erythrocytes just before they are needed. This is done
forty-five minutes after the experiment has been started
(page 394, step 6), that is to say, before the completion of
the first period of incubation, thus:
1. Measure out into a sterile test-tube (or flask) five c.c.
of erythrocyte solution.
2. Measure out twenty minimal haemolytic doses of haemolysin,
add to the erythrocyte solution on the test-tube.
3. Allow the erythrocyte and haemolysin to remain in contact
for fifteen minutes at room temperature. The red cells are
then sensitised and ready for use.
4. When the tubes are removed from the incubator at the end
of the first hour (i. e., step 7) add 1 c.c. sensitised
red cells to each tube by means of a graduated pipette.
5. Mix thoroughly, return the tubes to the incubator at
37 deg. C. and complete the experiment as previously described
(steps 8 onward).
XIX. POST-MORTEM EXAMINATIONS OF EXPERIMENTAL ANIMALS.
The post-mortem examination should be carried out as soon as possible
after the death of the animal, for it must be remembered that even in
cold weather the tissues are rapidly invaded by numerous bacteria
derived from the alimentary tract or the cavities of the body, and from
external sources.
The following outlines refer to a complete and exhaustive necropsy, and
in routine work the examination will rarely need to be carried out in
its entirety.
NOTE.--Throughout the autopsy the searing irons must be
freely employed, and it must be recollected that one
instrument is only to be employed to seize or cut one
structure. This done, it must be regarded as contaminated
and a fresh instrument taken for the next step.
~Apparatus Required~:
Water steriliser.
{ Scalpels.
Surgical instruments: { Scissors.
{ Forceps.
{ Bone forceps.
Spear-headed platinum spatula (Fig. 199).
Searing irons (Fig. 198).
Tubes of media--bouillon and sloped agar.
Surface plates in petri dishes (of agar or one of its derivatives).
Platinum loop.
Aluminium "spreader."
Grease pencil.
Sterile capillary pipettes (Fig. 13, a).
Sterile glass capsules, large and small.
Cover-slips or slides.
Bottles of fixing fluid (_vide_ page 114) for pieces of tissue intended
for sectioning.
1. Place the various instruments, forceps, scissors, scalpels, etc.,
needed for the autopsy inside the steriliser and sterilise by boiling
for ten minutes; then open the steriliser, raise the tray from the
interior and rest it crosswise on the edges.
2. Heat the searing irons to redness in a separate gas stove.
[Illustration: FIG. 197.--Apparatus for post-mortem examination, animal
on board.]
3. Drench the fur (or feathers) with lysol solution, 2 per cent. This
serves the twofold purpose of preventing the hairs from flying about and
entering the body cavities during the autopsy, and of rendering
innocuous any vermin that may be present on the animal.
[Illustration: FIG. 198.--Searing iron.]
4. Examine the cadaver carefully. Recollect that laboratory animals are
not always hardy; death may be due to exposure to heat or cold, to
starvation or over- or improper feeding or to the attack of rats--and
not to the bacterial infection.
5. Fasten the body of the animal, ventral surface upward (unless there
is some special reason for having the dorsum exposed), out on a board
by means of copper nails driven through the extremities.
6. With sterile forceps and scalpel incise the skin in the middle line
from the top of the sternum to the pubes. Make other incisions at right
angles to the first out to the axillae and groins, and reflect the skin
in two lateral flaps. (Place the now infected instruments on the board
by the side of the body or support them on a porcelain knife rest.)
~Seat of Inoculation.~--
7. Inspect the seat of inoculation. If any local lesion is visible, sear
its exposed surface and with the platinum loop, remove material from the
deeper parts to make tube and surface plate cultivations and cover-slip
preparations.
Collect specimens of pus or other exudation in capillary pipettes for
subsequent examination.
8. Inspect the neighbouring lymphatic glands and endeavour to trace the
path of the virus.
9. Sear the whole of the exposed surface of the thorax with the searing
irons.
~Pleural Cavity.~--
10. Divide the ribs on either side of the sternum and remove a
rectangular portion of the anterior chest wall with sterile scissors and
a fresh pair of forceps, exposing the heart. Place the infected
instruments by the side of the first set.
11. Observe the condition of the anterior mediastinal glands, the thymus
and the lungs. Collect a quantity of pleuritic effusion, if such is
present, in a pipette for further examination later.
12. Raise the pericardial sac in a fresh pair of forceps and burn
through this structure with a searing iron.
Collect a sample of pericardial fluid in a pipette for microscopical and
cultural examination.
