Chapter XIII: Appendix: 492 (11)

Eighteen to twenty-four-hour-old bouillon cultivation of the
organism to be tested (e. g., Bacillus typhi abdominalis)

Pipette end with the remainder of the specific serum
labelled ~s.s.~

Tubes containing the three solutions of the specific serum,
10, 1, and 0.1 per cent. respectively.

Pipette end with pooled normal serum labelled ~p.s.~

METHOD.--

1. Make five hanging-drop preparations, thus:

(a) One loopful of bouillon cultivation + one loopful pooled serum; label "Control."

(b) One loopful culture + one loopful undiluted specific serum; label 50 per cent.

Mount these two cover-slips on a double-celled slide.

(c) One loopful bouillon culture + one loopful 10 per cent. serum; label 5 per cent.

Mount this on single-cell slide.

(d) One loopful bouillon culture + one loopful 1 per cent. serum; label 0.5 per cent.

(e) One loopful bouillon culture + one loopful 0.1 per cent. serum; label 0.05 per cent.

Mount these two cover-slips on a double-celled slide.

2. Note the time: Examine the control to determine that the bacilli are motile and uniformly scattered over the field--not collected into masses.

3. Next examine the 50 per cent. serum preparation.

If agglutinin is present and the test is giving a positive reaction, the bacilli _will_ be collected in large clumps.

If the test is giving a negative reaction, the bacilli _may_ be collected in large clumps owing to the viscosity of the concentrated serum.

4. Observe the 5 per cent. preparation microscopically.

If the bacilli are aggregated into clumps, positive reaction.

If the bacilli are _not_ aggregated into clumps, observe until thirty minutes from the time of preparation before recording a negative reaction.

5. Examine the 0.5 and 0.05 per cent. preparations.

These may or may not show agglutination when the result of the examination of the 5 per cent. preparation is positive, according to the potency of the specific serum; and by the examination of a series of dilutions a quantitative comparison of the valency of specific sera from different sources, or of serum from the same animal at different periods during the course of active immunisation may be obtained.

NOTE.--The graduated pipettes supplied with Thoma's
haematocytometer (intended for the collection of the specimen
of blood required for the enumeration of leucocytes), giving
a dilution of 1 in 10--i. e., 10 per cent.--may be
substituted for the graduated capillary pipettes referred to
above, if the vessel in which the serum has been separated
is of sufficiently large diameter to permit of their use.

~The Macroscopical Reaction:~

Sterile graduated capillary pipettes to contain 90 c. mm.

Eighteen to twenty-four-hours-old bouillon cultivation of
the organism to be tested.

Three test-tubes containing the 10, 1, and 0.1 per cent.
solutions of specific serum (about 90 c. mm. remaining in
each).

Tube containing 50 per cent. solution of pooled serum.

Sedimentation pipettes (_vide_ page 17) or teat pipettes.

METHOD.

1. Pipette 90 c. mm. of the bouillon culture into each of the tubes containing the diluted serum; and the same quantity into the tube containing the pooled serum.

2. Fill a sedimentation tube (by aspirating) or a teat pipette from the contents of each tube. Seal off the lower ends of the sedimentation tubes in the Bunsen flame.

3. Label each tube with the dilution of serum that it contains--viz., 5, 0.5, and 0.05 per cent.

4. Place the pipettes in a vertical position, in a beaker, in the incubator at 37 deg. C., for one or two hours.

5. Observe the granular precipitate which is thrown down when the reaction is positive, and the uniform turbidity of the negative reaction as compared with the appearances in the control pooled serum.

OPSONIN.

Opsonin is the term applied by Wright to a substance, present in the serum of an inoculated animal, which is able to act upon or sensitise bacteria of the species originally injected, so as to render them an easy prey to the phagocytic activity of polymorphonuclear leucocytes. In the method for demonstrating opsonin about to be described, a comparison is made between the opsonic "power" of the pooled serum and the specific serum.

_Apparatus:_

Small centrifuge and tubes for same (made from the barrels
of broken capillary pipettes by sealing the conical ends in
the bunsen flame).

Capillary Pasteur pipettes.

India-rubber teats.

