Bichromate of potassium, 3 per cent. solution5partsOsmic acid, 1 per cent. solution1part
for three to five days. Excess of bichromate is removed from the sections by blotting paper, and they are transferred to thefreshly preparedstaining mixture:—
Phospho-molybdic acid (10 per cent.)1minim2drops.Nitrate of silver (1 per cent.)1ounce60c.c.
which must not be filtered. Stain for several days.
The sections should be cut at once after removal from the staining solution. It is claimed that the minute details of structure of the cell processes are better shewn by this method.
Corrosive sublimatemethod.—This method is similar in its mode of action to the last, mercury being deposited in the cell instead of silver. It is rather less certain and requires more practice. It seldom stains uniformly. One cell will be found exquisitely stained while those in its vicinity are unaffected.
Small pieces of cortex are hardened for several weeks in Müller’s fluid, or other bichromate solution, and are then transferred direct to a one-half per cent. aqueous solution of corrosive sublimate, in which they should be left from three to six weeks. Shorter periods will only give disappointing and inconstant results. Sections should becut, if possible, in gum. They may be mounted in Farrant, or dehydrated and mounted in balsam. Tal has proposed to render the effect sharper by transforming the deposit of mercury into mercuric sulphide, by treating the sections with a solution of sulphide of sodium, which he prepares by saturating a ten per cent. solution of caustic soda with sulphuretted hydrogen and then adding an equal quantity of fresh soda solution. They are stained in this for a few minutes and then thoroughly washed.
By this method the pyramidal cells and their delicate processes appear as black opaque objects on a light ground. The neuroglia cells with their fine delicate processes are often also beautifully stained.
Nissl’s aniline method.—This method is complementary to Golgi’s method. The latter impregnates the cell rendering it opaque and shewing its form with great definiteness.
Nissl’s method stains the protoplasm without greatly reducing its transparency and allows us to study details of cell structure. Small portions of tissue,removed as soon as possible after death, arehardened in alcohol. Sections are then cut, preferably in gum, as celloidin is inconvenient owing to its staining so deeply with aniline dyes.
Sections are transferred from alcohol to a one-half per cent. aqueous solution of methylene blue, which is heated in a watch glass till it steams freely, but short of the boiling point. Stain for about a quarter of an hour and allow to cool. Transfer the sections to a mixture containing one part of aniline oil and ten of absolute alcohol, and move them about till no more colour comes away. Transfer the section to a slide with a section lifter, drain, and dry well by pressing folded filter paper carefully on the section. Allow some origanum oil to flow over the section and remove excess of this by pressure with blotting paper. Moisten with benzine,1and add a drop of colophonium resin dissolved in benzine. The slide is warmed cautiously till the benzine is driven off and the colophonium liquefied by heat alone, and then the cover-glass is applied.
Magenta and other aniline dyes may also be employed in a similar manner.
It is impossible, within the limits of this work, to attempt any adequate description of the modern methods of bacteriological investigation. Some of these are very lengthy and complicated, and require much skill and practice before good results can be relied on. But those who do not desire to make a special study of bacteriology may often require to examine for the presence of organisms in sections, or in various excretions, and it is hoped that they may find the following short description of special methods sufficient for their purpose. For more elaborate work they must consult one of the many excellent textbooks on the subject.
The student should provide himself with the following dyes inpowder:—
Methylene blue.
Gentian violet.
Methyl violet.
Fuchsine.
Bismarck brown.
The following solutions of these dyes areused:—
1. Saturated alcoholic solutions which may be kept in stoppered bottles.
2. One per cent. aqueous solutions. These must be freshly made each time of using.
In filtering either alcoholic or aqueous solutions it is well to moisten the filter paper beforehand with alcohol or water as the case may be.
The following special solutions will also bewanted:—
Löffler’s methylene blue.—In this solution a weak solution of caustic potash is employed as amordant:—
Saturated alcoholic solution of methylene blue3volumes.Caustic potash, aqueous solution 1 : 10,00010volumes.
Filter.
This solution is perhaps the most generally useful stain. It colours most bacilli and micrococci,and while rapid in its action rarely overstains. It must be made up fresh on each occasion. It is the best counterstain after staining tubercle bacilli, &c., with fuchsine.
Ziehl’s carbol-fuchsine.
Carbolic acid(5 per cent. aqueous solution)100volumes.Fuchsine(saturated alcoholic solution)11volumes.
