Owing in some measure to the more complete knowledge of the subject gained by the experience of years, and the extreme value of micro-photography in the delineation of bacteria, and perhaps in a measure to the advent of the perfected dry-plate process, photography is being rapidly pressed forward in conjunction with the microscope. In the course of the year [1898] no less than six, more or less, new forms of micro-photographic apparatus have appeared; two are simple, one for daylight, one for lamp, one for electric, and one for lime-light illumination. Passing over the simpler forms, for a notice of which I am unable to find room, there is one piece of new apparatus, that of Mr. E. B. Stringer, which is not only new, but is in every way adapted to the work of micro-photography. It is in fact a well-arranged camera, fitted with a powerful condensing arrangement, each portion of which is capable of being independently centred and controlled. Indeed, the specially interesting feature of the apparatus is the control of the gas and the beautiful and uniformally illuminating disc of zircon, about a quarter of an inch in diameter.
Fig. 446.—Mr. E. B. Stringer’s Improved Micro-photography Apparatus.B. Oxyhydrogen jet with zirconium cylinder, covered by the cowl A when working.C. Doublet parallelising condenser, with centering screws.D. Iris diaphragm.E. Holder for trough and light-filtering media.F. Plano-convex lens, 41â„4ins. diameter, with centering screws GH. Plano-concave lens, with iris diaphragm T.K. Connecting pulleys between focussing rod of camera and fine adjustment of Microscope.L. Triangular frame in which Microscope feet are placed.M. Flap shutter.N. Door through which image is observed on card screen, etc.O. Solid block of mahogany on which camera body is fixed and supported.P. Dark slide.
Fig. 446.—Mr. E. B. Stringer’s Improved Micro-photography Apparatus.
B. Oxyhydrogen jet with zirconium cylinder, covered by the cowl A when working.C. Doublet parallelising condenser, with centering screws.D. Iris diaphragm.E. Holder for trough and light-filtering media.F. Plano-convex lens, 41â„4ins. diameter, with centering screws GH. Plano-concave lens, with iris diaphragm T.K. Connecting pulleys between focussing rod of camera and fine adjustment of Microscope.L. Triangular frame in which Microscope feet are placed.M. Flap shutter.N. Door through which image is observed on card screen, etc.O. Solid block of mahogany on which camera body is fixed and supported.P. Dark slide.
This efficient photo-micrographic apparatus (Fig. 446) is made by Messrs. W. Watson & Sons, under the instructions of Mr. E. B. Stringer. The illuminating condensing system is mounted on a square brass bar, the illuminant being oxygen-hydrogen light burning on zirconium. Immediately in front of this is a condenser, c,four and a half inches diameter, with an iris diaphragm,D, immediately in front of it. The holder,E, carries the light filtering media through which the beam passes and enters the condenser,F. It then goes through a tank of water contained in the cone,FtoH, and emerges a practically parallel beam of great intensity through a plano-concave lens, h, of such a diameter as to exactly fill the back lens of the substage condenser. There is an iris diaphragm,T, for cutting off stray light.
The whole of the apparatus is fitted with centring screws and clamps, and after having been once adjusted it is ready for use at any moment without preparation. By means of this apparatus, instantaneous pictures can be taken of living rotifers, so brilliant is the illumination, while photographs of such fine objects as the flagella of bacteria cannot be secured with the same amount of certainty by any other microphotographic apparatus with which I have made myself acquainted.
FORMULÆ AND METHODS:—CEMENTING, CLEARING, HARDENING AND MOUNTING.90
The object of employing a clearing agent is to replace the alcohol in the dehydrated section by a liquid which has a refractive index about the same as the balsam into which it is to be placed, and which will readily mix with it.
Oil of Bergamotwill clear quickly from 90 per cent. of alcohol. Clove oil clears more rapidly, but it dissolves out aniline colours to a considerable extent. Xylol is without action on aniline colours. This strength of alcohol is chosen because of its being that of the methylated spirit sold in London, and which is much used in washing and dehydrating on account of its cheapness.
Oil of Cedar Wood, although an essential oil, resembles xylol, but evaporates slowly. It has very little solvent action on the aniline colours. It clears rapidly from absolute alcohol, but not well from 90 per cent. Sections can be left in it for several days. It is a convenient medium in which to examine tissues before mounting them permanently. It clears celloidin without dissolving it; and as a connecting fluid between the object and objective nothing better has been discovered.
Other clearing agents have been tried, but as they dissolve out the aniline colours, are no longer used.
Grove’s Mastic and Bismuth.—Dissolve gum mastic in chloroform, and thicken with nitrate of bismuth. The solution of mastic should be nearly saturated.
Grove’s Oxide of Zinc, Dammar, and Drying Oil.—Rub up well-ground oxide of zinc, 2 ozs., with drying oil, to the consistence of thick paint. Then add an equal quantity of gum dammar, previously dissolved in benzoline, and of the thickness of syrup. Strain through close-meshed muslin. Keep in well-corked bottle, and, if necessary, thin with benzoline.
Isinglass Cement.—Heat the isinglass in a covered vessel on the water-bath with a little glacial acetic acid, until it is thoroughly softened and forms a stiff mass, then gradually add more acid until it produces a thick solution which is of uniform consistence, and just fluid while hot. Then run into wide-mouth bottles and close with good corks.
Kitton’s Cementof white lead and red lead in powder, and litharge powder in equal parts. Grind together with a little turpentine, until thoroughly incorporated, and mix with gold size. The mixture should be thin enough to use with a brush; in using, one coat should be allowed to dry before applying another. No more cement should be mixed with the gold size than is required for immediate use, as it sets quickly, and becomes unworkable.
Krönig’s Cement.—Gradually add ordinary resin, 7 to 9 parts, to melted beeswax, 2 parts, then steam and cool.
Shellac Cement.—Dissolve shellac in an equal weight of methylated spirit, then pour off the clear portion and add a few drops of balsam and castor oil.
Marine Glue.—Dissolve indiarubber in mineral naphtha, and add twice the quantity of powdered shellac; or make chloroform the solvent, and use mastic instead of shellac. For casting battery trays, use a composition of 4 parts resin and 1 of gutta percha, with a little boiled oil.
Selier(Cleaning Glass Slides).—New slides or cover-glasses must be placed for a few hours in a mixture of 1 part of potassium bichromate, 1 of sulphuric acid, and 25 of water. Subsequently wash with water and wipe dry with a linen rag, after draining off the excess of moisture. Covers that have been used should be previously immersed for a few days in a mixture of equal parts of alcohol and hydrochloric acid. Scrape old slides free of mounting medium before immersing them in the bichromate solution.
Elsching’s Celloidin Solution.—Allow the celloidin shavings to swell up for 24 hours in the necessary quantity of absolute alcohol, then add the proper amount of ether.
Koch’s Copal.—Stain small pieces of material in bulk, and dehydrate with alcohol, then immerse in a thin solution of copal in chloroform. Evaporate with a gentle heat until the solution is so far concentrated as to draw out into threads that are brittle on cooling. Then remove the objects and leave on a tile for a few days to dry. Sections may then be cut by means of a fine saw. If objects are imbedded unstained, remove copal from sections by soaking in chloroform, decalcify if necessary, and stain.
Eulenstein’s Cement.—Mix equal parts of Brunswick black and gold size with a very little Canada balsam.
