous inoculation--and under similar aseptic precautions.
1. Sterilise an all-glass syringe of suitable size, and when cool draw
into the syringe some sterile sodium citrate solution and moisten the
whole of the interior of the barrel; then eject all the citrate solution
if less than 5 c.c. blood are to be withdrawn; if more than 5 c.c. are
required retain about half a cubic centimetre of the fluid in the
syringe. This prevents coagulation of the blood.
The sodium citrate solution is prepared by dissolving:
Sodium citrate 10 gramme.
Sodium chloride 0.75 grammes.
In distilled water 100 c.c.
Sterilise by boiling.
2. Prepare the animal as for intravenous inoculation (see page 363) and
introduce the syringe needle into the lumen of the selected vein.
3. Slowly withdraw the piston of the syringe. When sufficient blood has
been collected direct the assistant to release the proximal compression
of the vein; and withdraw the needle.
4. Remove the needle from the nozzle of the syringe and deliver the
citrated blood into a small Ehlenmeyer flask containing about 250 c.c.
of nutrient broth.
5. Label, incubate and examine daily until growth occurs or until the
expiration of ten days.
c. The ~serological examination of the blood~ is directed to the
demonstration of the presence of certain specific antibodies in the sera
of experimentally infected animals, and within certain limits to an
estimation of their amounts.
The chief of these bodies are:
Antitoxin.
Agglutinin.
Precipitin.
Opsonin.
Immune body or Bacteriolysin.
None of these substances are capable of isolation in a state of purity
apart from the blood serum, consequently special methods have been
elaborated to permit of their recognition. In every instance the
behaviour of serum from the experimental animal, which may be termed
"specific" serum, is studied in comparison with that of serum from an
uninoculated animal of the same species, and which is termed "normal"
serum. In view of minor differences in constitution exhibited by the
serum of various individuals of the same series, it is usual to employ a
mixture of sera obtained from several different normal animals of the
same species as the inoculated animal, under the term "pooled serum."
The method of collecting blood (e. g., from the rabbit) for
serological tests is as follows:
~Collection of Serum.~
_Apparatus required:_
Razor.
Liquid soap.
Cotton-wool.
Lysol 2 per cent. solution, in drop bottle.
Ether in drop bottle.
Flat Hagedorn needles.
Blood pipettes (Fig. 16, page 12).
Centrifugal machine.
Centrifuge tubes.
Glass cutting knife.
Bunsen flame.
Writing diamond or grease pencil.
METHOD.
1. Shave the dorsal surface of the ear over the course of the posterior
auricular vein (see Fig. 192).
2. Sterilise the skin by washing with lysol.
The lysol should be applied with sterile cotton-wool and the ear
vigourously rubbed, not only to remove superficial scales of epithelium,
but also to render the ear hyperaemic and the vein prominent.
3. Remove the lysol with ether dropped from a drop bottle, and allow the
ether to evaporate.
4. Puncture the vein with a sterile Hagedorn needle.
5. Take a small blood-collecting pipette (Fig. 161) and hold it at an
angle to the ear, one end touching the issuing drop of blood, the other
depressed.
The blood will now enter the pipette at first by capillarity; afterward
gravity will also come into play and the pipette can be two-thirds
filled without difficulty.
6. Hold the tube by the end containing the blood, the clean end pointing
obliquely upward--warm this end at the bunsen flame to expel some of the
contained air; then seal the clean point in the flame.
[Illustration: FIG. 192.--Collecting blood from rabbit.]
7. Place the pipette down on a cool surface (e. g., a glass slide).
The rapid cooling of the air in the clean end of the pipette creates a
negative pressure, and the blood is sucked back into the pipette,
leaving the soiled end free from blood. Seal this end in the bunsen
flame.