Bacteria Cultivation, Sterilising, and Preparing for Microscopical Examination.

Fig. 245.—Sections of Wood.

That branch of mycology which is now looked upon as a separate department of science, termed bacteriology, took shape in the years 1875-9, when its founder, the veteran botanist Cohn, who recognised that the protoplasm of plants corresponded to the animal sarcode, published his exact mode of studying bacteria. But it was a pupilof his, Dr. Koch, who a year later discovered that a specific cattle disease, anthrax, was due to a bacillus, and it was he also who gave us the useful modification of gelatine as a medium in which to grow bacteria; he hit upon the method of pouring melted gelatine containing distributed germs on to plates, and thus isolating the colonies and ensuring the further isolation of the spores, and so facilitate the preparation of pure cultures on a large scale, and with great saving of time.

The difficulty of isolating a bacterium and tracing its life history under the microscope must at first sight appear great. A further objection that such work is slow and difficult has no more weight here than in any other department of science, as will be seen on proceeding to follow out the directions I am about to furnish for the use of the student.

A good microscope with a wide-angled sub-stage condenser, and objectives of an inch, ¼-inch, or1⁄6-inch, and a1⁄12-inch homogeneous oil-immersion.

A large bell-glass for covering the same when fuming acids are in use in the laboratory.

About a square foot of blackened plate-glass.

A white porcelain slab, or a shallow photographic dish of some size.

Glass bottles with ground stoppers for alcoholic solutions and aniline dyes.

Glass bottles with funnels for filtering solutions of stains, with pipettes.

A specialised form of pipette for the micro-chemical filtration of solutions (Fig. 246).

A small stoppered bottle of cedar oil (Fig. 247).

Set of small glass dishes or watch-glasses for section staining.

Stock of glass slides sterilised, together with round thin glass-covers, in boxes (Fig. 248).

Needle holders and platinum needles, with a packet of ordinary sewing needles (Fig. 249).

Platinum, or plated copper section-lifters (Fig. 250).

Glass rods, drawn out to a fine point, for manipulating sections when acids are employed.

Fig. 246.—Pipette for Micro-chemical Filtration.

Fig. 247.—Bottle and Dipper for Cedar Oil.

Fig. 249.—Needle Holders, fine Lifter and Hook for Manipulating Structure.

Fig. 248.—Box for keeping Glass-covers.

Fig. 250.—Section Lifters.

Fig. 251.—Spring Flat Forceps.

Fig. 251a.—Forceps with fine Points.

A pair of small spring steel platinum-pointed forceps for holding glass-covers (Fig. 251).

One or two pairs of fine-pointed forceps (Fig. 251a).

Collapsible tubes for containing Canada balsam and dammar.

Soft rags or old pocket handkerchiefs for removing cedar oil from lenses and cover-glasses. Chamois leather for wiping lenses and removing dust.

Reagents,alcohol, bergamot oil,celloidin, dissolved in equal parts of ether and alcohol.

Ebner’ssolution. (See Appendix.)

Formalin, glycerine, gelatine,Klebs’andKleinenberg’ssolutions. (See Appendix.) The latter consisting of a watery solution of picric acid 100 parts; strong sulphuric acid two parts; filter, and add distilled water 300 parts.

Mullerfluid. (See Appendix.)

Osmic acid, a five per cent. solution.

Paraffin,spermacetiandxylol,acetic acid,hydrochloric acid, a one per cent. solution withalcohol.

Ammonialiquid,ether,picro-lithium carmine,potash solution.

Safranine, concentrated alcoholic solution of, and a watery solution.

Turpentine,vesuvin, water distilled and sterilised.

Aqueous solutions of the several dyes may be kept in bottles ready for use.

To both aqueous and alcoholic solutions a few drops of phenol, or a crystal of thymol, should be added as a preservative. For the rapid staining of cover-glass preparations, it is convenient to have the most frequently used stains—fuchsine, methyl-violet, &c.—in bottles provided with pipette stoppers.

Clearing Agents.—Oils of cedar wood, cloves, origanum, aniline, terebene, toluol and xylol, benzol and spirits of turpentine.

Mounting Media.—Acetate of potash solution concentrated, benzole, balsam, glycerine jelly, Fanant’s medium, dammar and mastic, Canada balsam in xylol, Hollis’s glue, zinc white.

Cement for fixing small specimens temporarily to a glass slide. Remove all traces of moisture, place upon it a drop or two of a medium prepared as follows:—Dissolve over a water bath 15 grammes of white lac in 100 grammes of absolute alcohol, decant off the clear liquid, and stand it by for a while.