In the case of bony structures, or tissues so impregnated with calcium salts, the material should be decalcified by an acid capable of dissolving out the mineral matter. Hydrochloric acid with alcohol is in more general use. The older the bone the stronger will be the acid required, nitric with alcohol and chromic acid. Picric acid is preferred for fœtal bone.
Andeer, J. J., finds an aqueous solution of phloroglucin acts as a powerful decalcifying agent on the bones of animals, but is without action on the most delicate organic tissue. If treatment with hydrochloric acid be employed as well, the residual “ossein†will be without a trace of either calcium phosphate or carbonate.
Ebner’s Fluids.—(1) Mix 100 C.c. of cold saturated aqueous solution of sodium chloride, 100 C.c. of water, and 4 C.c. of hydrochloric acid. Preparations are placed in the fluid, and 1 to 2 C.c. of hydrochloric acid added daily until they are soft. (2) Mix 2·5 parts of hydrochloric acid (sp. gr. 1·16) with 500 of alcohol (90 per cent.), 100 of water, and 2·5 of sodium chloride.
Fol’s Liquid.—Mix 70 volumes of 1 per cent. chromic acid, 3 of nitric acid, and 200 of water.
Mayer’s Desilification Process.—Place the objects in alcohol contained in a glass vessel coated internally with paraffin, then add hydrofluoric acid drop by drop until desilification is complete, avoiding the fumes meanwhile.
Marsh’s Chlorine Method.—Chlorine is generated in a small bottle by treating crystals of potassium chlorate with strong HCl., and the gas is led through a piece of glass tubing, bent twice at right angles, to the bottom of a bottle containing the sections immersed in water.
Ranvier’s Fluid.—Use 50 per cent. hydrochloric acid with the addition of sodium chloride to counteract its swelling action.
Squire’s Fluid.—(1) Mix 95 parts of glycerine with 5 parts of hydrochloric acid; used for softening teeth. (2) Use a 4 per cent. aqueous solution of arsenic acid at a temperature of 30° to 40° C. After softening tissues in this solution, keep them in alcohol.
Waldeyer.—To a 0·1 per cent. solution of palladium chloride, add one-tenth its volume of hydrochloric acid.
Altmann(Fixing Solution).—A mixture of equal parts of 5 per cent. potassium bichromate solution and 2 per cent. osmic acid.
Alcohol.—Strengths of alcoholic solutions, as given by Squire, will be found of practical value. Absolute alcohol (sp. gr. O·797) containing about 98 per cent. of ethylic alcohol is taken as the basis in most instances. Alcohol of 90 per cent. (sp. gr. 0·823) is prepared by mixing 14 volumes of absolute alcohol and 1 volume of distilled water; 84 per cent. alcohol (sp. gr. 0·838) is rectified spirit B.P.; 70 per cent. alcohol (sp. gr. 0·872) may be obtained by adding 1 volume of distilled water to 3 volumes of absolute alcohol, 6 volumes of rectified spirit, or 4 volumes of methylated spirit; 50 per cent. alcohol (sp. gr. 0·918) is prepared by adding 4 volumes of distilled water to 5 volumes of absolute alcohol, 3 volumes of water to 5 volumes of rectified spirit, or 3·5 volumes of water to 5 volumes of methylated spirit. Absolute alcohol, 75 C.c., mixed with acetic acid, 25 C.c., serves as an excellent fixing agent for nuclei. Immerse tissues in it for 6 to 12 hours, then transfer to 90 per cent. alcohol until hardened, afterwards preserving in 70 per cent. alcohol till wanted.
Betz’s Hardening Fluid.—A mixture of equal parts of sulphuric ether and alcohol. This is used for hardening the brain of insects prior to cutting sections.
Cole’s Freezing Process.—Dissolve picked gum acacia, 4 ozs., in distilled water, 6 ozs., and to each 5 parts of the resulting mucilage add 3 parts of syrup made by dissolving loaf sugar, 1 lb., in distilled water, 1 pint. To each ounce of the medium add 5 grains of pure carbolic acid, and soak the tissues in it prior to freezing. For tissues liable to come to pieces, mix 4 parts of syrup with 5 of mucilage.
Flemming’s Fixing Solution.—Osmic acid (1 per cent. solution), 80 C.c.; chromic acid (10 per cent. solution), 15 C.c.; glacial acetic acid, 10 C.c.; distilled water, 95 C.c.
Fol’s Fixing—Osmic acid (1 per cent. solution), 4 C.c.; chromic acid (10 per cent. solution), 5 C.c.; glacial acetic acid, 10 C.c.; distilled water, 181 C.c.
Fischer’s Imbedding Mass.—Dissolve 15 parts of transparent soap in 17·5 parts of 96 per cent. alcohol.
Klein’s Hardening.—Mix 1 C.c. of 10 per cent. chromic acid solution with 60 C.c. of water, and add 30 C.c. of 90 per cent. alcohol.
Müller’s Fluid Formula, see page 288.—This solution is sometimes mixed with one-third its volume of 90 per cent. alcohol, its hardening action being then much more rapid.
Rabl’s Hardening Fluid.—Chromic acid solution (10 per cent.), 7 C.c.; water, 200 C.c.; formic acid (sp. gr. 1·2), 5 drops.
Rollett’s Freezing Process.—Small portions of tissue placed on the stage of microtome, after immersion in the white of an egg, then frozen and cut with a very cold knife.
Ryder(Double Embedding).—After the celloidin bath, soak objects in chloroform, then remove into a mixture of chloroform and paraffin, heated to not more than 40° C., and finally into a bath of pure paraffin.
Stricker(Imbedding Mass).—Prepare the objects in alcohol and imbed in a concentrated solution in gum arabic in a paper case, then throw the whole into alcohol and cut after 2 or 3 days.
Webb(Dextrin Freezing).—A thick solution of dextrin (1:40) in aqueous solution of carbolic acid is used for imbedding, and subsequently frozen.
Sections are usually mounted in balsam, dammar, glycerine, &c., but it is not a necessity that the cover-glass should be fixed or cemented down. Some cements (caoutchouc by preference) should be employed when glycerine or aqueous (Farrant’s) media are used.
Alleger’s Gelatine Process.—Add a few drops of formalin to each gramme of 0·5 to 1 per cent. gelatine solution. After mounting the section in this, apply heat to the slide until the paraffin is softened, and allow the superfluous gelatine to drain from the edge of the slide.
Apáthy’s Mounting Medium.—Picked gum arabic, 50 Gm.; cane-sugar, 50 Gm.; distilled water, 50 Gm.; dissolve over a warm bath and add 0·05 Gm. of thymol. This medium sets very hard, and combined with a paper cell it may be used for ringing glycerine mounts.
Cole’s Slow or Exposure Method of Mounting.—Dissolve dried Canada balsam, 3 ozs., in benzole, 3 fl. ozs., and filter. Apply a clean cover-glass to a slide that has been moistened by breathing on it, and place a few drops of the balsam solution on the cover-glass. Then remove a section from turpentine, and put it into the balsam. Put aside for 12 hours to allow the benzole to evaporate, and havingwarmed a slide and added a drop of fresh balsam solution to that on the cover-glass, bring the fluid balsam in contact with the warmed slide. Press the cover down carefully to avoid the inclusion of air bubbles, and when the excess of balsam is squeezed out, put the slide aside to cool, after which it may be cleaned with a camel-hair brush or soft rag moistened with methylated spirit.