Grease pencil.

Bunsen burner with peep flame.

Electrical signal clock (see page 39) stop watch, or watch.

Rectangular glass box or tray to hold pipettes.

Incubator regulated at 37 deg. C.

3 x 1 slides.

Piece of light rubber tubing.

Rectangular block of plasticine.

Flask of normal saline solution.

Flask of sodium citrate (1.5 per cent.) in normal saline
solution.

_Materials required_, and their preparation:

Small tube of "washed cells" (red blood discs and
leucocytes); human cells are used in estimating the
opsonising power of the serum of experimental animals.

Small tube of emulsion of bacteria of the species
responsible for the infection of the experimental animal.

Blood pipette containing specific serum.

Blood pipette containing "pooled" serum.

_Washed Cells._--

1. Take a small centrifuge tube and half fill it with sodium citrate solution. Mark with the grease pencil the upper limit of the fluid.

2. Cleanse the skin of the distal phalanx of the second finger of the left hand above the root of the nail with lint and ether. Wind the rubber tubing tightly round the second phalanx; puncture with a sterile Hagedorn needle through the cleansed area of skin.

3. Take up a sufficiency of the issuing blood (more or less according to the number of tests to be performed) with a teat pipette, transfer it to the tube of citrate solution and mix thoroughly. Make a second mark on the tube at the upper level of the mixed citrate solution and blood.

4. Place the tube in the centrifuge, counterpoise accurately and centrifugalise until the blood cells are thrown down in a compact mass occupying approximately the same volume as is included between the two pencil marks.

The column of fluid in the tube now shows clear supernatant fluid (citrate solution and blood plasma) separated from the sharp cut upper surface of the red deposit of corpuscles by a narrow greyish layer of leucocytes.

5. Remove the supernatant column of citrate solution by means of a teat pipette, fill normal saline solution into the tube up to the upper pencil mark, and distribute the blood cells throughout the saline by means of the teat pipette. Centrifugalise as before.

6. Again remove the supernatant fluid and fill in a fresh supply of saline solution and centrifugalise once more.

7. Remove the supernatant saline solution as nearly down to the level of the leucocytes as can be safely done without removing any of the leucocytes.

8. Next distribute the leucocytes evenly throughout the mass of red cells by rotating the tube between the palms of the hands--just as is done with a tube of liquefied medium prior to pouring a plate.

9. Set the tube upright in the plasticine block near to one end.

_Bacterial Emulsion._--

1. Take an 18- to 24-hour culture of the required bacterium (e. g., Diplococcus pneumoniae) grown upon sloped blood agar at 37 deg. C. Pour over the surface of the medium some 5 c.c. of normal saline solution.

2. With a platinum loop emulsify the growth from the surface of the medium as evenly as possible in the saline solution.

3. Allow the tube to stand for a few minutes so that the large masses of growth may settle down; transfer the upper portion of the saline suspension to a centrifuge tube and centrifugalise thoroughly.

4. Examine a drop of the supernatant opalescent emulsion microscopically to determine its freedom from clumps and masses. If unsatisfactory prepare another emulsion, this time scraping up the surface growth with a platinum spatula, transferring it to an agate mortar and grinding it up with successive small quantities of normal saline. If satisfactory insert the tube in the plasticine block next to that containing the washed cells.

~Specific Serum.~--

~Pooled Serum.~--

These sera are collected and treated as already described (see page 379), and the portions of the blood pipettes containing them are arranged in the remaining space in plasticine block.

The plasticine block now presents the appearances shown in Fig. 194.

METHOD FOR DETERMINING THE OPSONIC INDEX.--

1. Take a capillary pipette fitted with a teat, cut the distal end _square_ and make a pencil mark about 2 cm. from the end.

2. Aspirate into the pipette one volume of washed cells, air index, one volume of bacterial emulsion, air index, and one volume of specific serum (see Fig. 195).

3. Mix thoroughly on a 3 by 1 slide by compressing the teat and ejecting the contents of the pipette on to the surface of the slide, relaxing the pressure and so drawing the fluid up into the pipette again. These two processes should be repeated several times; finally take up the mixture in an unbroken column to the central portion of the capillary stem.