The solution must be filtered immediately before being used.
Gram’s iodine solution.—Sections are placed in this solution after being stained with aniline dyes. The iodine in some way fixes the dye in the organisms, so that they are not decolourised along with the rest of the tissues.
It is madethus:—
Iodine1grm.11/2grains.Iodide of potassium2grms.3grains.Distilled water300c.c.1ounce.
Ten per cent. aqueous solutions ofnitricandsulphuricacids should be prepared and may be kept indefinitely.
The following are the general methods of employing these reagents for the purpose of staining organisms in sections. Special methods are required for special organisms, but one or two only can be given.
Weigert’s method.—The sections must be placed in a freshly made one per cent. aqueous solution of methyl violet, gentian violet, fuchsine, &c. The solution may be kept at the temperature of the body in an incubator. The organisms will often stain more readily if the section be passed through a 1 in 2000 solution of corrosive sublimate before putting it into the staining fluid. After staining the section is washed in distilled water and then in methylated spirit until it appears almost decolourised. Some prefer to decolourise the tissues by washing in a half per cent. solution of acetic acid instead of methylated spirit. Practice is required before the correct time for decolourising is accurately estimated. The beginner should float a section rapidly on the slide now and then, put on a cover-glass and examine it under a low power to see if the decoloration has been carried far enough. A contrast stain maythen be used, such as picrocarmine, after which the section may be mounted in Farrant’s medium: or a weak solution of another aniline colour may be used as a counter stain, after which the section is clarified in xylol, and mounted in balsam dissolved in xylol.
Gram’s method.—Place some aniline oil in a test tube and add ten times its volume of distilled water. Close the end with the thumb and shake very thoroughly. Filter ninety drops into another clean test tube, and add ten drops of a saturated solution of gentian violet or some similar dye. Filter the mixture into a watch glass. Stain sections in it for from three minutes to half-an-hour according to the temperature,—the shorter time for the incubator at 100°, the longer when the sections are stained at the ordinary temperature of the room. Wash in distilled water, and transfer to Gram’s iodine solution until they become black, usually in a few minutes. They are then decolourised in absolute alcohol. This often takes some time. It may be hastened, as Crookshank suggests, by placing the section in clove oil, returning to alcohol, and so on.
Ehrlich’s modificationof Gram’s method. The contrast stain is here used first.
Stain the section (e.g., that of a mitral valve in a case of ulcerative endocarditis), in an alcoholic solution of eosine (1 in 1500). Transfer to a solution of some aniline dye, such as gentian violet, dissolved in aniline oil water, exactly as in Gram’s method. The section floats on the surface and spreads out, owing to the alcohol diffusing out. Stain for about twenty minutes. Wash the section in water, and float out (p.55) on a glass slide. Allow the water to drain off and add Gram’s iodine slowly from a pipette so as not to disarrange the section. When the section has become quite black pour off the Gram’s solution. Remove all superfluous fluid from the slide with blotting paper, and dry the section by carefully and firmly pressing on it a folded piece of blotting paper. If this is done with care the section need not be injured in the least. Decoloration is effected on the slide with aniline oil, instead of alcohol as in the preceding method. The slide is rocked about so that the colour may be evenly discharged by the aniline. When no more colourcomes away, the aniline oil is poured off, the section clarified in xylol, and mounted in Canada balsam.
As soon as the section is decolourised it may be treated with a contrast stain, the most suitable being alcoholic solutions of eosine or Bismarck brown if a blue stain has been employed, or methylene blue if fuchsine has been the first stain used.
The following will be found the most useful stains and contraststains:—
Stains.Contrast Stains.Gentian violet.Methyl violet.Methylene blue.Picrocarmine.Eosine.Bismarck brown.Safranine.Magenta.Fuchsine.Methylene blue.Andvice versâ.
Much practice is required in using either of the methods before one can judge accurately how long to leave sections in the staining reagents or decolourising agents, and the beginner must not be discouraged if at first he is unable to obtaingood results although he follows the book directions most minutely.
Ehrlich method for tubercle bacilli.—Sections are stained for six to twenty-four hours in a one per cent. solution of gentian violet, methyl violet, methyl blue or fuchsine. They will stain more rapidly if the staining fluid be kept in an incubator at the body temperature. They should be removed from the staining fluid, and washed in distilled water, and then transferred (preferably on a glass section lifter) to a ten per cent. solution of nitric acid in distilled water until they are nearly decolourised. They should then be very thoroughly washed in distilled water. They may then be treated with some suitable contrast stain and mounted in Canada balsam.