Farrant’s Solution.—Take of gum arabic 5 parts; water 5 parts; when the gum is fairly dissolved add 10 parts of a 5 per cent. solution of carbolic acid.
Flemming’s Glycerine Preservative.—Mix equal parts of alcohol, glycerine, and water. Lee recommends the addition of 0·5 to 0·75 per cent. of acetic acid.
Lee’s Turpentine Colophonium Mounting Medium.—This is highly recommended for general work, and is prepared by adding small pieces of colophonium to rectified oil of turpentine, heating in a stove, and when the solution is sufficiently thick filtering twice in the stove.
Seaman(Glycerine Jelly).—Dissolve isinglass in water so as to make a jelly that remains stiff at the ordinary temperature of the room, and add one-tenth part of glycerine, together with a little solution of borax, carbolic acid, or camphor water. Filter through muslin whilst warm and add a little alcohol.
Seiler(Alcohol Balsam).—Heat Canada balsam until it becomes brittle when cold, then dissolve in warm absolute alcohol and filter through absorbent cotton-wool. This is chiefly useful as a mounting medium for objects stained with carmine.
Squire(Farrant’s Medium).—Dissolve in 200 C.c. of distilled water 1 Gm. of arsenious acid and 130 Gm. of gum arabic, then add 100 C.c. of glycerine. Filter through fine Swedish filter paper upon which has been deposited a thin layer of talc.
Squire(Glycerine and Gum).—Dissolve 130 Gm. of gum arabic in 200 C.c. of chloroform water (1 in 200), then add 100 C.c. of glycerine and filter.
Squire(Glycerine Jelly).—Soak 100 Gm. of French gelatine in chloroform water, drain when soft, and dissolve with heat in 750 Gm. of glycerine. Add 400 Gm. of chloroform water, with which has been incorporated about 50 Gm. of fresh egg albumen, mix thoroughly, and heat to boiling point for about 5 minutes. Make up the total weight to 1550 Gm. with chloroform water and filter in a warm chamber.
Squire(Canada Balsam).—Dry the balsam over a water bath until brittle when cooled, then to each 200 Gm. add 100 C.c. of benzole or rather less xylol.
Squire(Dammar Solution).—(1) Dissolve 100 Gm. of dammar in 100 C.c. of benzole. (2) Dissolve 100 Gm. of dammar in 200 C.c. of turpentine oil, and add 50 Gm. of mastic dissolved in 200 C.c. of chloroform.
Squire(Potassium Acetate Solution).—Dissolve 250 Gm. of potassium acetate in 100 C.c. of water, by the aid of gentle heat, and filter. This is used as a mounting medium.
Squire(Treatment of Sections).—Imbed tissues to be cut in paraffin melting between 45° and 50° C., according to the temperature of the room and the nature of the material. Afterwards preserve the sections, prior to staining and mounting, in 50 per cent. alcohol, or in a mixture of equal volumes of glycerine and thymol water (1 in 1500). Sections may be conveniently washed in alcohol, dehydrated, and cleared, in small wide-mouthed bottles.
Topping’s Solution.—-Mix 1 part of absolute alcohol with 5 parts of water, or 4 parts of water and 1 part of aluminium acetate. Add an equal volume of glycerine before use.
Apáthy’s Hæmatoxylin Stain.—After staining in 1 per cent. solution of hæmatoxylin in 70 or 80 per cent. alcohol, wash out in 1 per cent. solution of potassium bichromate in alcohol of the same strength. The bichromate solution should be freshly made by mixing 1 part of a 5 per cent. aqueous solution with about 4 parts of 80 to 90 per cent. alcohol.
Alferow(Silver Staining).—An acid solution of silver picrate, lactate, acetate, or citrate, is prepared by adding to 800 C.c. of the solution 10 to 15 drops of a concentrated solution of the acid of the salt taken.
Bethe’s Stain for Chitin.—Place series of mounted sections on slides in a freshly prepared 10 per cent. solution of aniline hydrochloride, containing 1 drop of hydrochloric acid for each 10 C.c., for 3 or 4 minutes, then rinse in water, and put the slide with sections downwards in a 10 per cent. solution of potassium bichromate. The process may be repeated if the stain is not sufficiently intense, but the sections must be well rinsed with water after each immersion.
Beale’s Ammonia Carmine.—Carmine, 10 grs.; strong solution of ammonia, 30 mins.; distilled water, 2 ozs.; alcohol, 0·5 oz.; glycerine, 2 ozs. Dissolve the carmine in the ammonia by the aid of heat, boil for a few seconds, and let the solution cool. Then allow the excess of ammonia to evaporate, add the other ingredients, and filter. If any carmine should deposit on keeping add one or two drops of ammonia solution to redissolve it.
Benda’s Copper Hæmatoxylin.—Harden the material with chromic acid or Flemming’s solution and leave sections for 24 hours in a 5 per cent. solution of neutral copper acetate at a temperature of about 40° C., wash out well with distilled water, and stain to a dark grey or blackish tint in a saturated aqueous hæmatoxylin solution. Decolourise the sections in 0·2 per cent. hydrochloric acid until light yellow, put back into the copper solution until they turn bluish-grey, then wash, dehydrate, clear, and mount in balsam.
Bismarck Brown.—Vesuvine 0·5 Gm., rectified spirit 2, and distilled water 80 C.c.; or a concentrated alcoholic solution may be kept ready for dilution.
Bochmer’s Hæmatoxylin.—Dissolve (a) crystallised hæmatoxylin, 1 Gm., in absolute alcohol, 10 C.c., and (b) alum ammonia, 10 Gm., in distilled water, 200 C.c. Mix the two solutions, and allow to ripen for some days before use. Filter after standing a week. Wash out with aqueous solution of alum (0·5 per cent.) or with acids.
Calberla’s Indulin Stain.—Dilute a concentrated aqueous solution with 6 volumes of water and stain sections for 5 to 20 minutes. Afterwards wash in water or alcohol, and examine in glycerine or clove oil.
Calberla’s Macerating Mixture(for nerve and muscle of embryos).—Dissolve potassium chloride, 0·4 Gm., sodium chloride, 0·3 Gm., sodium phosphate, 0·2 Gm., and calcium chloride, 0·2 Gm., in water, 100 Gm., saturated with carbon dioxide just before using. Mix one volume of this solution with half a volume of Müller’s solution and one volume of water. The Müller’s solution may be replaced by a 2·5 per cent. solution of ammonium chromate. Tissues macerated in this mixture are isolated by teasing and shaking, and mount specimens in concentrated potassium acetate solution.
Canoy’s Salt Solution.—Add a trace of osmic acid to a 0·75 per cent. solution of sodium chloride in water.
Chenzinsky’s Methylene Blue and Eosine.—Mix saturated aqueous solution of methylene blue, 40 parts, with 0·5 per cent. solution of eosine in 70 per cent. alcohol, 20 parts, and distilled water or glycerine, 40 parts.
Cohnheim’s Gold Method.—Place pieces of tissue in 0·5 per cent. gold chloride solution until quite yellow, then expose to light in water acidulated with acetic acid until the gold is thoroughly reduced. Mount specimens in acidulated glycerine.
Crookshank’s Method of Staining Flagella.—Cover-glass preparations are stained with a drop of concentrated alcoholic solution of gentian violet, then rinsed in water, allowed to dry, and mounted in balsam.