4. Seal the point of the pipette in the peep flame of the bunsen burner and remove teat.

5. Mark the pipette (with the grease pencil) with the distinctive number of the serum and place it in the glass box or tray.

6. Take another similarly prepared pipette and aspirate into it equal volumes of washed cells, bacterial emulsion and pooled serum. Treat precisely as in 3 and 4, label it "control" or "N.S." (normal serum) and place in the box by the side of the specific serum preparation.

7. Place the box with the pipettes in the incubator and set the signal clock to ring at 15 minutes (or start the stop watch).

8. At the expiration of the incubation time remove the pipettes from the incubator.

9. Cut off the sealed end of the specific serum preparation. Mix its contents thoroughly as in step 3, and then divide the mixture between two 3 by 1 slips and carefully spread a blood film (_vide_ page 376) on each in such a way that only one-half of the surface of each slide is covered with blood--the free edge of the blood film approximating to the longitudinal axis of the slide.

Allow films to dry and label the slides with writing diamond.

10. Treat the contents of the control pipette in similar fashion.

11. Select the better film from each pair for fixing and staining.

12. Fixing and staining must be carried out under strictly comparable conditions, and to this end the slides are best handled by placing in a glass staining rack which can be lowered in turn into each of a series of glass troughs containing the various reagents (Fig. 196). Place the rack in the first trough which contains the alcoholic solution of Leishman's stain for two minutes to fix.

Transfer to the second trough containing the diluted stain for ten minutes.

Transfer to the third trough containing distilled water, and holding the trough over a sink, run in a stream of distilled water until washing is complete. Remove slides from the rack and dry.

Leishman's stain is the best for routine work for all bacteria other than B. tuberculosis. Films containing tubercle bacilli must of course be stained by the Ziehl Neelsen method.

13. Examine specific serum slide microscopically with 1/12 inch oil immersion. Find the edge of the blood film--along this the bulk of the leucocytes will be collected. Starting at one end of the film move the slide slowly across the microscope stage and as each leucocyte comes into view count and record the number of ingested bacteria. The sum of the contents of the first 50 consecutive polymorphonuclears that are encountered is marked down. (The _average_ number of bacilli ingested per leucocyte = the "_phagocytic index_.")

14. In precisely similar manner enumerate the bacteria present in the first 50 cells of the control preparation. This number is recorded as the denominator of a vulgar fraction of which the numerator is the number recorded for the specific serum. This fraction, expressed as a percentage of unity = the _opsonic index_.

IMMUNE BODY.

Immune body or amboceptor is the name given to a substance present in the serum of an infected animal that has successfully resisted inoculation with some particular micro-organism, and which possesses the power of linking the complement normally present in the serum to bacteria of the species used as antigen in such a manner that the micro-organisms are rendered innocuous, and ultimately destroyed. The presence of the immune body in the serum can be demonstrated _in vitro_ by the reaction elaborated by Bordet and Gengou, known as the complement fixation test, the existence or the absence of the phenomenon of complement fixation being rendered obvious macroscopically by the absence or presence of haemolysis on the subsequent addition of "sensitised" red blood corpuscles, (e. g., a mixture of crythrocyte solution and the appropriate haemolysin--two of the three essentials in the haemolytic system, _vide_ page 326).

_Apparatus Required:_

Sterile pipettes 1 c.c., (graduated in tenths).

16 x 2 cm. test-tubes.

9 x 1 cm. test-tubes.

Test-tube racks for each size of test-tube.

_Reagents Required:_

Normal saline solution.

Erythrocyte solution (human red cells, page 329) = E.

Haemolytic serum (for human cells) = H.S.

Complement (fresh guinea-pig serum) = C.

Specific serum from inoculated animal, inactivated = S.S.

Control pooled serum from normal animals of same species,
Inactivated = P.S.

_Antigen_ (cultivation upon solid medium of the organism
(e. g., B. typhosus) which has already served as antigen
in the inoculation of the experimental animal) = A.

To prepare the antigen for use, emulsify the whole of the bacterial growth in 5 c.c. normal saline solution.