13. Grasp the apex of the heart in the forceps and sear the surface of
the right ventricle.
14. Plunge the open point of a capillary pipette through the seared area
into the ventricle and fill with blood.
Make cultivations and cover-slip preparations of the heart blood.
15. Collect a further sample of blood or serum for subsequent
investigation as to the presence of antibodies.
~Peritoneal Cavity.~--
16. Sear a broad track in the middle line of the abdominal wall; open
the peritoneal cavity by an incision in the centre of the seared line.
Observe the condition of the omentum, the mesentery, the viscera and the
peritoneal surface of the intestines.
17. Collect a specimen of the peritoneal fluid (or pus, if present) in a
capillary pipette. Make cultivations, tube and surface plate, and
cover-slip preparations from this situation.
18. Collect a specimen of the urine from the distended bladder in a
large pipette (in the manner indicated for heart blood), for further
examination, by cultivations, microscopical preparations, and chemical
analysis.
19. Collect a specimen of bile from the gall bladder in similar manner.
20. Excise the spleen and place it in a sterile capsule. Later, sear the
surface of this organ; plunge the spear-headed spatula through the
centre of the seared area, twist it round between the finger and thumb,
and remove it from the organ. Sufficient material will be brought away
in the eye in its head to make cultivations. A repetition of the process
will afford material for cover-slip preparations.
21. Seize one end of the spleen with sterile forceps. Sear a narrow band
of tissue, right around the organ and divide the spleen in this
situation with a pair of scissors. Holding the piece of spleen in the
forceps, dab the cut surface on to a surface plate in a number of
different spots.
22. In like manner examine the other organs--liver, lungs, kidneys,
lymphatic glands (mesenteric, hepatic, lumbar, etc), etc. Prepare
cultivations and cover-slip preparations.
23. Dissect out a long bone from one upper and one lower limb and one of
the largest ribs. Prepare cultures from the bone marrow in each case.
Set aside these bones for the subsequent preparation of marrow films.
24. Film preparations of bone marrow are best made by the Price-Jones
method. Seize the bone in a pair of pliers and squeeze out some of the
marrow; receive it in a platinum loop, and transfer to a watch glass of
dissociating fluid and emulsify. The dissociating fluid is a neutral 10
per cent. solution of glycerine prepared as follows:--
Measure out 10 c.c. Price's best glycerine and 90 c.c.
sterile ammonia-free distilled water. Mix. Titrate against
n/10 sodic hydrate solution using phenolphthalein as the
indicator. The initial reaction is usually + 0.1 to + 0.5;
add the calculated amount of n/10 sodic hydrate solution to
neutralise.
25. Place a loopful of fresh desiccating fluid on a 3 x 1 glass slide;
add a similar loopful of the marrow emulsion, and spread very gently
over the surface of the slip.
26. Allow film to dry in the air (protected from dust) without heating.
27. Stain with Jenner's polychrome stain (page 97) for two and a half
minutes.
28. Wash with ammonia-free distilled water, dry thoroughly and mount in
xylol balsam.
~Cranial and Spinal Cavities.~--
29. In some instances it may be necessary (e. g., experimental
inoculation of rabies) to examine the cranial cavity or to remove the
spinal cord. Return the viscera to the abdominal cavity; draw the flaps
of skin together and secure with Michel's steel clips. Draw the copper
nails securing the limbs to the board, reverse the animal and again nail
the limbs down--the body now being dorsum uppermost.
30. Make a longitudinal incision in the mesial line from snout to root
of tail, and four transverse incisions--one joining the roots of the two
ears, one across the body at the level of the spinis of the scapulae,
another at the level of the costal margin and the last across the upper
level of the pelvis. Reflect these flaps of skin.
31. With forceps and scalpel dissect out the muscles lying in the furrow
on either side of the spinal processes.
32. Cut through the bases of the transverse processes with bone forceps.
Cut away the vault of the skull, cut through the roots of the nerves and
remove the brain and spinal cord, place in a large glass dish for
examination. Prepare cultivations from the cerebro-spinal fluid. The
removal of the brain and cord is a tedious process and during the
dissection it is difficult to avoid injury to these structures.
The operation is, however, carried out very expeditiously and neatly
with the aid of the surgical engine (_vide_ page 361). A small circular
saw is fitted to the hand piece. The bones of the skull are cut through
and the whole of the vault removed, exposing the entire vertex of the
brain. Similarly all the spinous processes can be removed in one string
by running the saw down first one side of the spinal column and then the
other. In this way ample space for the removal of the nervous tissues is
obtained with a minimum of labour.