Czoker’s Alum Cochineal.—Dissolve alum 1 Gm. in distilled water, 100 C.c., add powdered cochineal, 1 Gm., and boil; evaporate down to half of its original bulk, filter, and add ½ C.c. of liquid carbolic acid.
Delafield’s Hæmatoxylin.—Dissolve hæmatoxylin, 4 Gm., in absolute alcohol, 25 C.c., and add the solution to 400 C.c. of a saturated aqueous solution of ammonia alum. Expose the mixture to light and air for 3 or 4 days, then filter and add glycerine, 100 C.c., and methylic alcohol, 100 C.c. Again expose the solution to light until it becomes dark-coloured, then filter and preserve in a stoppered bottle.
Ehrlich’s Acid Hæmatoxylin.—Dissolve hæmatoxylin, 2 Gm., in absolute alcohol, 100 C.c., and add glycerine, 100 C.c., distilled water, 100 C.c., ammonia alum, 2 Gm., glacial acetic acid, 10 C.c. Expose to daylight for at least a month before use, removing the stopper at intervals.
Ehrlich’s Hæmatoxylin (Ammoniated).—Dissolve ammonium carbonate, 0·4 Gm., and hæmatoxylin, 2 Gm., in proof spirit, 40 C.c., and expose to the air in a shallow dish for 24 hours. Then make up the volume to 40 C.c. with proof spirit (warming if necessary to re-dissolve any separate crystals), and add ammonia alum, 2 Gm., dissolved in distilled water, 80 C.c., together with glycerine, 100 C.c., rectified spirit, 80 C.c., and glacial acetic acid, 10 C.c.
Ehrlich-Biondi Mixture(or Ehrlich-Biondi-Heidenheim mixture).—Dissolve (a) methyl green, 0·5 Gm., in distilled water, 100 C.c.; (b) acid fuchsine, 0·5 Gm., in distilled water, 40 C.c.; (c) orange, 2 Gm., in distilled water, 200 C.c. Mix the three solutions and filter before use. Stain sections for 12 hours, then wash, dehydrate, clear, and mount.
Ehrlich-Weigert-Koch’s Gentian-Violet-Aniline-Water.—Aniline water,100 C.c., concentrated alcoholic solution of gentian violet, 11 C.c.; absolute alcohol, 10 C.c.
Everard, Demoor, and Massart’s Hæmatoxylin-Eosine.—Dissolve alum, 20 Gm., in water, 200 Gm., by the aid of heat, then filter, and after 24 hours add a solution of hæmatoxylin, 1 Gm., in alcohol, 10 Gm. Let the solution stand for 8 days, again filter, and mix with an equal volume of the following solution:—Eosine, 1 Gm., alcohol, 25 Gm., water, 75 Gm., glycerine, 50 Gm.
Flemming’s Gentian Violet Method.—Use a concentrated alcoholic solution of Gentian Violet diluted with about one half its bulk of water. Differentiate the stained objects in alcohol acidulated with about 0·5 per cent. of hydrochloric acid, followed by pure alcohol and clove oil.
Flemming’s Orange Method.—Stain for days or weeks in strong alcoholic safranine solution diluted with half its bulk of aniline water (saturated); then rinse in distilled water, differentiate in absolute alcohol containing 0·1 per cent. of hydrochloric acid, stain for 1 to 3 hours in strong aqueous gentian violet solution, again wash in distilled water, and finally treat with concentrated aqueous solution of Orange. After a few minutes transfer sections to absolute alcohol, then clear in clove or bergamot oil, and mount in dammar or balsam.
Fol’s Ferric Chloride Fixing and Staining Process.—Preparations are treated with tincture of ferric chloride diluted with 5 to 10 times its bulk of 70 per cent. alcohol, and then transfer for 24 hours to alcohol containing a trace of gallic acid.
Frey’s Fuchsine Solution.—A solution of 0·01 Gm. of crystallised fuchsine, 20 to 25 drops absolute alcohol, and 15 C.c. of water.
Friedlaender’s Staining Methods.—Cover-glass preparations are treated for 3 minutes with a 1 per cent. solution of acetic acid, and allowed to dry after removal of excess of liquid by filter paper. Next place them in gentian violet aniline water (aniline water, 100 C.c., concentrated alcoholic solution of gentian violet, 11 C.c.; absolute alcohol, 10 C.c.) for half a minute, wash in water, mount and dry in balsam. Sections are kept for 24 hours in a warm place, in the following solution:—Concentrated alcoholic solution of gentian violet, 50 C.c.; distilled water, 100 C.c.; glacial acetic acid, 10 C.c. Then treat for 1 or 2 minutes with 0·1 per cent. acetic acid, dehydrate, clear, and mount in balsam.
Gaffky’s Staining Methods.—Sections of material hardened in alcohol are left for 20 to 24 hours in a deep blue opaque solution, freshly made by adding saturated alcoholic solution of methylene blue to distilled water. Then wash in distilled water, dehydrate in absolute alcohol, clear in turpentine oil, and mount in balsam.
Giacomi’s Staining Method.—Stain cover-glass preparations for a few minutes in a hot solution of fuchsine, then place in water containing a few drops of ferric chloride solution, and afterwards decolourise in strong ferric chloride solution. If any precipitate be formed with the iron solution, complete the decolourisation in alcohol. Counterstain with vesuvine.
Gibbes’ Double Staining Method.—Well mix magenta, 2 Gm., and methylene blue, 1 Gm., then add slowly aniline oil, 3 C.c., dissolve in rectified spirit, 15 C.c. Subsequently add 15 C.c. of distilled water and keep the stain in a stoppered bottle. Cover-glass preparations are placed for 4 minutes in the slightly heated stain and sections left for some hours in the stain at the ordinary temperature. Afterwards, wash in methylated spirit until no more colour comes away, then dehydrate, clear in cedar oil, and mount in balsam.
Gibbes’ Magenta Stain.—Mix magenta, 2 Gm.; aniline oil, 3 Gm.; rectified spirit, 20 C.c.; and distilled water, 20 C.c.
Golgi’s Sublimated Method.—Small cubes of tissue are hardened for 15 to 30 days in Müller’s fluid, which should be frequently changed. Then transfer for 8 to 10 days to 0·25 to 1 per cent. aqueous mercuric chloride solution, which must be changed, as it becomes coloured. If desired, treat subsequently with weak sodium sulphide solution to darken the stain and make it sharper. After cutting sections from material thus prepared they must be well washed with water.
Gram’s Stain for Bacteria.—This is prepared by shaking 15 drops of aniline oil with 15 Gm. of water, filtering the solution and adding to the filtrate 4 to 5 drops of saturated alcoholic solution of gentian violet. Or shake 3·3 C.c. of aniline with 100 C.c. of distilled water and, after filtering, add 11 C.c. of concentrated alcoholic solution of gentian violet and 10 C.c. of absolute alcohol. After preparations have been stained for 1 to 3 minutes in one of the above they are quickly rinsed in absolute alcohol and then placed in Gram’s solution of iodine in potassium iodine (iodine, 1 Gm.; potassium iodine, 2 Gm.; water, 300 C.c.), until they have acquired a browncolour. This takes about 1 to 3 minutes, and they are next washed in 90 per cent. alcohol until they become pale yellow, then dehydrated, cleared, and mounted in balsam. Counterstain with eosine or vesuvine if desired.
Gram’s Solution.—Iodine, 1 Gm.; potassium iodine, 2 Gm.; distilled water, 300 Gm.
