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:

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:

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 hyperæmic 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.

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.

8. Mark the distinctive title of the specimen (e. g., animal's number) upon the pipette with a writing diamond or grease pencil.

9. When the sealed ends are cold and the blood has clotted, place the pipette on the centrifuge, clean end downward; counterpoise and centrifugalise thoroughly. On removing the pipette from the centrifuge, the red cells will be collected in a firm mass at one end, and above them will appear the clear serum.

10. By marking the blood pipette above the level of the serum with the glass cutting knife and snapping the tube at that point, the blood-serum becomes readily accessible for testing purposes.

If larger quantities of blood are required, the animal, after puncturing the vein, should be inverted, an assistant holding it up by the legs. Blood to the volume of several cubic centimetres will now drop from the punctured vein, and should be caught in a tapering centrifuge tube, the tube transferred to the incubator at 37° C. for two hours, then placed in the centrifugal machine, counterpoised and centrifugalised thoroughly. The three most important of the antibodies referred to which can be demonstrated with a certain amount of facility are agglutinin, opsonin and bacteriolysin; and the methods of testing for these bodies will now be considered.

AGGLUTININ.

Agglutinin is the name given to a substance present in the blood-serum of an animal that has successfully resisted inoculation with a certain micro-organism. This substance possesses the power of collecting together in clumps and masses, or agglutinating watery suspensions of that particular microbe.

Dilution of the Specific Serum:

Apparatus required:

Method.—

1. Take three sterile test-tubes and number them 1, 2 and 3.

2. Pipette 0.9 c.c. sterile normal saline solution into each tube, and stand tubes upright in the sand in the capsule, or in the plasticine block.

3. Make a scratch with the glass cutting knife on the blood pipette above the upper level of the clear serum, and snap off and discard the empty portion of the tube.

4. Remove 0.1 c.c. of the serum from the blood pipette tube, and mix it thoroughly with the fluid in tube No. 1; and label s.s., (specific serum), 10 per cent.

5. Remove 0.1 c.c. of the solution from tube No. 1 by means of a fresh pipette, and mix it with the contents of tube No. 2; and label s.s., 1 per cent.

6. Remove 0.1 c.c. of the solution from tube No. 2 by means of a fresh pipette, and mix it with the contents of tube No. 3; and label s.s., 0.1 per cent.

When the yield of serum from the specimen of blood which has been collected, or is available, is small, the above method of diluting is not practicable, and the dilution should be carried out by Wright's method in a capillary teat pipette.

Dilution of Serum by Means of a Teat Pipette.

Materials required:

Method:

1. Mark three small test-tubes 10 per cent., 1 per cent. and 0.1 per cent. respectively, and stand them upright in the plasticine block.

2. Take a Pasteur pipette, nick the capillary stem just above the sealed end with a glass cutting knife, and snap off the sealed end with a quick movement so that the fracture is clean cut and at right angles to the long axis of the capillary stem—cut "square", in fact. Prepare several, say a dozen, in this manner.

3. Fit a rubber teat to the barrel of each of the pipettes.

4. Make a mark with the grease pencil on the stem of one of the pipettes about 2 or 3 cm. from the open extremity.

5. Compress the teat between the finger and thumb (Fig. 193) to such an extent as to drive out the greater part of the contained air.

6. Maintaining the pressure on the teat pass the stem of the pipette into the capsule holding the saline solution, until the open end of the pipette is below the level of the fluid.

7. Now cautiously relax the pressure on the teat and let the fluid enter the pipette and rise in the stem until it reaches the level of the grease pencil mark. As soon as this point is reached, check the movement of the column of fluid by maintaining the pressure on the teat, neither relaxing nor increasing it.

8. Withdraw the point of the pipette clear of the fluid, and again relax the pressure on the teat very slightly. The column of saline solution rises higher in the stem, and a column of air will now enter the pipette and serve as an index to separate the first volume of fluid drawn into the stem from the next succeeding one.

9. Again introduce the end of the pipette into the fluid and draw up a second volume of saline to the level of the grease pencil mark, and follow this with a second air index.

10. In like manner take up seven more equal volumes of saline solution and their following air bubbles. There are now nine equal volumes of normal saline in the pipette.

11. Now pass the point of the pipette into the blood tube and dip the open end below the surface of the serum. Proceeding as before, aspirate a volume of serum into the capillary stem up to the level of the pencil mark.

12. Eject the contents of the pipette into the small tube marked 10 per cent. by compressing the rubber teat between thumb and finger.

13. Mix the one volume of serum with the nine volumes of saline solution very thoroughly by repeatedly drawing up the whole of the fluid into the pipette and driving it out again into the test-tube.

14. Now take a clean pipette and proceed precisely as before, 4 to 10.

15. Having aspirated nine equal volumes of saline into this second pipette, now take up one similar volume of the fluid in the "10 per cent. tube."

16. Eject the contents of this pipette into the second tube marked 1 per cent. and mix thoroughly as before.

17. In similar fashion make the 0.1 per cent. solution and transfer to the third tube.

18. Further dilutions in multiples of ten can be prepared in the same way, and by varying the number of volumes of diluting fluid or serum any required dilution can be made (see Appendix, Dilution Tables).

The Microscopical Reaction:

Apparatus Required:

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.

The Macroscopical Reaction:

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°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.

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 pneumoniæ) grown upon sloped blood agar at 37° 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 hæmolysis on the subsequent addition of "sensitised" red blood corpuscles, (e. g., a mixture of crythrocyte solution and the appropriate hæmolysin—two of the three essentials in the hæmolytic system, vide page 326).

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° 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° C. for a period of one hour.

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

8. Mix thoroughly and return the tubes to the incubator at 37° 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 hæmolysis; tube 2 should give no evidence whatever of hæmolysis.

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

In tube No. 1 the absence of hæmolysis 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 hæmolytic system; on the other hand the presence of hæmolysis 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 hæmolytic serum, and was ready to combine with those reagents to complete the hæmolytic system.

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

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.

Apparatus Required:

Water steriliser.

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 axillæ 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:—

25. Place a loopful of fresh desiccating fluid on a 3 × 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 scapulæ, 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.