As the alcohol evaporates from the warmed surface of the glass slide a hard transparent coating is left. This may be slightlysoftened at any time by means of a drop of oil of lavender. After arranging the objects the heat of a spirit-lamp will cause the oil to evaporate, leaving them firmly attached. Objects may be mounted on cover-glasses in a similar way. A resinous mounting medium may then be employed in the usual manner. If glycerine or glycerine jelly is the mounting medium employed, collodion diluted with two or three times its volume of oil of lavender may be found preferable as the fixing agent. The section should be placed in position before the preparation dries and the oil is evaporated.

Methylated spirit is often so largely adulterated with rock-oil as to render it unsuitable for technical purposes. Even to varnishes it imparts a fluorescent appearance as it dries off.

Fig. 252.—Iron Box for holding Sterilised Instruments and Glass Plates.

The needles and instruments used must not be passed through a Bunsen burner flame, which is most destructive, but enclosed in a sheet-iron box made for the purpose (Fig. 252), and placed in the hot-air steriliser for an hour at 150°C. The box can be opened at the side, and each instrument withdrawn with a pair of sterilised forceps when required for use.

Glass platesare sterilised in the same iron box, and theplatinum needlesfor inoculating nutrient media, examining cultivations, &c., are served in the same manner before being used. The needles consist of two or three inches of platinum wire fixed to the end of a glass rod. Several of these needles should be made by fixing pieces of wire into a glass rod about six inches long. The glass rod must be heated at the extreme end in the flame of a Bunsen burner, or blow-pipe, and the platinum wire held near one extremity with forceps, and fused into the end of the glass rod. Some of these rods should be straight, and some bent, and others provided with a loop, and kept especially ready for inoculating test-tubes of nutrient jelly.

Fig. 253.—Damp Chamber for Plate-cultivations.

Glass Dishes.—Several shallow glass dishes are required for preparing damp chamber cultivations, the upper covers fitting over the under (as inFig. 253), in the centre of which culture-plates are stacked one above the other, and when necessary placed in the incubator.

Lister’s Flasks.—Lister devised a globe-shaped flask with two necks, a vertical and a lateral one, the lateral being a bent spout, tapering towards the extremity. When the vessel is restored to the erect position after pouring out some of its contents, a drop of liquid remains behind in the end of the nozzle, and thus prevents the regurgitation of air through the spout. A cap of cotton-wool is tied over the orifice, and the residue left in the flask for future use. The vertical neck of the flask is plugged with sterilised cotton-wool in the ordinary way.

Fig. 254.—Pasteur’s Bulb Pipette.Fig. 255.—Storing Cultivation Tube.

Fig. 254.—Pasteur’s Bulb Pipette.

Fig. 255.—Storing Cultivation Tube.

Sternberg advocates the use of a glass bulb, provided with a slender neck drawn out to a fine point and hermetically sealed. Special forms of tubes, bulbs, and pipettes were devised by Pasteur,and are still in use at the Bacteriological Institute, Paris, and known as the Pasteur’s bulb pipette (Fig. 254).

Others are provided with lateral or with curved arms, one of which is drawn out to a fine point, and the slender neck plugged with cotton-wool, as inFig. 255.

Fig. 256.—Pfeiffer’s Warm Chamber.

The Warm Chamber.—This is an accessory of importance in bacteriological work. For the continuous heating of specimens during cultivation it is an absolute necessity. Pfeiffer’s warm chamber (Fig. 256) is suitable for microscopical work generally. Itconsists of a hard-wood box, made air-tight, with doors and glass windows to allow of the specimen being moved from time to time, and kept under constant observation. The box is mounted on a metal plate tripod stand, and is heated from below by a small gas burner, with a thermo-regulator. A paraffin lamp will do as well, so long as it maintains a temperature of from 25° to 45°C., and without danger of injury to the stand and lenses of the microscope. A thermometer is placed in the air space to mark the temperature.

Fig. 257.—Crookshank’s Incubator.

Hot-air Incubators and Sterilisersare usually made of sheet-iron, in the form of a cubical chest, with double walls, supported on four legs, as that of Dr. Crookshank’s (Fig. 257). They are heated by gas or a lamp from below, while the temperature is indicated by a thermometer inserted through a hole in the top, as in that of the Hearson’s incubator. Test-tubes, flasks, funnels, cotton-wool, &c., must be sterilised by exposure to a temperature of 150°C. for an hour or more.