Shake the emulsion in a test-tube with some sterilised glass beads to ensure a homogenous emulsion, and sterilise by heating to 60 deg. C. in a water-bath for one hour.

METHOD.--

1. Take five small test-tubes, and number them 1 to 5 with a grease pencil.

2. Into tubes Nos. 1, 3, 4 and 5 pipette 0.1 c.c. of complement.

3. Into tubes Nos. 1 and 2 pipette 0.2 c.c. of the serum to be tested.

4. Into tube No. 4 pipette 0.2 c.c. of control serum.

5. Into tubes Nos. 1, 2, 3 and 4 pipette 1 c.c. of the bacterial emulsion which forms the antigen.

6. Place the whole set of tubes in the incubator at 37 deg. C. for a period of one hour.

7. Remove the tubes from the incubator and pipette 1 c.c. erythrocyte solution and 4 minimal haemolytic doses of the corresponding haemolysin into each tube.

8. Mix thoroughly and return the tubes to the incubator at 37 deg. C. for further period of one hour.

9. At the expiration of that time transfer the tubes to the ice chest, and allow them to stand for three hours.

10. Examine the tubes.

Tubes 3, 4 and 5 should show complete haemolysis; tube 2 should give no evidence whatever of haemolysis.

These tubes form the controls to the first tube, which contains the serum to be tested.

In tube No. 1 the absence of haemolysis would indicate the presence in the serum of the inoculated animal of a specific antibody to the micro-organism used in the inoculations; since it shows that the complement has been bound by the immune body to the bacterial antigen, and none has been left free to enter into the haemolytic system; on the other hand the presence of haemolysis would show that no appreciable amount of antibody has yet been formed in response to the inoculations. In other words, there is an absence of infection, since the complement remained unfixed at the time of the addition of the erythrocyte solution and haemolytic serum, and was ready to combine with those reagents to complete the haemolytic system.

The method may be shown diagramatically as under using the symbols already indicated

Test-tubes.

1 2 3 4 5

0.1 c.c. C. ........ 0.1 c.c. C. 0.1 c.c. C. 0.1 c.c. C.

0.2 c.c. S.S. 0.2 c.c. S.S. ......... 0.2 c.c. P.S. ........

A. A. A. A. ........
--------------------------------------------------------------------------
Incubate at 37 deg. C. for one hour.
--------------------------------------------------------------------------

1 c.c. E. 1. c.c. E. 1 c.c. E. 1 c.c. E. 1 c.c. E.

H.S.^{4} H.S.^{4} H.S.^{4} H.S.^{4} H.S.^{4} -------------------------------------------------------------------------- Incubate at 37 deg. C. for one hour. -------------------------------------------------------------------------- (?) No haemolysis. |__________________________________|

Haemolysis.

NOTE.--It is sometimes more convenient to _sensitise_ the
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.

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.

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.

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. | | |

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.

~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.

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._

Bacillus diphtheriae (Klebs-Loeffler).
Bacillus Hoffmanni.
Bacillus xerosis.
Bacillus septus.

VIII. _Coli-typhoid group._

B. typhi abdominalis (B. typhosus).
B. coli communis.
B. enteritidis (Gaertner).
to contrast with
B. aquatilis sulcatus.

IX. _Escherich group._

B. coli communis (Escherich).
B. coli communior.
B. lactis aerogenes.
B. cloacae.

X. _Gaertner group._

Bacillus enteritidis (Gaertner).
B. paratyphosus A.
B. paratyphosus B.
Bacillus cholerae suum (Hog Cholera).
B. psittacosis.

XI. _Eberth group._

B. typhosus (Eberth).
B. dysenteriae (Shiga).
B. dysenteriae (Flexner).
B. faecalis alcaligines.

XII. _Spirillum group._

Vibrio cholerae.
Vibrio metschnikovi.
to contrast with
Vibrio proteus (Finkler and Prior).
Spirillum rubrum.
Spirillum rugula.

XIII. _Anthrax group._

Bacillus anthracis.
to contrast with
Bacillus subtilis.
Bacillus mycoides.
Bacillus mesentericus fuscus.