Grenacher’s Alum Carmine.—Dissolve 5 Gm. of ammonium alum in 100 C.c. of distilled water, add 1 Gm. of carmine, and boil for 20 minutes, filter when cool, and add distilled water to make up to 100 C.c.
Grenacher’s Alcoholic Borax Carmine.—Dissolve 4 Gm. of borax in 100 C.c. of distilled water, then add 3 Gm. of carmine, and heat gently. Finally, add 100 C.c. of 70 per cent. alcohol, filter the solution, if necessary, before use. Pieces of tissues are stained in this for 1 to 3 days, and then transferred to 70 per cent. alcohol, containing 0·5 to 1 per cent. of hydrochloric acid.
Heidenhain’s Hæmatoxylin Method.—Dissolve (a) hæmatoxylin, 1 Gm., in distilled water, 300 C.c.; (b) potassium chromate, 1 Gm., in distilled water, 200 C.c. Small pieces of tissue hardened in alcohol or picric acid are placed in (a) for 12 to 24 hours, and then transferred for a similar length of time to (b). Wash thoroughly in water, dehydrate in alcohol, and imbed in paraffin.
Henle’s Stain(for nervous tissue).—Sections are left in palladium chloride solution (1:300 to 1:600) till they are of a straw colour, then rinsed in water and stained with strong ammonia carmine.
Henneguy’s Alum Carmine.—Excess of carmine is boiled in saturated solution of potash alum, and 10 per cent. of glacial acetic acid added on cooling. Allow to settle for some days, and then filter.
Henneguy’s Permanganate Method.—Treat sections for 5 minutes with 1 per cent. potassium permanganate solution, then wash in water and stain with safranine, rubin, gentian violet, vesuvine, preference being given to a safranine solution prepared with aniline water.
Hermann’s Platino-aceto-osmic Mixture.—Mix 15 parts of 1 per cent. platinic chloride solution, 1 part of glacial acetic acid, and 2 or 4 parts of 2 per cent. osmic acid.
Hertwig’s Macerating Fluid.—Mix equal parts of 0·05 per cent. osmic acid, and 0·2 per cent. acetic acid. Medusæ are treated with this mixture for 2 or 3 minutes, then washed in 0·1 per cent. acetic acid until free from osmic acid. Leave them for 24 hours in the dilute acetic acid, then wash in water, stain with Beale’s carmine, and mount in glycerine. For Actiniæ use 0·04 per cent. osmic acid and make both solutions with sea water. Wash out with 0·2 per cent. acetic acid, and stain with picro-carmine.
Hessert’s Method for Staining Flagella.—Fix the film by treating cover-glass preparations with a saturated alcoholic solution of mercuric chloride, wash, and stain for 30 or 40 minutes in a hot 10 per cent. aqueous solution of saturated alcoholic solution of fuchsine.
Hoffmann’s Blue Stain.—Dissolve 1 Gm. of Hoffmann’s blue in 20 C.c. of rectified spirit and 80 C.c. of distilled water, then add 0·5 C.c. of glacial acetic acid. As a nuclear stain immerse sections for 10 minutes or more, rinse in water, wash in 90 per cent. alcohol, dehydrate, clear, and mount in balsam. To stain sieve areas, less time is required, 5 to 10 minutes, rinse in distilled water, and mount in glycerine; or dehydrate, clear, and mount in balsam.
Hoyer’s Shellac Injection Mass.—Dissolve shellac in 80 per cent. alcohol to the consistency of a thin syrup, and strain through muslin of medium thickness. Colour with aniline colours in alcoholic solution, or by means of vermilion or other pigment suspended in alcohol.
Hoyer’s Silver Nitrate Gelatine Mass.—Mix a concentrated solution of gelatine with an equal volume of a 4 per cent. silver nitrate solution and warm, then add a very small quantity of aqueous pyrogallic acid solution to reduce the silver salt, and add chloral and glycerine as in the carmine gelatine mass.
Hoyer’s Silver Stain.—Add ammonia to a solution of silver nitrate of known strength, until the precipitate formed just re-dissolves, then dilute the solution until it contains 0·75 to 0·50 per cent. of the salt.
Kaiser’s Bismarck Brown Stain.Sections are stained for 48 hours, at a temperature of 60 C., in a saturated solution of Bismarck brown in 60 per cent. alcohol, and washed out in 60 per cent. alcohol containing 2 per cent. of H.C.L., or 3 per cent. of acetic acid.
Kaiser’s Nerve Stain.—This is a modification of Weigert’s process. The material is hardened in Müller’s solution for 2 or 3 days, then cut into slices 2 to 4 Mm. thick,and treated with the solution for 5 or 6 days more. Subsequently immerse in Marchi’s solution for 8 days, then wash, pass through alcohol, and imbed in celloidin. Sections are mordanted for 5 minutes in the following mixture:—Solutions of ferric chloride, 1 part; distilled water, 1 part; rectified spirit, 8 parts. Next wash in Weigert’s hæmatoxylin, and warm in a fresh quantity of the same for a few minutes, wash with water, differentiate in Pal’s solution, and neutralise the oxalic acid by washing in water containing a little ammonia.
Kaiser’s Stain for the Spinal Cord.—Sections are stained for a few hours in solution of náphthylamine brown, 1 part, in water, 200 parts, and alcohol, 100 parts. Afterwards wash with alcohol and clear with origanum oil.
Kallin’s Neurological Method.—Dissolve hydroquinone, 5 Gm., sodium sulphite, 40 Gm., and potassium carbonate, 75 Gm., in 25 Gm. of distilled water. At the time of using, dilute this solution with one-third to one-half its bulk of absolute alcohol; immerse sections of silvered material for several minutes until reduction is complete. Then place them in 70 per cent. alcohol for 10 to 15 minutes, and subsequently leave in aqueous solution of sodium hyposulphite (1:5) for 24 hours or more. Finally dehydrate and mount. Carmine may be used as an afterstain.
Kleinenberg’s Solution(Improved Formula).—Hæmatoxylin, 2½ Gm.; crystallised calcium chloride, 20 Gm. in 10 C.c. of distilled water; alum, 3 Gm. in 16 C.c. of distilled water; rectified spirit, 240 C.c. Dissolve the calcium chloride and alum in their respective quantities of water by the aid of heat; mix the solutions and immediately dilute with rectified spirit; after an hour filter and add the hæmatoxylin. This makes a good working solution which keeps well. Of course it contains the alumina in solution, not as alum but aluminium chloride. If in special cases the colour is considered too strong, the dilution (when staining in bulk) must be made with some of the solution to which hæmatoxylin has not been added.
Koch’s Method for Staining Flagella.—Immerse cover-glass preparations in a 1 per cent. aqueous solution of hæmatoxylin, then transfer to a 5 per cent. solution of chromic acid or to Müller’s fluid; dry and mount in balsam.