Wire Cagesor crates are used for containing test-tubes, especially when they are to be sterilised in the hot-air steriliser, or for loweringtubes of nutrient jelly into the steam steriliser. All instruments, needles, scalpels, &c., before using must be carefully sterilised.

Fig. 258.—Dr. Koch’s Steam Steriliser.

Steam Sterilisersare made either of iron or tin, jacketed with thick felt, and provided with a conical cap or lid perforated at the apex to receive a thermometer (Fig. 258). Inside the vessel is an iron grating or diaphragm about two-thirds of the way down, which divides the interior into two chambers, the upper or steam chamber, and the lower or water chamber. A gauge outside marks the level of the water in the lower chamber; this should be kept about two-thirds full. The apparatus stands upon three legs, and is heated from below with a Bunsen burner or a lamp. It is employed for sterilising nutrient media in tubes or flasks, for cooking potatoes or hastening the filtration of agar-agar. When the thermometer indicates 100° C. the lid is removed, and test-tubes are lowered in a wire-basket by means of a hook and string, and the lid quickly replaced. Potatoes or small flasks are lowered into the cylinder in a tin receiver with a perforated bottom, which rests upon the grating, and admits of the contents being exposed to the steam generated.

One of the most efficient forms of incubators introduced into the bacteriological laboratory is that known as Hearson’s (Fig. 259). This consists of a chamber surrounded by a water-jacket, with water space below, to afford room for the pipe, L, which conveys the heated products from the flame of the lamp, T, through the water and back again to the lantern. A is the water-jacket surrounding the chamber containing the cultures; O, the pipe through which the water supply is admitted; N, the tap for employing the same; M, the overflow pipe; S, the capsule in a case attached by a tube to the lower plate outside; D, a lever pivoted on the left, carrying at its free end a damper, F, which, when resting on the chimney, V, effectually closes it; P, a screwfor adjusting the damper when starting the apparatus; H, a lead weight for bringing more pressure on the capsule; K, a thermometer, the bulb of which is inside and the scale outside the chamber.

Fig. 259.—The Baird-Hearson Biological Incubator.

The treated products of combustion move in the direction indicated until the water and chamber are sufficiently heated to distend the capsule. When this point is reached the wire between S and P is pushed up by the capsule, and the lever causes the damper to rise more or less off the chimney, V, and on examining the thermometer the inside of the chamber is at length found to remain steadily at the required temperature.

When the thermometer registers the desired temperature, the lead weight must be damped to the lever by means of the milled-head screw which goes through it. After having been once adjusted the heat in the interior will remain constant, notwithstanding the utmost changes of temperature occurring in these latitudes, nor will very great alterations in the size of the lamp-flame seriously interfere with the results. The milled-head screw, P, must be turned, after the first adjustment, during the whole time that the incubator is in use. Observe the temperature before opening the door; observations taken afterwards are worthless.

Fig. 260.—Plate Cultivation Showing Colonies.

To cultivate micro-organisms artificially they must be supplied with the proper nutrient material, perfectly free from pre-existing organisms. The secret of Koch’s methods greatly depends upon the possibility, in the case of starting with a mixture of micro-organisms, of being able to isolate them completely one from another, and to obtain an absolutely pure growth of each cultivable species. When sterile nutrient gelatine has been liquefied in a test-tube and inoculated with a mixture of bacteria in such a way that the individual micro-organisms are distributed throughout it, and the liquid is poured out on a glass plate and allowed to solidify, the individual bacteria, instead of moving about freely as in a liquid medium, are fixed to one spot, where they develop their own species.In this way colonies are formed, each possessing its own biological characteristics and morphological appearances (Fig. 260).

To maintain individuals isolated from each other during growth, and free from contamination, it is only necessary to thin out the cultivation to protect the plates from the air, and to have facilities for examining them from time to time, and observing the characteristic microscopical appearances. The colonies on nutrient gelatine examined with a low power (Fig. 260), if micro-organisms such asBacillus anthracesandProteus mirabilis, the naked eye appearances in test-tubes of the growth of the bacilli of anthrax and tubercle, and the brilliant growth of micro-coccus prodigiosus, may be given as examples in which the appearances are often very striking and sometimes quite characteristic. I must, however, first direct attention to a well-recognised fact, that bacteriology only touches animal pathology at a few points, and that so far from bacteria being synonymous withdisease germs, the majority of these remarkable organisms appear to be beneficent rather than inimical to man. This is of immense importance to science, as I shall attempt to show further on; although even a brief description of all the useful ferments due to bacteria and brought into use would occupy a volume to themselves, and call for a school of bacteriology quite apart from that involved in the medical aspect of the question, for the purpose of fully investigating problems raised by the agriculturist, the forester, the gardener, the dairyman, brewer, dyer, tanner, and other industries, which open up vistas of practical application, and to some extent are already being taken advantage of in commerce.