Neelsen’s stain for tubercle bacilli.—Sections are placed in Ziehl’s carbol-fuchsine solution (p.103) which should be warmed for ten minutes to half-an-hour. They are then decolourised in a solution of sulphuric acid. Twenty-five per cent. is the strength originally recommended, but a ten per cent. solution does equally well and injures the section less. They are then verythoroughly washed in a large quantity of water, and afterwards may be treated with a contrast stain.
Gibbes’ double stain for tubercle bacilli.—
(1)Rosaniline hydrochlorate2grms.25grs.Methyl blue1grm.12·5grs.
Triturate in a glass mortar,
(2)Aniline oil3c.c.37·5grs.Rectified spirit15c.c.31/2drms.
Dissolve and add slowly to (1).
(3)Lastly add slowly to the mixtureDistilled water15c.c.31/2drms.
Some of the solution is filtered into a watch glass and warmed. The sections are placed in it and left for some hours. They are then washed in methylated spirit till they are sufficiently decolourised, and then rapidly passed through absolute alcohol and oil of cloves and mounted in balsam and xylol. It is a very useful stain for examining the sputum for tubercle bacilli.
In order to stain fluids, such as blood, pus, or sputum, for organisms, a very thin layer should be obtained by placing a little of the fluid betweentwo clean cover-glasses and pressing them together. They are then separated and allowed to dry. The film is fixed by holding the cover-glass in a pair of forceps, and passing it slowly through the flame of a spirit lamp two or three times. Films of pus should be ‘cleared’ after fixing by placing them in a twenty per cent. solution of acetic acid for three minutes.
For clinical purposes it is often necessary to examine urine, fæces or vomited matter for bacilli. Films are prepared in the usual way and allowed to evaporate slowly, and then fixed by passing through the flame, and then washed in distilled water before staining. In the case of vomited matter and fæces this is usually done without difficulty. In the case of urine however it is often difficult to get the urine to evaporate completely. A syrupy layer remains, and if more heat be applied it decomposes and chars, and the products cause precipitation of aniline during subsequent staining processes. This may be partly avoided by gently washing the film in distilled water before staining.
Another plan is to mix the urinary deposit witha little gelatine free from organisms, such as that in unused culture tubes. The gelatin is liquefied by heat, and mixed with the deposit. Films are made from this mixture, and allowed to set, and then thoroughly washed in distilled water. The film is then dried thoroughly, and the cover-glass laid flat with the film uppermost, and a few drops of the staining fluid filtered on to it. After it has been stained sufficiently the stain is drained off, and the slip gently washed. The film may then be stained with some contrast stain in exactly the same way as sections, again washed, dried between folds of blotting paper, and mounted in balsam.
It is sometimes difficult to tell which is the side of the cover-glass which bears the film. This is readily done by holding the glass obliquely so that light from a window is reflected from its surface. The side which is coated appears dull; while the other is smooth and bright.
In all these methods blood is obtained by pricking the skin of one of the fingers, or the lobule ofthe ear, preferably the latter. The skin must previously be washed with soap and water or ether, to remove any grease or epithelial scales. The puncture should be made firmly so that blood may escape freely. The finger or ear must not be squeezed. Specimens must be made rapidly before red corpuscles have run into rouleaux. The slides and coverslips employed must be scrupulously clean, or it is impossible to get really good films. They should be cleaned with nitric acid and alcohol according to the directions on page57.
Fresh specimens should be examined. The coverslip is made just to touch the drop of blood at one edge, so as to transfer a small quantity only, and is at once lowered on to the slide with the aid of a mounted needle. If slide and coverslip be perfectly clean the blood will spread out into a thin film, the corpuscles lying quite flat. If there be any delay, or if the cover-glass be not quite clean the red corpuscles will run into masses and the specimen will be useless for minute examination. Another specimen may be mixed with a little of Ferrier’s solution (p.129) before mounting. Permanent coverslip films may also be prepared.