33. Having completed the preparation of cultures remove small portions
of various organs at leisure and place each in separate bottles of
fixing fluid for future sectioning. Affix to each bottle a label bearing
all necessary details as to its contents.
34. If necessary, remove portions of the organs for preservation and
display as museum specimens (_vide_ page 404).
35. Gather up all the infected instruments, return them to the
steriliser, and disinfect by boiling for ten minutes.
[Illustration: FIG. 199.--Spear-headed platinum spatula (actual size.)]
36. Sprinkle dry sawdust into the exposed body cavities to absorb blood
and fluid. Cover the body with blotting or filter paper, moistened with
2 per cent. lysol solution. Place in a galvanised iron pail, provided
with a lid, ready for transport to the crematorium.
37. Cremate the cadaver together with the board upon which it is fixed.
38. Stain the cover-slip preparations by suitable methods and examine
microscopically.
39. Incubate the cultivations and examine carefully from day to day.
40. Make full notes of the condition of the various body cavities and of
the viscera immediately the autopsy is completed; and add the result of
the microscopical and cultural investigation when available.
As part of the card index system in use in the author's laboratory
already referred to (_vide_ page 335) there is a special yellow card for
P-M notes. On the face of the card are printed headings for various
data--some of which are sometimes unintentionally omitted--and on the
reverse is a schematic figure which can be utilised for indicating the
position of the chief lesions in the cadaver of any of the laboratory
animals.
AUTOPSY CARD Laboratory No. _________
Date ________
Animal ______ No. in Series ______ [Symbols: male female] Weight ________
+------------------------------------------------------------------------+
Died (or killed) _____ o'clock ____ m. Autopsy made _____ o'clock ____ m.
+------------------------------------------------------------------------+
Notes on Post Mortem Examinations.
_General._
A. Seat of Inoculation.
B. Thoracic Cavity.
C. Abdominal Cavity.
D. Cranial Cavity.
+-------------------+---- -------------+--------------------------+
_Bacteriological_ | _Histological_ | _Organs Preserved._ |
_Examination._ | _Examination._ | |
A. | | |
| | |
B. | | |
| | |
C. | | |
| | |
D. | | |
[Illustration: FIG. 200.--Front of post-mortem card.]
41. Finally, the results of the action of the organism or organisms
isolated may be correlated with the symptoms observed during life and
the observations summarised under the following headings:
Tissue changes:
1. Local--i. e., produced in the neighbourhood of the bacteria.
Position: (a) At primary lesion.
(b) At secondary foci.
Character: (a) Vascular changes and tissue } Acute
reactions. } or
(b) Degeneration and necrosis. } chronic.
2. General (i. e., produced at a distance from the bacteria, by
absorption of toxins):
(a) In special tissues--e. g., nerve cells and fibres, secreting
cells, vessel walls, etc.
(b) General effects of malnutrition, etc.
Symptoms:
(a) Associated with known tissue changes.
(b) Without known tissue changes.
[Illustration: FIG. 201.--Back of post-mortem card.]
~Permanent Preparations--Museum Specimens.~--
_I. Tissues._--The naked-eye appearances of morbid tissues may be
preserved by the following method:
1. Remove the tissue or organ from the cadaver as soon after death as
possible, using great care to avoid distortion or injury.
2. Place it in a wide-mouthed stoppered jar, large enough to hold it
conveniently, resting on a pad of cotton-wool, and arrange it in the
position it is intended to occupy (but if it is intended to show a
section of the tissue or organ, do not incise it yet).
3. Cover with the Kaiserling fixing solution, and stopper the jar; allow
the tissues to remain in this solution for from forty-eight hours to
seven days (according to size) to fix. Make any necessary sections.
Kaiserling modified solution is prepared as follows:
Weigh out
Potassium acetate 30 grammes.
Potassium nitrate 15 grammes.
and dissolve in
Distilled water 1000 c.c.
then add
Formalin 150 c.c.
Filter.
This fixing solution can be used repeatedly so long as it remains clear.
Even when it has become turbid, if simple filtration is sufficient to
render it clear, the filtrate may be used again.
4. Transfer the tissue to a bath of methylated spirit (95 per cent.) for
thirty minutes to one hour.
5. Remove to a fresh bath of spirit and watch carefully. When the
natural colours show in their original tints, average time three to six
hours, remove the tissues from the spirit bath, dry off the spirit from
the cut surfaces by mopping with a soft cloth, then transfer to the
mounting solution.