XIV. _Acid fast group._

Bacillus tuberculosis (human).
" " (bovine).
" " (avian).
" " (fish).
to contrast with
Bacillus phlei (Timothy grass bacillus).
Butter bacillus of Rabinowitch.

XV. _Plague group._

Bacillus pestis.
B. septicaemiae haemorrhagicae.
B. suipestifer.

XVI. _Influenzae group._

B. influenzae.
Bacillus aegypticus (Koch-Weeks).
Bacillus pertussis.

XVII. _Miscellaneous._

Bacillus leprae.
Bacillus mallei.
Micrococcus melitensis.

XVIII. _Streptothrix group._

Streptothrix actinomycotica.
Streptothrix madurae.
to contrast with
Cladothrix nivea.

XIX. _Tetanus group._

Bacillus tetani.
Bacillus oedematis maligni.
Bacillus chauvei (symptomatic anthrax).

XX. _Enteritidis sporogenes group._

Bacillus enteritidis sporogenes.
B. botulinus.
B. butyricus.
B. cadaveris.

FOOTNOTES:

[15] See note on Vivisection License, page 334.

XXI. BACTERIOLOGICAL ANALYSES.

Each bacteriological or bacterioscopical analysis of air, earth, sewage, various food-stuffs, etc., includes, as a general rule, two distinct investigations yielding results of very unequal value:

1. Quantitative.
2. Qualitative.

The first is purely quantitative and as such is of minor importance as it aims simply at enumerating (approximately) the total number of bacteria present in any given unit of volume irrespective of the nature and character of individual organisms.

The second and more important is both qualitative and quantitative in character since it seeks to accurately identify such pathogenic bacteria as may be present while, incidentally, the methods advocated are calculated to indicate, with a fair degree of accuracy, the numerical frequency of such bacteria, in the sample under examination.

The general principles underlying the bacteriological analyses of water, sewage, air and dust, soil, milk, ice cream, meat, and other tinned stuffs, as exemplified by the methods used by the author, are indicated in the following pages, together with the methods of testing filters and chemical germicides; and the technique there set out will be found to be capable of expansion and adaptation to any circumstance or set of circumstances which may confront the student.

~Controls.~--The necessity for the existence of adequate controls in all experimental work cannot be too urgently insisted upon. Every batch of plates that is poured should include at least one of the presumably "sterile" medium; plate or tube cultures should be made from the various diluting fluids; every tube of carbohydrate medium that is inoculated should go into the incubator in company with a similar but uninoculated tube, and so on.

BACTERIOLOGICAL EXAMINATION OF WATER.

The bacteria present in the water may comprise not only varieties which have their normal habitat in the water and will consequently develop at 20 deg. C., but also if the water has been contaminated with excremental matter, varieties which have been derived from, or are pathogenic for, the animal body, and which will only develop well at a temperature of 37 deg. C. In order to demonstrate the presence of each of these classes it will be necessary to incubate the various cultivations at each of these temperatures.

Further, the sample of water may contain moulds, yeasts, or torulae, and the development of these will be best secured by plating in wort gelatine and incubating at 20 deg. C.

~1. Quantitative.~--

_Collection of the Sample._--The most suitable vessels for the reception of the water sample are small glass bottles, 60 c.c. capacity, with narrow necks and overhanging glass stoppers (to prevent contamination of the bottle necks by falling dust). These must be carefully sterilised in the hot-air steriliser (_vide_ page 31).

(a) If the sample is obtained from a ~tap~ or ~pipe~, turn on the water and allow it to run for a few minutes. Remove the stopper from the bottle and retain it in the hand whilst the water is allowed to run into the bottle and three parts fill it. Replace the stopper and tie it down, but _do not seal it_.

(b) If the sample is obtained from a ~stream~, ~tank~, or ~reservoir~, fasten a piece of stout wire around the neck of the bottle, remove the stopper, and retain it in the hand. Then, using the wire as a handle, plunge the bottle into the water, mouth downward, until it is well beneath the surface; then reverse it, allow it to fill, and withdraw it from the water. Pour out a few cubic centimetres of water from the bottle, replace the stopper, and tie it down.