The Preparation of Nutrient Gelatine and Agar-agar.—Take half a kilogramme (one pound) of beef as free as possible from fat, chop finely, transfer to a flask or cylindrical vessel, and shake up well with a litre of distilled water. Place the vessel in an ice-pail, or ice-cupboard, or in winter in a cold cellar, and leave for the night. Next morning commence with the preparation of all requisite apparatus. Thoroughly wash and rinse with alcohol about 100 test-tubes, and allow them to dry. Plug the mouths of the test-tubes with cotton-wool, place them in their wire cages in the hot-air steriliser, to be heated for an hour at a temperature of 150°C. In the same manner cleanse and sterilise several flasks, and a smallglass funnel. In the meantime, the meat infusion must be well shaken, and the liquid portion separated by filtering and squeezing through a linen cloth or a meat press. The red juice thus obtained must be brought up to a litre by transferring it to a large measuring glass and adding distilled water. It is then poured into a sufficiently large and strong beaker, and set aside after the addition of ten grammes of peptone, five grammes of common salt, and 100 grammes of best gelatine.

In about half an hour the gelatine is sufficiently softened, and subsequent heating in a water bath causes it to be completely dissolved.

The next process requires the greatest care and attention. Some micro-organisms grow best in a slightly acid, others in a slightly alkaline, medium. For example, for the growth and characteristic appearances of thecomma bacillusof Asiatic cholera a faintly alkaline soil is absolutely essential. This slightly alkaline medium will be found to answer best for most micro-organisms, and may be obtained as follows:—With a clean glass rod dipped in the mixture, the reaction upon litmus-paper may be obtained, and a concentrated solution of carbonate of soda must be added drop by drop until red litmus-paper becomes faintly blue. If it is too alkaline, it can be neutralised by the addition of lactic acid.

Finally, the mixture is heated for an hour in the water-bath. Ten minutes before the boiling is completed the white of an egg beaten up with the shell is added, and the liquid is then filtered while hot.

During filtration the funnel should be covered over with a plate of glass, and the process of filtering must be repeated, if necessary, until a pale straw-coloured, perfectly transparent filtrate results. The sterilised test-tubes are filled to about a third of their depth by pouring in the gelatine carefully and steadily. The object of this care is to prevent the mixture touching the part of the tube with which the plug comes into contact; otherwise, when the gelatine sets, the cotton-wool adheres to the tubes and becomes a source of embarrassment to subsequent procedures. As the tubes are filled they are placed in a basket, and then sterilised. They are either lowered into the steam steriliser, when the thermometer indicates 100 cc., for twelve minutes, for four or five successive days, or they may be transferred to the test-tube water-bath, and heated for an hour or two for three successive days.

If the gelatine shows any turbidity after, it must be poured back into a flask, boiled for ten minutes, and filtered again, and the process of sterilisation repeated.

Nutrient Agar-agaris a substance prepared from seaweed which grows on the coasts of Japan and India, and is supplied in long crinkled strips. It boils at 90° C., and remains solid up to a temperature of about 45° C. It is therefore substituted for gelatine in the preparation of a jelly for the cultivation of those bacteria which will grow best in the incubator at the temperature of the blood, and also at ordinary temperature for bacteria which lignify gelatine. The preparation is conducted on much the same principles as those already described. Instead, however, of 100 grammes of gelatine, only about twenty grammes of agar-agar (1·5 to 2 per cent.), and to facilitate the solution it must be allowed to soak in salt water overnight. Flannel is substituted for filter paper. The hot-water apparatus is invariably employed. The final results, when solid, should be colourless and clear; but if slightly milky, it may still be employed.

Wort-gelatineis used in studying the bacteria of fermentation. It is made by adding from five to ten per cent. of gelatine to beer-wort.

Glycerine Agar-agar.—This is made by adding five per cent. of glycerine to nutrient agar-agar, after the boiling and before the filtration.

Fig. 261.—Pure Cultivation in Tubes (Crookshank).

