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.

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:

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

and dissolve in

then add

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

Equally good but much cheaper is Frost's mounting solution:

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

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 rôle.

The organisms included in this group are:

and in certain special tissues

The group may with advantage be subdivided as indicated in the following pages:

I. Pyogenic cocci.

1. Prepare subcultivations from each:

Compare the naked-eye appearances of the cultures from day to day. Note M. agilis refuses to grow at 37°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.

III. Pyogenic cocci.

IV. Pyogenic bacilli.

V. Pyogenic bacilli.

VI. Pneumonia group.

VII. Diphtheroid group.

VIII. Coli-typhoid group.

IX. Escherich group.

X. Gaertner group.

XI. Eberth group.

XII. Spirillum group.

XIII. Anthrax group.

XIV. Acid fast group.

XV. Plague group.

XVI. Influenzæ group.

XVII. Miscellaneous.

XVIII. Streptothrix group.

XIX. Tetanus group.

XX. Enteritidis sporogenes group.

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:

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° 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° 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 torulæ, and the development of these will be best secured by plating in wort gelatine and incubating at 20° 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:

Method.—

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

23. Count and record the number of colonies that have developed upon the agar at 37° 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.

26. Calculate and record the number of organisms present per cubic centimetre of the original water from the average of the six gelatine plates at the latest date possible up to seven days—the presence of liquefying bacteria may render the calculation necessary at an earlier date, hence the importance of daily observations.

Method of Counting.—The most accurate method of counting the colonies on each of the plates is by means of either Jeffery's or Pakes' counting disc. Each of these discs consists of a piece of paper, upon which is printed a dead black disc, subdivided by concentric circles and radii, printed in white. In Jeffery's counter (Fig. 207), each subdivision has an area of 1 square centimetre; in Pakes' counter (Fig. 208), radii divide the circle into sixteen equal sectors, and counting is facilitated by concentric circles equidistant from the centre.

(a) In the final counting of each plate, place the plate over the counting disc, and centre it, if possible, making its periphery coincide with one or other of the concentric circles.

(b) Remove the cover of the plate, and by means of a hand lens count the colonies appearing in each of the sectors in turn. Make a note of the number present in each.

(c) If the colonies present are fewer than 500, the entire plate should be counted. If, however, they exceed this number, enumerate one-half, or one-quarter of the plate, or count a sector here and there, and from these figures estimate the number of colonies present on the entire plate. In practice it will be found that Pakes' disc is more suitable for the former class of plate; Jeffery's disc for the latter. It should be recollected however that unless the plates have been carefully leveled and the medium is of equal thickness all over it is useless to try and average from small areas—since where the medium is thick all the bacteria will develop, where the layer is a thin one, only a few bacteria will find sufficient pabulum for the production of visible colonies.

It will be noted that the quantities of water selected for addition to each set of tubes of nutrient media have been carefully chosen in order to yield workable results even when dealing with widely differing samples. Plates prepared in agar with 0.1 c.c. and in gelatin with 0.02 c.c. can be counted even when large numbers of bacteria are present in the sample; whereas if micro-organisms are relatively few, agar plate 4 and gelatine plate 1 will give the most reliable counts. Again the counts of the plates in a measure control each other; for example, the second and third plates of each gelatine series should together contain as many colonies as the first, and the second should contain about half as many more than the third and so on.

2. Qualitative Examination.—

Collection of Sample.—The water sample required for the routine examination, which it will be convenient to consider first, amounts to about 110 c.c. It is collected in the manner previously described (vide page 416); similar bottles are used, and if four are filled the combined contents, amounting to about 240 c.c., will provide ample material for both the qualitative and quantitative examinations. Unless the examination is to be commenced at once, the ice-box must be employed, otherwise water bacteria and other saprophytes will probably multiply at the expense of the microbes indicative of pollution, and so increase the difficulties of the investigation.

In the routine examination of water supplies it is customary to limit the qualitative examination to a search for

A. B. coli and its near allies.

B. Streptococci,

organisms which are frequently spoken of as microbes of indication, as their presence is held to be evidence of pollution of the water by material derived from the mammalian alimentary canal, and so to constitute a danger signal.

C. Some observers still attach importance to the presence of B. enteritidis sporogenes, but as the search for this bacterium, (relatively scarce in water) necessitates the collection of a fairly large quantity of water it is not usually included in the routine examination.

In the case of water samples examined during the progress of an epidemic, of new supplies and of unknown waters the search is extended to embrace other members of the coli-typhoid group; and on occasion the question of the presence or absence of Vibrio choleræ or (more rarely) such bacteria as B. anthracis or B. tetani, may need investigation.

When pathogenic or excremental bacteria are present in water, their numbers are relatively few, owing to the dilution they have undergone, and it is usual in commencing the examination, to adopt one or other of the following methods:

A. Enrichment, in which the harmless non-pathogenic bacteria may be destroyed or their growth inhibited, whilst the growth of the parasitic bacteria is encouraged.

This is attained by so arranging the environment, (i. e., Media, incubation temperature, and atmosphere) as to favor the growth of the pathogenic organisms at the expense of the harmless saprophytes.

B. Concentration, whereby all the bacteria present in the sample of water, pathogenic or otherwise, are concentrated in a small bulk of fluid.

This is usually effected by filtration of the water sample through a porcelain filter candle, and the subsequent emulsion of the bacterial residue remaining on the walls of the candle with a small measured quantity of sterile bouillon.

A. Enrichment Method.

(Dealing with the demonstration of bacteria of intestinal origin.)

Apparatus Required (Preliminary Stage):