(c) If the sample is obtained from a ~lake~, ~river~ or the ~sea~; or when it is desired to compare samples taken at varying depths, the apparatus designed by v. Esmarch (Fig. 203) is employed. In this the sterilised bottle is enclosed in a weighted metal cage which can be lowered, by means of a graduated line, until the required depth is reached. At this point the bottle is opened by a thin wire cord attached to the stopper; when the bottle is full (as judged by the air bubbles ceasing to rise) the pull on the cord is released and the tension of the spiral spring above the stopper again forces it into the neck of the bottle. When the apparatus is taken out of the water, the small bottles are filled from it, and packed in the ice-box mentioned below.

An inexpensive substitute for Esmarch's bottle can be made in the laboratory thus:

Select a wide-mouthed glass stoppered bottle of about 500 c.c. capacity (about 20 cm. high and 8 cm. in diameter).

Remove the glass stopper and insert a rubber cork with two perforations in its place.

Through one perforation pass a piece of glass tubing about 5 cm. long and through the other a piece 22 cm. long, reaching to near the bottom of the bottle, each tube projecting about 2.5 cm. above the rubber stopper. Plug the open ends of the tubes with cotton wool. Secure the stopper in place with thin copper wire.

Sterilise the fitted bottle in the autoclave. Remove the cotton wool plugs and connect the projecting tubes by a piece of loosely fitting stout rubber pressure tubing about 5 cm. long, previously sterilised by boiling.

Take a piece of stout rubber cord about 33 cm. long, and of 10 mm. diameter (such as is used for door springs) thread a steel split ring upon it and secure the free ends tightly to the neck of the bottle by cord or catgut.

Attach the cord used for lowering the bottle into the water to the split ring on the rubber suspender. The best material for this purpose is cotton insulated electric wire knotted at every metre.

Connect the split ring also with the short piece of rubber tubing uniting the two glass tubes by a piece of catgut (or thin copper wire) of such length that when the bottle is suspended there is no pull upon the rubber tube, but which, however, will be easily jerked off when a sharp pull is given to the suspending cord.

Now wind heavy lead tubing about 1 cm. diameter around the upper part of the bottle, starting at the neck just above the shoulder. This ensures the sinking of the bottle in the vertical position (Fig. 204).

The apparatus being arranged is lowered to the required depth, a sharp jerk is then given to the suspending cord, which detaches the rubber tube and so opens the two glass tubes. Water enters through the longer tube and the air is expelled through the shorter tube. The bubbles of air can be seen or heard rising through the water, until the bottle is nearly full, a small volume of compressed air remaining in the neck of the bottle.

As the apparatus is raised, the air thus imprisoned expands, and prevents the entry of more water from nearer the surface.

_Transport of Sample._--If the examination of the sample cannot be commenced immediately, steps must be taken to prevent the multiplication of the bacteria contained in the water during the interval occupied in transit from the place of collection to the laboratory. To this end an ice-box such as that shown (in Fig. 205) is essential. It consists of a double-walled metal cylinder into which slides a cylindrical chamber of sufficient capacity to accommodate four of the 60 c.c. bottles; this in turn is covered by a metal disc--the three portions being bolted together by thumb screws through the overhanging flanges. When in use, place the bottles, rolled in cotton-wool, in the central chamber, pack the space between the walls with pounded ice, securely close the metal box by screwing down the fly nuts, and place it in a felt-lined wooden case. (It has been shown that whilst bacteria will survive exposure to the temperature of melting ice, practically none will multiply at this temperature.)

On reaching the laboratory, the method of examination consists in adding measured quantities of the water sample to several tubes of nutrient media previously liquefied by heat, pouring plate cultivations from each of these tubes, incubating at a suitable temperature, and finally counting the colonies which make their appearance on the plates.

_Apparatus Required_:

Plate-levelling stand.
Case of sterile plates.
Case of sterile pipettes, 1 c.c. (in tenths of a cubic centimetre).
Case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre).
Case of sterile capsules, 25 c.c. capacity.
Tubes of nutrient gelatine.
Tubes of nutrient agar.
Tubes of wort gelatine.
One 250 c.c. flask of sterile distilled water.
Tall cylinder containing 2 per cent. lysol solution.
Bunsen burner.
Grease pencil.
Water-bath regulated at 42 deg. C.