Test-tube Cultivations.—To inoculate test-tubes containing nutrient jelly, the cotton-wool plug is removed. A sterilised needle, charged,for example, with blood or pus containing bacteria, is thrust once in the middle line into the nutrient jelly, and steadily withdrawn. The tube should be held horizontally or with its mouth downwards, and the plug replaced as quickly as possible, and an india-rubber cap fitted over the mouth of the tube.

The appearance produced by the growths in the test-tubes can be in most cases sufficiently examined with the naked eye (Fig. 261). In some cases the jelly is partially liquefied, while in others it remains solid. The growths may be abundant or scanty, coloured or colourless. When liquefaction slowly takes place in the needle tracts, the appearances which result are often very delicate and in some very characteristic. The appearance of a simple white thread with branching lateral filaments, of a cloudiness, or of a string of beads in the track of the needle, may be given as examples. In some cases much may be learnt by means of a magnifying-glass.

Beneke recommends that gelatine culture tubes should be inoculated by making a puncture quite at the side of the medium, close to the glass. The advantage of this method over the plan of inoculating the mass in the middle is that the growing culture can be microscopically examined from the outside, and various details made out, such as the nature of the growth, the comparative appearance of colonies near the surface and those situated more deeply, and the presence of one or more distinct organisms. If the tubes used have the opposite sides flat and parallel, such examinations will be still further facilitated.

Plate Cultivations.—By this method a mixture of bacteria, whether in fluids, excreta, or in cultivations on solid media, can be so treated that the different species are isolated one from the other, and perfectly pure cultivations of each of the cultivable bacteria in the original mixture established in various nutrient media. We are enabled also to examine under a low power of the microscope the individual colonies of bacteria. The same process, with slight modification, is also employed in the examination of air, soil, and water.

In order to spread out the liquid jelly evenly on the surface of a glass plate, and to hasten its solidification, it is necessary to place the plate upon a level and cool surface. The glass plates are sterilised in an iron box placed in the hot-air steriliser, at 150° C., from one to two hours.

The damp chambers for the reception of the inoculated plates are prepared by cleansing and washing out with one in twenty carbolic acid the shallow glass dish and bell-cover (Fig. 253). A piece of filter-paper should cover the bottom of dish, moistened with the same solution.

“In a glass-beaker with pad of cotton-wool at bottom place tube containing cultivation, the three tubes to be inoculated, three glass rods which have to be sterilised, and a thermometer. Liquefy the gelatine in the three tubes by placing them in a beaker containing water 30° C. Keep the tubes, both before and after the inoculation, in the warm water to maintain the gelatine in a state of liquefaction. Remove the plug from the culture and also the plug of test-tube with liquefied jelly. With the needle take up a droplet of the cultivation and stir it round in the liquefied jelly. Replace both plugs, and set aside the cultivation. Hold the freshly-inoculated tube almost horizontally, then raise it to the vertical, so that the liquid gelatine gently flows back. By repeating this motion, and rolling the tube, the micro-organisms which have been introduced are distributed throughout the gelatine. Any violent shaking, and consequent formation of bubbles, must be carefully avoided. Inoculate the second tube, and also third, in the same way, but with three droplets from a sterilised needle. The next process consists in pouring out the gelatine on glass plates and allowing it to solidify.

“Remove cover of box containing sterilised plates, withdraw a plate with sterilised forceps, and rapidly transfer it to the filter-paper under the bell-glass and quickly replace cover of box. Remove plug from the test-tube which was first inoculated, and the contents are poured out on the plate. With a glass rod the gelatine must be then rapidly spread out in an even layer within about half an inch of the margin of the plate, the bell-glass is replaced, and the gelatine is allowed to set. Meanwhile a glass bench is placed in damp chamber, upon which the plate is placed when the gelatine is quite solid; precisely the same process is repeated with the other tubes.

“The colonies will be found to develop in the course of a day or two, the time varying with the temperature of the room. The lower plate will contain a countless number of colonies, which, if the micro-organisms liquefy gelatine, speedily commingle, and produce in a very short time a complete liquefaction of the whole gelatine. On themiddle plate the colonies will also be very numerous, but retain their isolated positions for a longer time; while on the uppermost plate the colonies are completely isolated from one another, with an appreciable surface of gelatine intervening.

“The microscopical appearances of the colonies are best studied by placing the plate on a slab of blackened glass, or on a porcelain slab if the colonies are coloured. A small diaphragm is used, and the appearances studied principally with a low power. A much simpler method of plate-cultivation is to pour the liquefied jelly into shallow flat dishes; they take up much less room, and in many ways are more convenient.