Koch-Ehrlich, Bacilli.—Place sections, or cover-glass preparations, for at least 12 hours in gentian violet, or fuchsine aniline water (aniline water, 100 C.c.; concentrated alcoholic solution of gentian violet, or fuchsine, 11 C.c.; absolute alcohol, 10 C.c.), then immerse in a mixture of pure nitric acid (sp. gr. 1·42), 10 C.c., and distilled water, 30 C.c., for some seconds. Rinse in 60 per cent. alcohol for a few minutes, and then counterstain with vesuvine (vesuvine, 0·5 Gm.; rectified spirit, 20 C.c.; distilled water, 80 C.c.) after gentian violet; or methylene blue (methylene blue, 0·25 Gm.; rectified spirit, 20 C.c.; distilled water, 80 C.c.) after fuchsine. Finally rinse in water, dehydrate, clear, and mount in balsam. According to Squire, who points out that nitric acid is apt to injure delicate sections, Watson Cheyne recommends that sections should be transferred from fuchsine aniline water to distilled water, then rinsed in alcohol, and placed in the following contrast stain for 1 or 2 hours:—Saturated alcoholic solution of methylene blue, 20 C.c.; distilled water, 100 C.c.; formic acid (sp. gr. 1·2), 1 C.c.
Kühne’s Carbolic Methylene Blue.—Rub up 1·5 Gm. of methylene blue with 10 C.c. of absolute alcohol, and add 100 C.c. of a 5 per cent. aqueous solution of carbolic acid.
Kühne’s Methyl Violet Solution.—Dissolve 1 Gm. of methyl violet in 90 C.c. of distilled water and 100 C.c. of alcohol.
Kühne’s Aniline Oil Solutions.—Rub up as much methylene blue, methyl green, or safranine as will go upon the point of a knife, with 10 C.c. of aniline, and allow to settle.
Kühne’s Carbolic Fuchsine or Black Brown.—Dissolve 1 Gm. of fuchsine or black brown in 10 C.c. of absolute alcohol, and add 100 C.c. of a 5 per cent. aqueous solution of carbolic acid.
Kühne’s Modification of Gram’s Method.—Stain nuclei with carmine, then treat sections for 5 minutes in methyl violet solution, diluted one-sixth with a 1 per cent. aqueous solution of ammonium carbonate, or in a solution of Victoria blue, 0·25 Gm., in rectified spirit, 20 C.c., and distilled water, 80 C.c. Next rinse thoroughly in water and transfer to Grain’s solution for 2 to 3 minutes; again rinse in water and extract excess of stain with solution of yellow fluorescine, 1 Gm., in absolute alcohol, 50 C.c. Finally, pass through pure alcohol, aniline, terebene, and xylol, and mount in balsam.
Löffler’s Solution.—Concentrated alcoholic solution of methylene blue, 30 C.c.; solution of (caustic potash) potassium hydrate (1:10,000), 100 C.c. Mix and filtershortly before use. Sections are stained for a few minutes (tubercle sections for some hours), and excess of stain can be removed by immersion for a few seconds in 0·5 per cent. acetic acid. Dehydrate in absolute alcohol, clear in cedar oil, and mount in balsam. Löffler found that most bacteria stained better in this solution than in the weaker solutions used by Koch for turbercle bacillus.
Lavdowsky’s Bilberry Juice Stain.—Well wash the fresh berries ofVaccinium myrtillus, then express the juice and mix with twice its bulk of distilled water, mixed with a little 90 per cent. alcohol. Heat for a short time and filter whilst warm. Dilute the stain with 2 or 3 volumes of distilled water before use.
Lee’s Formaldehyde Solutions.—(1) Mix 1 part of 40 per cent. formaldehyde solution with two parts of 1 per cent. chromic acid solution, and add 4 per cent. of acetic acid. (2) Mix 1 part of 40 per cent. formaldehyde solution with 4 parts of 1 per cent. platinic chloride solution, and add 2 per cent. of acetic acid.
Lee’s Osmic Acid and Pyrogallol Stain.—Fix the tissues in Hermann’s mixture or Flemming’s mixture for half an hour, then place in a weak solution of pyrogallol, which may be prepared with alcohol in some cases. Safranine may be used as a second stain.
Martinotti’s Picro-nigrosine Stain.—Pathological objects are stained for 2 or 3 hours or days, in a saturated solution of nigrosine in saturated alcoholic picric acid solution. Then wash out in a mixture of 1 part of formic acid with 2 parts of alcohol until the grey matter appears clearly differentiated from the white to the naked eye.
Mayer’s Aluminium Chloride Carmine.—Dissolve 1 Gm. of carminic acid and 3 Gm. of aluminium chloride in 200 C.c. of water.
Mayer’s Berlin Blue Injection.—Add a solution of 10 C.c. of tincture of ferric chloride in 500 C.c. of water, to a solution of 20 Gm. of potassium ferrocyanide in 500 C.c. of water, allow to stand for 12 hours, decant, wash the deposit for 1 or 2 days with distilled water until the washings come through dark blue, then dissolve the blue in about a litre of water.
Mayer’s Carmalum.—Dissolve 1 Gm. of carminic acid and 10 Gm. of alum in 200 C.c. of distilled water; decant, or filter, and add a few crystals of thymol, 0·1 per cent. of salicylic acid, or 0·5 per cent. of sodium salicylate. A weaker solution contains 3 to 5 times as much alum and 5 times as much water.
Merbel’s Carmine and Indigo Fluids(give a blue and red stain, and are very selective).—To prepare the red fluid, take—Carmine, 2 dr.; borax, 2 dr.; distilled water, 4 ozs. For the blue fluid, take—Indigo carmine, 2 dr.; borax, 2 dr.; distilled water, 4 ozs. Mix each in a mortar, and allow it to stand, then pour off the supernatant fluid. If the sections have been hardened in chromic acid, picric acid, or a bichromate, they must be washed in water till no tinge appears. Place them in alcohol for fifteen or twenty minutes, then in the two fluids mixed in equal proportions, after which wash them in a saturated aqueous solution of oxalic acid, where they should remain a rather shorter time than in the staining fluids. When sufficiently bleached, wash them in water, to get rid of the acid, then pass them through spirit and oil of cloves, and mount in balsam or dammar.
Mitrophanow’s Gold Process for Prickle-Cells and Intercellular Canals.—Wash the tail of an axolotl larva with distilled water, place for an hour in a watch-glassful of 0·25 per cent. solution of gold chloride, containing 1 drop of hydrochloric acid; wash, and reduce in a mixture of 1 part of formic acid with 6 parts of water.
Mitrophanow’s Maceration Method for Epithelium.—Fix the embryo for 15 minutes in 3 per cent. nitric acid; then place for an hour in a mixture of alcohol, 1 volume, and water 2 volumes, and finally treat with stronger alcohol for 24 hours to separate the epidermis.
Müller’s Berlin Blue for Injections.—Precipitate a concentrated solution of Berlin blue by means of 90 per cent. alcohol. The precipitate is very finely divided, whilst the fluid is perfectly neutral and much easier to prepare than that of Beale.
Neilsen’s Solution of Methyl Violet.—Dissolve fuchsine, 1 part, in alcohol, 10 parts, and add a 5 per cent. watery solution of carbolic acid, 100 parts.
Neisser’s Double-Staining for Spore-Bearing Bacilli.—Cover-glass preparations are immersed for 20 minutes in fuchsine aniline water (concentrated alcoholic solution of fuchsine, 11 C.c.; absolute alcohol, 10 C.c.; aniline water, 100 C.c.; then heat to 80° or 90° C.; next rinse in water, alcohol, or weak acid, according to the nature of the bacilli, counterstain with aqueous solution of methylene blue, rinse in water, dry and mount in balsam). The spores are stained red and the rest of the bacilli blue.