Jore's mounting solution (modified) consists of
Glycerine 500 c.c.
Distilled water 750 c.c.
Formalin 2 c.c.
Equally good but much cheaper is Frost's mounting solution:
Potassium acetate 160 grammes.
Sodium fluoride 80 grammes.
Chloral hydrate 80 grammes.
Cane sugar (Tate's cubes) 3,500 grammes.
Saturated thymol water 8,000 c.c.
6. After twenty-four hours in this solution, or as soon as the tissue
sinks, transfer to a museum jar, fill with fresh mounting solution, and
seal.
_6a._ Or transfer to museum jar and fill with liquefied gelatine, to
which has been added 1 per cent. formalin. Cover the jar and allow the
gelatine to set. When solid, seal the cover of the jar in place.
7. To seal the museum preparation first warm the glass plate which forms
the cover. This is most conveniently done by placing the cleaned and
polished cover-plate upon a piece of asbestos millboard over a bunsen
flame turned low.
8. Smear an even layer of hot cement over the flange of the jar. The
cement is prepared as follows:
Weigh out and mix in an iron ladle
Gutta percha (pure) 4 parts.
Asphaltum 5 parts.
and melt together over a bunsen flame, stirring with an iron rod until
solution is complete.
9. Invert the glass plate over the jar and press down firmly into the
cement. Place a piece of asbestos board on the top and on that rest a
suitable weight until the cement is cold and has thoroughly set.
10. Trim off any projecting pieces of cement with an old knife, burr
over the joint between jar and cover-plate with a hot smooth piece of
metal (e. g., the searing iron).
11. Paint a narrow band of Japan black to finish off, round the joint,
overlapping on to the cover-plate.
_II. Tube Cultivations of Bacteria._--When showing typical appearances
these may be preserved, if not permanently, at least for many years, as
museum specimens, by the following method:
1. Take a large glass jar 25 cm. high by 18 cm. diameter, with a firm
base and a broad flange, carefully ground, around the mouth. The jar
must be fitted with a disc of plate glass ground on one side, to serve
as a lid.
2. Smear a thick layer of resin ointment (B.P.) on the flange around the
mouth of the jar.
3. Cover the bottom of the jar with a layer of cotton-wool and saturate
it with formalin.
4. Remove the cotton-wool plug from the culture tubes and place them,
mouth upward, inside the jar. (If water of condensation is present in
any of the culture tubes, it should be removed by means of a capillary
pipette before placing the tubes in the formalin chamber.)
5. Adjust the glass disc, ground side downward, over the mouth of the
jar and secure it by pressing it firmly down into the ointment, with a
rotary movement.
6. Remove the tubes from the formalin chamber after the lapse of a week,
and dry the exterior of each.
[Illustration: FIG. 202.--Bulloch's tubes.]
7. Seal the open mouth of each tube in the blowpipe flame and label.
If the cultivations are intended for museum purposes when they are first
planted, it is more convenient to employ Bulloch's tubes. These are
slightly longer than the ordinary tubes, and are provided with a
constriction some 2 cm. below the mouth (Fig. 202)--a feature which
renders sealing in the blowpipe flame an easy matter.
XX. THE STUDY OF THE PATHOGENIC BACTERIA.
The student, who has conscientiously worked out the methods, etc.,
previously dealt with, is in a position to make accurate observations
and to write precise descriptions of the results of such observations.
He is, therefore, now entrusted with pure cultivations of the various
pathogenic bacteria, in order that he may study the life-history of each
and record the results of his own observations--to be subsequently
corrected or amplified by the demonstrator. In this way he is rendered
independent of text-book descriptions, the statements in which he is
otherwise too liable to take for granted, without personally attempting
to verify their accuracy.
During the course of this work attention must also be directed, as
occasion arises, to such other bacteria, pathogenic or saprophytic, as
are allied to the particular organisms under observation, or so resemble
them as to become possible sources of error, by working them through on
parallel lines--in other words the various bacteria should be studied in
"groups." In the following pages the grouping in use in the author's
elementary classes for medical and dental students and for candidates
for the Public Health service is adopted, since a fairly long experience
has completely vindicated the value and utility of this arrangement, and
by its means a fund of information is obtained with regard to the
resemblances and differences, morphological and cultural, of a large
number of bacteria. The fact that some bacteria appear in more than one
of these groups, so far from being a disadvantage, is a positive gain to
the student, since with repetition alone will the necessary familiarity
with the cultural characters of important bacteria be acquired. The
study of the various groups will of course vary in detail with
individual demonstrators, and with the student's requirements--the
general line it should take is indicated briefly in connection with the
first group only (pages 410-411). This section should be carefully
worked through before the student proceeds to the study of
bacterioscopical analysis.