“Nutrient agar-agar can also be employed for the preparation of plate-cultivations, but it is much more difficult to obtain satisfactory results.”

Bacteria in Liquids, Cultures, and Fresh Tissues.—In conducting bacteriological researches, the importance of absolute cleanliness cannot be too strongly insisted upon. All instruments, glass vessels, slides, and cover-glasses should be thoroughly cleansed before use. The same applies to the preparation and employment of culture media; any laxity in the processes of sterilisation, or insufficient attention to minute technical details, will be followed with disappointing results by contamination of the cultures, resulting in the loss of much time.

For the preparation of microscopical specimens it will be found convenient to use a platinum inoculating needle, sterilised, as before directed, in the sheet-iron box; in a few moments it will be cool enough not to destroy the bacteria with which it is brought into contact.

Unstained Bacteria.—The bacteria in liquids, such as blood and culture-fluids, can be investigated in the unstained condition by transferring a drop with a looped platinum needle, or a capillary pipette, to a slide, covering it with a clean cover-glass, and examining without further treatment. If it is desirable to keep the specimen under prolonged observation, a drop of sterilised water or salt solution must be run in at the margin of the cover-glass to counteract the tendency to dry.

Cultures on the solid media can be examined by transferring a small portion with a sterilised needle to a drop of sterilised water on a slide, thinning it out, and covering with cover-glass as already described. Tissues in the fresh state may be teased out with needles (Fig. 249) in sterilised salt solution, and pressed out into a sufficiently thin layer between the slide and cover-glass. Glycerine may in many cases be substituted for salt solution, especially for such as actinomyces and mould fungi.

Very small bacilli and micro-cocci are distinguished from granular matter or fat-crystals, orvice versâ, by the fact that the latter are altered or dispersed by the addition of acetic acid, and changed by solution of potash; ether dissolves out fatty particles, while micro-organisms remain unaffected. Baumgarten demonstrated tubercle bacilli in sections by treating them with potash, which clarified the tissues and brought the bacilli clearly into view. In examining unstained bacteria the iris-diaphragm should be used, and the sub-stage condenser carefully centred and focussed.

His’s Method of Staining.—A slide is prepared as for bacteria in the fresh state; the reagents are then applied by placing them with a pipette drop by drop at a margin of the cover-glass, and causing them to flow through the preparation by means of a strip of filter-paper placed at the opposite margin.

Babès’ Method is as follows: A little of the growth spread out on a cover-glass into as thin a film as possible; when almost dry, apply a drop or two of a weak aqueous solution of methyl-violet from a pipette to the film; any excess of the stain must be removed by gentle pressure with a strip of filter-paper.

Cover-glass Preparations.—A cover-glass is smeared with the substance to be examined spread out into a sufficiently thin layer; in the case of cultures on solid media, diffuse the bacteria in a little sterilised water. By means of another cover-glass the juice or fluid is squeezed out from between them into a thin layer, and on sliding them apart each cover-glass bears on it a thin film of the material. The cover-glass is then placed with its film side upwards and allowed to dry. After a few minutes it is passed from above downwards through the flame of a Bunsen burner three times. Apply two or three drops of an aqueous solution of fuchsine or methyl-violet to cover the film, wash away any surplus stain after a few minuteswith distilled water. The cover-glass is then allowed to dry, when the preparation may be mounted in Canada balsam, or while still wet, turned over on a slide, and the excess of water removed with filter-paper.

If necessary to apply stain for a much larger period, pour staining solution into a watch glass and allow cover-glass to swim on surface with prepared side downwards.

Crookshank, instead of watery solutions of aniline dyes, prefers to use stronger solutions, and to reduce the staining by a momentary immersion in alcohol. The method is as follows: cover-glass preparations are stained with carbolised fuchsine (Neelsen’s solution) for about two minutes, rinsed in alcohol for a few seconds, and quickly washed in water. This method is specially valuable for sarcinæ and streptococci.

Gram’s Method.—The whole film is first stained violet with gentian-violet, fixed by a solution of iodine, in iodide of potassium in the bacilli, but not in any débris, pus cells, or tissue elements present. Transfer cover-glass to alcohol, the bacilli alone remain stained, the violet colour being changed to blue. By employing a contrast colour, such as eosin, a double staining is obtained.