Nissl’s Fuchsine Stain for Nerve Cells.—(1) Fresh material in pieces measuring 1 C.c. are hardened in a “chromic solution in 70 per cent. alcohol†for 2 days, then transferred to absolute alcohol for 5 days, and afterwards cut. Stain the sections singly in a saturated solution of fuchsine, warming in a deep watch-glass until vapours begin to be given off. Next plunge the section into absolute alcohol for 1 or 2 minutes, then place it on a slide, flood with clove oil, and when no more colour is given off, drain and mount in balsam.
Ohlmacher’s Formaldehyde Staining.—Formalin in a 2 to 4 per cent. solution is used as a mordant for tar colours. The tissues may be mordanted separately by treatment for 1 minute or longer, or the formalin may be added to the stain. Dissolve 1 Gm. of fuchsine in 10 C.c. of absolute alcohol, and add to 100 C.c. of 4 per cent. formalin solution. Or, add saturated alcoholic solution of gentian violet or methyl violet 5 B. to the formalin solution, in the proportion of 1:10. In the case of methylene blue, dissolve 1 G.m. in 100 C.c. of the formalin solution. Sections stain in half a minute, and are said to resist alcohol much more than if formalin were not used.
Oppitz’s Silver Staining.—Reduction is very rapidly effected by placing the preparations for 2 or 3 minutes in a 0·25 to 0·5 per cent. solution of chloride of tin.
Pal’s Hæmatoxylin Stain.—Dissolve 0·75 Gm. of hæmatoxylin in 90 C.c. of distilled water and 10 C.c. of absolute alcohol. Just before use add saturated solution of lithium carbonate in the proportion of 3 drops to each 10 C.c. of hæmatoxylin solution. (See Weigert.)
Pal’s Hæmatoxylin Method.—Proceed at first as in Weigert’s process for nerve fibre, omitting the copper bath, and stain in Pal’s hæmatoxylin solution (see above) for 5 or 6 hours. Then wash the sections in distilled water (containing a trace of lithium carbonate if the sections are not deep blue), next treat for 15 to 30 seconds with a 0·25 per cent. potassium permanganate solution, rinse in water, and decolourise in Pal’s bleaching solution. (If black spots appear replace in the permanganate solution, again bleach, and wash for 15 minutes in water.) The grey substance of the sections is decolourised in a few sections; the sections should then be well washed out, and may be double-stained with picro-carmine or acetic acid carmine (see Schneider), Magdala red, or eosine. The nuclei may be stained with alum carmine. Finally dehydrate, clear, and mount.
Pal-Exner’s Osmic Acid Method.—Spinal cord or brain in 0·25 inch cubes is immersed in 0·5 per cent. osmic acid solution for 2 days, the solution being changed each day; then wash in water, transfer to absolute alcohol, and imbed in celloidin or paraffin. Place sections as cut in glycerine, then wash in water, treat with potassium permanganate and Pal’s solution, as in Pal’s hæmatoxylin method, counter-stain with carmine, dehydrate, clear, and mount in balsam.
Plant’s Method of Staining Actinomycosis.—Sections are placed for 10 minutes in Gibbes’ magenta solution or carbolic fuchsine, at 45° C.; next they are rinsed in water and placed in saturated aqueous solution of picric acid, mixed with an equal volume of absolute alcohol, for 5 or 10 minutes; they are then washed once more, passed through 50 per cent. alcohol into absolute alcohol, cleared in cedar oil, and mounted in balsam.
Ranvier’s Lemon Juice Method.—Soak pieces of fresh tissue in fresh lemon juice until transparent (5 to 10 minutes), then rapidly wash in distilled water, treat for 10 to 60 minutes with 1 per cent. gold chloride solution, again wash and expose to light in a bottle containing 50 C.c. of distilled water and 2 drops of acetic acid. Reduction is complete in 24 to 48 hours. If it is not desired to retain the superficial epithelium, reduction may be more completely effected in the dark, by treatment with formic acid (sp. gr. 1·2), diluted with 3 times its volume of water. The lemon juice in the above process may be replaced by an aqueous solution of citric acid (40 grains in each ounce).
Ranvier’s Picro-Carmine.—Carmine, 1 part; distilled water, 10 parts; solution of ammonia, 3 parts; mix and add of a cold saturated solution of picric acid 200 parts.
Renaut’s Hæmatoxylic Eosine.—Mix 30 C.c. of concentrated aqueous solution of eosine, 40 C.c. of saturated alcoholic solution of hæmatoxylin (which has been kept for some time and precipitated), and 130 C.c. of saturated solution of potash alum in glycerine (sp. gr. 1·26). Stand for 5 or 6 weeks in a partially covered vessel, protected from dust, until the alcohol is evaporated, and then filter. The filtrate can be diluted with glycerine if desired. Mount objects in this fluid diluted with 1 or 2 volumes of glycerine, or, stain separately for some days or weeks and mount in balsam, after washing in alcohol charged with a sufficient quantity of eosine.
Ranvier and Vignal’s Osmium Mixture.—Fix tissues in a freshly-prepared mixture of equal volumes of 1 per cent. osmic acid and 90 per cent. alcohol, then wash out in 80 per cent. alcohol, next with water, and stain for 48 hours with picro-carmine or hæmatoxylin. This method has been applied to the histology of insects.
Renaut’s Glycerine Hæmatoxylin.—To a saturated solution of potash alum in glycerine, add a saturated solution of homatoxylin in 90 per cent. alcohol drop by drop, so as to form a deeply coloured solution. Expose to daylight for a week, and then filter. This solution, like Renaut’s hæmatoxylic cosine, may be used for mounting unstained sections, which after some time absorb the colour from the liquid and become stained.
Safranine.—Safranine, 0·5 Gm.; rectified spirit, 20 C.c.; distilled water, 80 C.c.
Schäfer’s Acid Logwood Solutionis especially useful for certain structures, as tendon, cells, &c. It is thus prepared:—A 1 per cent. solution of acetic acid is coloured by the addition of 1·3 of its volume of logwood solution.
Schäfer’s Aniline Dyes, whether in aqueous or alcoholic solutions, give good results, and are prepared as follows:—Roseanilin or magenta (1 gr. to 1 oz. of alcohol), red; acetate of mauvein (4 gr., alcohol 1 oz., acid nitric 2 drops), blue; aniline black (2 gr., water 1 oz.), grey-black; Nicholson’s soluble blue (1-6 gr., alcohol 1 oz., and nitric 2 m.), blue. These stains should be used weak; and after sections are stained they should be passed through alcohol and oil of cloves as rapidly as possible; otherwise the colour will dissolve out before they can be mounted in balsam.
Schultze(Staining Bacilli).—Stain sections and cover-glass preparations for some hours in aqueous methylene blue solution, differentiate in 0·5 per cent. acetic acid, dehydrate in alcohol, clear in cedar oil, and mount in balsam.
Sclavo’s Stain for Flagella.—Leave the preparations for 1 minute in a solution of 1 Gm. of tannin in 100 C.c. of 50 per cent. alcohol; wash in distilled water; transfer for 1 minute to 50 per cent. phospho-molybdic acid; again wash, and stain for 3 to 5 minutes in a hot saturated solution of fuchsine in aniline water. Then wash in water, dry on filter paper, and mount in balsam.