METHOD.--

1. Arrange the plate-levelling platform with its water compartment filled with water, at 45 deg. C.

2. Number the agar tubes, consecutively, 1 to 6; the gelatine tubes, consecutively, 1 to 6, and the wort tubes, 1, 2, and 3. Flame the plugs and see that they are not adherent to the lips of the tubes.

3. Place the agar tubes in boiling water until the medium is melted, then transfer them to the water-bath regulated at 42 deg. C. Liquefy the nutrient gelatine and wort gelatine tubes by immersing them in the same water-bath.

4. Remove the bottle containing the water sample from the ice-box, distribute the bacterial contents evenly throughout the water by shaking, cut the string securing the stopper, and loosen the stopper, but do not take it out.

5. Remove one of the 1 c.c. pipettes from the case, holding it by the plain portion of the tube. Pass the graduated portion twice through the Bunsen flame. Tilt the bottle containing the water sample on the bench holding the neck between the middle and ring fingers of the left hand; grasp the head of the stopper between the forefinger and thumb, and remove it from the bottle.

6. Pass the pipette into the mouth of the bottle, holding its point well below the surface of the water (Fig. 206). Suck up rather more than 1 c.c. into the pipette and allow the pipette to empty; this moistens the interior of the pipette and renders accurate measurement possible. Now draw up exactly 1 c.c. into the pipette. Withdraw the pipette from the bottle, replace the stopper, and stand the bottle upright.

7. Take the first melted agar tube in the left hand, remove the cotton-wool plug, and add to its contents 0.5 c.c. of the water sample from the pipette; replug the tube and replace it in the water-bath. In a similar manner add 0.3 c.c. water to the contents of the second tube, and 0.2 c.c. to the contents of the third.

8. In a similar manner add 1 c.c. of the sample to the contents of the fourth tube.

9. Similarly, add 0.5 c.c. and 0.1 c.c. respectively to the contents of the fifth and sixth tubes.

10. Drop the pipette into the cylinder containing lysol solution.

11. Mix the water sample with the medium in each tube in the manner described under plate cultivations; pour a plate from each tube. Label each plate with (a) the distinctive number of the sample, (b) the quantity of water sample it contains, and (c) the date.

12. Pour the contents of a tube of liquefied agar--not inoculated--into a Petri dish to act as a control to demonstrate the sterility of the batch of agar employed.

13. Allow the plates to set, and incubate at 37 deg. C.

14. Empty the water chamber of the levelling apparatus and refill it with ice-water.

15. By means of the sterile 10 c.c. pipette deliver 9.9 c.c. sterile distilled water into a sterile glass capsule.

16. Add 0.1 c.c. of the water sample to the 9.9 c.c. sterile water in the capsule. This will give a dilution of 1 in 100.

17. Plant the six tubes of nutrient gelatine in the following manner: To the first tube add 0.5 c.c. of the water sample direct from the bottle; to the second, 0.3 c.c.; and to the third, 0.2 c.c.; and pour a plate of each tube. To the fourth tube add 0.5 c.c. of the diluted water sample from the capsule; to the fifth, 0.3 c.c.; and to the sixth, 0.2 c.c.; and pour a plate from each.

18. Label each plate with the quantity of the water sample it contains--that is, 0.5 c.c., 0.3 c.c., 0.2 c.c., 0.005 c.c., 0.003 c.c., and 0.002 c.c.

19. Pour a control (uninoculated) gelatine plate.

20. Allow the plates to set, and incubate at 20 deg. C.

21. To the first tube of liquefied wort gelatine add 0.5 c.c. water sample; to the second, 0.3 c.c.; and to the third, 0.2 c.c.

22. Label the plates, allow them to set, and incubate at 20 deg. C.

23. Count and record the number of colonies that have developed upon the agar at 37 deg. C. after forty-eight hours' incubation.

24. Note the number of colonies present on each of the gelatine and wort gelatine plates after forty-eight hours' incubation.

25. Replace the gelatine and wort plates in the incubator; observe again at three days, four days, and five days.