For staining preparations with gentian-violet Crookshank employs the following useful method:—Place four or five drops of pure aniline in a test-tube, add distilled water to three-quarters full, close mouth with thumb, shake thoroughly. Filter the emulsion twice, pour filtrate into watch-glass. To the perfectly clear aniline water thus obtained, add, drop by drop, a concentrated alcoholic solution of gentian-violet till precipitation commences. Cover-glasses must be left in this solution ten minutes, transferred to iodine-potassic-iodide until the film becomes uniformly brown, then rinsed in alcohol. The decolourisation may be hastened by dipping the cover-glass in clove oil and returning to alcohol. Again immerse cover-glass in clove oil, dry by gently pressing between two layers of filter-paper, and mount in Canada balsam.

Double-stainingof cover-glass preparations.—They can be treated by Ehrlich’s method for staining tubercular sputum, or by Neelsen’s modification, or by staining with eosin after treatment by the method of Gram.

Ehrlich’s Method is as follows: Five parts of aniline oil are shakenup with one hundred parts of distilled water, and the emulsion filtered through moistened filter-paper. A saturated alcoholic solution of fuchsine, methyl-violet, or gentian-violet, is added to filtrate in watch-glass, drop by drop, until precipitation commences. Cover-glass preparations are floated in this mixture for fifteen minutes to half an hour, then washed for a few seconds in dilute nitric acid (one part of nitric acid to two of water), then rinsed in distilled water.

Neelsen’s Solution and Methylene Blue.—Ziehl suggested the use of carbolic acid as a substitute for aniline blue. Neelsen recommended a solution of carbolic acid, absolute alcohol and fuchsine. (See Appendix.)

Gram’s Solution and Eosin.—After using Gram’s method as above and decolourising in alcohol, the cover-glass is placed in a weak solution of eosin for two or three minutes, washed in alcohol, immersed in clove oil, dried, and mounted in balsam.

Staining of Spores.—The cover-glass preparation must be heated to 210° C. for half an hour, or passed about twelve times through the flame of a Bunsen burner, or exposed to the action of strong sulphuric acid for several seconds, then a few drops of a watery solution of aniline dye applied in the usual way. To double-stain spore-bearing bacilli the cover-glass preparation must be floated from twenty minutes to an hour on Ehrlich’s fuchsine-aniline-water, or on the Ziehl-Neelsen solution. The stain must be heated until steam arises.

Koch first stained flagella by floating the cover-glass on a watery solution of hæmatoxylin, transferring them to a five per cent. solution of chromic acid, or to Müller’s fluid, by which they obtained a brownish-black coloration.

Löffler’s Method.—Add together aqueous solutions of ferrous-sulphate and tannin (twenty per cent.) until the mixture turns a violet-black colour, then add three or four cc. of a one-in-eight aqueous solution of logwood; a few drops of carbolic acid may be added before transferring to a stoppered bottle; that is the mordant. The dye consists of 1 cc. of a one per cent. solution of caustic soda, added to 100 cc. of aniline water, in which four or five grammes of either methyl-violet, methylene blue, or fuchsine, are dissolved. Acover-glass preparation is made in the usual way, then the film is covered with mordant, and cover-glass held over flame until steam rises, the mordant is then washed off with distilled water. The stain is filtered and a few drops allowed to fall on film, after a few minutes the cover-glass is again warmed until steam rises. The stain is then washed off with distilled water, and the preparation is ready to be mounted for examination.

As Löffler’s process is somewhat complicated, a modification has been said to afford more satisfactory results. A specimen is taken from a recent gelatine culture and diluted with water. A little of the fluid is then transferred to a warm cover-glass by means of a pipette and allowed to dry, after which a drop of the following mordant is applied:—Aqueous solution of tannin (twenty per cent.), ten cc.; cold saturated solution of ferrous sulphate, five cc.; saturated solution of fuchsine in absolute alcohol, one cc. The cover is next heated gently for a short time until vapours are given off, then washed carefully. This process is repeated two or three times, and the specimen washed after each application. Subsequently, staining is effected by means of Ziehl’s fuchsine solution, the cover is afterwards warmed once or twice for about fifteen seconds, then washed, and the specimen examined in water to ascertain if the colour is sufficiently intense. If satisfactory, the preparation may then be dried and finally mounted in Canada balsam or dammar.

Preservation of Preparations.—After examining a cover-glass preparation with an oil-immersion objective the cedar oil must be carefully wiped off, and the slide set aside for the Canada balsam to set. At a convenient time these preparations should be sealed with a ring of Hollis’s glue.

Method of Hardening and Decalcifying Tissues.—To harden small organs, such as the viscera of a mouse, they should be placed on a piece of filter-paper at the bottom of a small wide-mouthed glass jar, and covered with about twenty times their volume of absolute alcohol. Larger organs are treated in the same way, but must be cut up into small pieces. Müller’s fluid, methylated spirit, or formalin may be used.