Squire’s Picro-Carmine.—(1) Dissolve 1 Gm. of carmine with a gentle heat in 3 C.c. of strong solution of ammonia, and 5 C.c. of distilled water, then add 200 C.c. of saturated aqueous solution of picric acid, heat to boiling, and filter. (2) Dissolve 10 Gm. of carmine in a solution of 1 Gm. of caustic soda in 1000 C.c. of distilled water; boil, filter and make up to 1000 C.c. with water. Mix the solution with an equal quantity of water, and add 1 per cent. aqueous solution of picric acid so long as the turbidity produced disappears on agitation.
Squire’s Blueing of Sections.—After staining with hæmatoxylin, treat for a few seconds with a solution of sodium bicarbonate (1:1000) in distilled water.
Valentine(Fuchsine).—Ether shaken with a solution containing fuchsine is coloured violet after adding ferrous iodide, but not before.
Victoria Blue.—Victoria blue, 0·25 Gm.; rectified spirit, 20 C.c.; distilled water, 80 C.c.
Wedl’s Orseille or Orchella Stain.—Mix 5 C.c. of acetic acid, 20 C.c. of absolute alcohol, and 40 C.c. of distilled water; then add sufficient archil, from which excess of ammonia has been driven off, to form a dark reddish fluid.
Weigert’s Hæmatoxylin.—Dissolve 1 part of hæmatoxylin in 10 parts of absolute alcohol; then add 90 parts of distilled water and 1 part of aqueous solution (1:70) of lithium carbonate.
Weigert(Gram’s Method).—In this modification aniline is substituted for alcohol, in order to avoid prolonged washing with the latter, and the process is conducted on a slide. The section is placed on a slide, stained with a few drops of gentian violet aniline water, prepared as in Gram’s method, the excess of fluid removed, and a few drops of Gram’s solution applied. Subsequently remove the liquid by gently blotting it off, then wash the section by allowing aniline to flow’ backwards and forwards over it, and when colour ceases to come away, repeat the operation with xylol for about 1 minute, then mount in balsam.
Weigert(Staining in Actinomycosis).—Immerse sections for 1 hour in Wedl’s Orseille stain, then quickly rinse with alcohol and counterstain with gentian violet. If it be desired to stain the mycelium also, afterwards submit the sections to Weigert’s modification of Gram’s method. See page 335.
Weigert(Staining Brain Tissue).—Pieces of brain and spinal cord are hardened in bichromate solution, followed by alcohol, then imbedded in celloidin or gum. If imbedded in celloidin, the pieces are subsequently taken from the spirit in which they are immersed, and placed for one or two days in saturated aqueous solution of copper acetate, diluted with an equal bulk of water, the mixture being kept at about40° C. Afterwards transfer the pieces to 80 per cent. alcohol until required for cutting. Or, the sections can be cut first, and then treated with copper acetate. To stain the sections, after being well washed in 90 per cent. alcohol, they are transferred to Weigert’s hæmatoxylin and left from a few hours to two days, according to the differentiation required. When opaque and of a deep blue-black colour, they should be well washed for two or three days in distilled water. Next decolourise for 0·5 to 2 hours in a solution of 2 Gm. of borax and 2·5 Gm. of potassium ferrocyanide in 200 C.c. of water. As soon as the grey and white substances are sharply defined, again wash the sections in water for half an hour, then dehydrate, clear, and mount in balsam.
Woodhead’s Method of Staining Tubercle Bacilli.—Take a small quantity of sputum rich in bacilli, and spread it out by pressure between two cover-glasses, so that a fairly thin film remains on each. Then carefully slip one over the other until they come apart. Thoroughly dry the covers, and pass them rapidly three times through the flame of a spirit lamp, care being taken not to scorch the film, then float them face downwards on the staining solution, which has been previously prepared and filtered into a watch-glass. The stain should consist of saturated alcoholic solution of basic fuchsine, 1 part; absolute alcohol or rectified spirit, 10 parts; carbolic acid solution (5 per cent.), 10 parts. Leave the preparations in the watch-glass for 12 to 24 hours, unless time is an object. In the latter case heat the fluid gently until vapour is given off, then drop the films on the surface, and leave them for 3 to 5 minutes only. Next transfer the covers to an aqueous solution of sulphuric acid (25 per cent.), and when decolourisation is complete, as evidenced by the pink colouration not returning when the specimens are plunged into a bowl of tap-water containing a single drop of ammonia solution, thoroughly rinse in the slightly alkaline water and counter-stain in an aqueous solution of methylene blue. Finally, wash in water, carefully dry and mount in Canada balsam. The bacilli should stand out as bright red rods on a blue background of cells.
Ziehl-Neelsen(Staining Bacilli).—Sections are removed from weak spirit into Neelsen’s carbolic fuchsine and left for 10 or 15 minutes; next decolourise in sulphuric acid (sp. gr. 1·84) or nitric acid (sp. gr. 1·42) diluted with 3 volumes of water, rinse in 60 per cent. alcohol, and wash in a large volume of water to remove the acid. Tubercle and leprosy bacilli are the only micro-organisms that can retain the stain after treatment with acid. If the presence of traces of nitrous acid in the nitric acid be suspected, Squire recommends the use of saturated aqueous solution of sulphanilic acid mixed with one-third its bulk of nitric acid. The sulphanilic acid destroys any free nitrous acid, which would otherwise exercise a bleaching action on the fuchsine-stained bacilli. The sections may be counterstained with a solution of 0·5 Gm. of methyl green (or 0·25 Gm. of methylene blue) in 20 C.c. of rectified spirit and 80 C.c. of distilled water. Finally dehydrate in absolute alcohol, clear in cedar oil, and mount in balsam.
The initial unit of the Metric System is the Metre or unit of length, which represents one ten millionth part of the earth’s quadrant, or one forty-millionth part of the circumference of the earth around the poles. The multiples and sub-divisions of this and all the other units are obtained by the use of decimals, and for this reason the system is also known as thedecimal system. The multiples are designated by the Greek prefixes,deca= 10;hecto= 100;kilo= 1000;myria= 10,000. For the sub-divisions Latin prefixes are employed, as follows:deci=1â„10;centi=1â„100;milli=1â„1000. Thus for measures of length we have the following expressions, showing the abbreviations commonly employed, and the equivalents in the ordinary English standards of measurement—
From the unit of linear measure of metre is derived the unit of the measure of capacity orLitre. This represents the cube of one-tenth part of a metre, or a cubic decimetre, and its multiples and sub-divisions with their corresponding equivalents in Imperial fluid measure are as follows:—
The unit of weight in the metric system is theGramme. This is also derived from the metre, and represents the weight of one cubic centimetre, of water, or the quantity of distilled water, at its maximum density, 4° C. (39·2° F.), which would fill the cube of one-hundredth part of a metre. The relative value of the gramme, together with its multiples and sub-divisions, as compared with the English standards of weight, may be seen from the following table:—
The expressionmicro-millimetreis used for microscopic measurements, and denotes the thousandth part of a millimetre. Of the measures of capacity, the terms most commonly employed are the litre and the cubic centimetre. Thus a decalitre may also be expressed as 10 litres, a centilitre as 10 cubic centimetres, etc. Of the metric weights the gramme and its fractional parts, with their respective prefixes, are much used in analytical work. The kilogramme is largely employed in commercial transactions, and is commonly abbreviatedkilo.
As a comparison of the values of some of the more frequently employed expressions of the metric and English systems, the following may be found convenient for reference:—
It may be well to bear in mind that on the Continent liquids are always weighed, not measured.