It is customary to commence the study of the pathogenic bacteria with
the Organisms of Suppuration. This is a large group, for all the
pathogenic bacteria possess the power, under certain conditions, of
initiating purely pyogenic processes in place of or in addition to their
specific lesions, (e. g., Bacillus tuberculosis, Streptococcus
lanceolatus, Bacillus typhosus, etc.). There are, however, a certain few
organisms which commonly express their pathogenicity in the formation of
pus. These are usually grouped together under the title of "pyogenic
bacteria," as distinct from those which only occasionally exercise a
pyogenic role.
The organisms included in this group are:
1. Staphylococcus pyogenes albus.
2. Staphylococcus pyogenes aureus.
3. Staphylococcus pyogenes citreus.
4. Streptococcus pyogenes longus.
5. Micrococcus tetragenus.
6. Bacillus pyocyaneus.
7. Bacillus pneumoniae.
and in certain special tissues
8. Micrococcus gonorrhoeae.
9. Micrococcus intracellularis meningitidis (Meningococcus).
10. Micrococcus catarrhalis.
11. Bacillus aegypticus (Koch-Weeks Bacillus).
The group may with advantage be subdivided as indicated in the following
pages:
I. _Pyogenic cocci._
Staphylococcus pyogenes albus.
Staphylococcus pyogenes aureus.
Staphylococcus pyogenes citreus.
to contrast with
Micrococcus candicans.
Micrococcus agilis.
1. Prepare subcultivations from each:
Bouillon, }
Agar streak, }
Blood serum, }
Litmus milk. } and incubate at 37 deg. C.
Agar streak, }
Gelatine stab, }
Potato. } and incubate at 20 deg. C.
Compare the naked-eye appearances of the cultures from day to day. Note
M. agilis refuses to grow at 37 deg. C.
2. Make hanging-drop preparations from the bouillon and agar
cultivations after twenty-four hours' incubation. Examine
microscopically and compare. Note the locomotive activity of M. agilis
and the Brownian movement of the remaining micrococci.
3. Prepare cover-slip films from the agar cultures, after twenty-four
hours' incubation. Stain for flagella by the modified Pitfield's method.
Note M. agilis is the only micrococcus showing flagella.
4. Make microscopical preparations of each from all the various media
after twenty-four and forty-eight hours and three days' incubation.
Stain carbolic methylene-blue, carbolic fuchsin, and Gram's method.
Examine the films microscopically and compare. Note in the Gram
preparation, the Gram negative character of certain individual cocci in
each film prepared from the three days' growth--such cocci are dead.
5. Stain section of kidney tissue provided (showing abscess formation
by Staphylococcus aureus) by Gram's method, and counterstain with cosin.
6. Stain film preparation of pus from an abscess (containing
Staphylococcus pyogenes aureus) with carbolic methylene-blue and also by
Gram's method, counterstained with cosin.
7. Inoculate[15] a white mouse subcutaneously with three loopfuls of a
forty-eight-hour agar cultivation of the Staphylococcus aureus,
emulsified with 0.2 c.c. sterile broth.
Observe carefully during life, and when death occurs make a careful
post-mortem examination.
II. _Pyogenic cocci._
Micrococcus gonorrhoeae.
Micrococcus intracellularis meningitidis (meningococcus).
Micrococcus catarrhalis.
Micrococcus tetragenus.
Micrococcus paratetragenus.
III. _Pyogenic cocci._
Streptococcus pyogenes longus.
Streptococcus of bovine mastitis.
Streptococcus lanceolatus (Diplococcus pneumoniae or pneumococcus).
to contrast with
Streptococcus brevis.
Streptococcus lebensis.
IV. _Pyogenic bacilli._
Bacillus pneumoniae (Friedlaender).
Bacillus rhinoscleromatis.
Bacillus lactis aerogenes.
V. _Pyogenic bacilli._
Bacillus pyocyaneus.
to contrast with
Bacillus fluorescens liquefaciens.
Bacillus fluorescens non-liquefaciens.
VI. _Pneumonia group._
Streptococcus lanceolatus (pneumococcus).
Bacillus pneumoniae (Friedlaender).
Streptococcus pyogenes longus.
VII. _Diphtheroid group._