Teeth, or osseous structures, must first be placed in a decalcifying solution, as Kleinenberg’s. When sufficiently softened, soak in water, to wash out picric acid, and transfer to weak spirit. Ebner’s solution gives good results.

Methods of embedding, fixing, and cutting.—Crookshank finds that after hardening, the pieces of tissue are embedded in a mixture of ether and alcohol for an hour or more, then transferred to a solution of celloidin in equal parts of ether and alcohol, and left there for several hours.

The piece of tissue is then placed in a glass capsule, and some of the celloidin solution poured over it. The capsule can be placed bodily in 60 to 80 per cent. alcohol, and left there until the following morning. The celloidin should be of the consistency of wax. The piece of tissue is next cut out, and after trimming is put into water until it sinks, then transferred to gum, and cut with the freezing microtome.

Sections of fresh tissues are to be floated in ·8 per cent. salt solution, and then carefully transferred by a platinum lifter to a watch-glass containing absolute alcohol.

Staining Bacteria in Tissue Sections.—Weigert’s method is as follows:—Place sections for from six to eighteen hours in a one per cent. watery solution of any of the basic aniline dyes. To hasten, place the capsule containing solution in the incubator, or heat it to 45° C., or a stronger solution may be used. In the latter case the sections must be treated with a half-saturated solution of carbonate of potash, as they are easily over-stained. In either case the sections are next washed with distilled water, passed through sixty per cent. alcohol into absolute alcohol. When almost decolourised, spread out on a platinum lifter and transfer to clove oil, or stain with picro-carmine solution (Weigert’s) for half an hour, wash in water, alcohol, and treat with clove oil, and transfer to clean glass slide.

Gram’s Method.—Sections are stained for ten minutes in a capsule containing aniline-gentian-violet solution, then placed in the iodine and iodide solution until uniformly brown, then placed in absolute alcohol, and washed by carefully moving sections in the liquid with a glass rod. When completely decolourised, they are transferred to clove oil and then to a slide.

Double-staining is obtained by transferring the sections after decolourisation to eosin, Bismarck brown, or vesuvin (Crookshank).

Formalinis an excellent preservative fluid; one part to 20,000 is sufficient to prevent fermentation. For the preservation of vegetable sections, a one per cent. solution is required; even the fresh appearance of vegetable structures is preserved for some time when immersed in it. In the nutrient gelatine for biological specimens, if used early, will arrest the liquefaction of the gelatine by bacteria. For hardening it saves time, and is even better than alcohol, chromic acid, pot. bich., and many others. It does not cause shrinkage of the cells. Tissue ½ to ¾ inch thick hardens in twenty-four hours in pure formalin; five to ten per cent. is best for loose tissue. In another method, by which time can be saved, instead of placing the specimen in theformalinand afterwards in mucilage, prior to cutting sections, make the mucilage with two per cent. (or stronger) formalin water, and it will then answer both purposes at the same time.

Various materials are required for preparing and mounting microscopic objects, as slips of glass, patent flatted plate measuring 3 × 1 inch, thin glass covers, glass cells, preservative media, varnishes, cements, a glazier’s diamond, and a Shadbolt’s turn-table.

The glass slides and covers, although sent out packed ready for use, should be immersed in an alkaline solution to ensure perfect freedom from any greasiness derived from touching by the fingers. Dr. Seller recommends a particular solution for this purpose. (SeeFormulæ, Appendix.)

Varnishes and cements must be selected with care, as these are not only expected to adhere firmly to the glass slide, but also to resist the action of the preservative fluid in which the specimen may be mounted. Among the numerous preparations employed, I may enumerate Canada balsam, gum dammar, Venice turpentine, Japanners’ gold size, used for closing up cells, asphalte varnish, Brunswick black, shellac, glue and honey, Hollis’ liquid glue, and marine glue. To give a finish to the mounted specimen, coloured varnishes are sometimes resorted to. A red varnish of sealing-wax ismade by digesting powdered sealing-wax in strong alcohol. Filter, and place the solution in a dish, and evaporate by means of a sand bath to reduce it to a proper consistency. This is said to resist the action of cedar oil. For white, zinc, cement is the best. This is made of benzole, gum dammar, oxide of zinc, and turpentine. Cole gives another formula, but either of these may be obtained of Squire, who supplies every kind of staining and mounting material.

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