Here again the use of absolutely clean coverslips is essential, and the blood must be taken immediately it escapes from the puncture. A little blood is taken on a cover-glass which is held horizontally. Another cover-glass is lowered on to this and by its weight and by capillary attraction, the drop of blood quickly becomes transformed into a thin film. The two covers are separated as soon as the film is formed by rapidly sliding them off one another. This manœuvre requires a little practice and dexterity. The movement of the slips must be in an exactly parallel direction otherwise the coating left will be uneven, just as when two pieces of bread and butter are pulled apart. Even with practice it is difficult to get more than one good film, the lower being usually best. There are four ways of fixing the film.
1. Exposure toosmic acid vapour.
The film while still moist is held over the mouth of a bottle containing at least one per cent. solution of osmic acid. In a minute or two the fixation will be complete, and the film becomes of adirty brown colour. It is then left exposed to the air to get rid of all traces of osmic acid, and may afterwards be stained as described below.
2. Treatment withsaturated aqueous solution of corrosive sublimate(Muir’s method).
The cover-glass on which the film has been spread, is floated before the latter has time to dry, film downwards on a saturated solution of corrosive sublimate in a watch glass for half an hour. The cover-glass is placed in distilled water and then in alcohol to remove excess of corrosive sublimate, and then stained. A little care is required when washing the film to prevent it sliding bodily off the cover-glass.
3. By drying and passing rapidly through the flame of a Bunsen burner, exactly as in preparing specimens of sputum, &c. (p.111). This method is handy for ordinary clinical purposes.
4. By keeping the coverslips at a temperature of about 200° F. (Ehrlich’s method).
Ehrlich uses for this purpose a strip of copper about two inches wide and a foot long which is supported on a retort stand in a horizontal position. One end is heated by a Bunsen’s burnerbeneath. The point in the copper strip at which the temperature is at boiling point is readily ascertained by dropping a little water on. The point at which a drop of water assumes the spherical state indicates a temperature there of 212° F. The coverslips are placed an inch or two further than this point, and kept there at a temperature of about 200° F. for some hours.
Fresh blood may be stained by mixing with Ferrier’s fuchsinesolution:—
Fuchsine1grm.Distilled water150c.c.
Dissolve and add
Alcohol (80 per cent.)50c.c.Neutral glycerine200c.c.
A spot of this solution is mixed with the blood on a slide by means of a mounted needle, and covered with a clean cover-glass. The red corpuscles are slightly stained, while the nuclei of the white corpuscles are stained a bright crimson, and the “blood plates” a deep pink colour.
Stained preparations may also be obtained by usingToison’s fluid, which serves also for diluting the blood in order to determine the exact number of red and white corpuscles present by means of Gowers’ or the Thoma-Zeiss hæmocytometer. It is preparedthus:—
Glycerine30c.c.1oz.Sodium sulphate8grms.2drms.Sodium chloride1grm.15grs.Methyl violet·25grm.4grs.Distilled water160c.c.5oz.
It stains the nuclei and blood plates, but does not alter the shape of the red cells. It requires to be made up fresh occasionally as torulæ are apt to form and multiply in it.
Dried films may be stained with hæmatoxyline, picrocarmine, or any of the general stains. The nuclei of the leucocytes may be stained rapidly in a couple of minutes in a one per cent. solution of methyl violet, washing in water, drying between blotting paper and mounting in balsam. The best method for general purposes is to stain with a saturated aqueous solution of methyl blue for half an hour or longer. Wash in water, and thenstain for ten minutes in a half saturated aqueous solution of eosine. In this way the eosinophile granules of the leucocytes and the red corpuscles, are stained by the eosine, while the nuclei of the leucocytes are stained by the methyl blue.
Kanthack and Drysdale recommend that the film should first be stained with a half per cent. solution of eosine in 50 per cent. alcohol, then washed, dried and fixed in the flame, and stained for a short time in Löffler’s solution of methylene blue (p.104).
These films may be stained for micro-organisms in the way described for cover-glass preparations (p.112).
Injection of blood vessels may be performed on small animals, or on individual human organs after removal from the body. The object is to fill the vessels with a coloured fluid which will solidify afterwards. It is possible in the same organ to inject the arteries with a red medium, the veins blue, and secretory ducts, such as bile ducts, yellow or blue.
The most convenient basis for an injection mass is gelatine, as its solutions liquefy at a temperature of about 100° F., and solidify a little below that point, and when solidified cut readily, and do not tend to become brittle. The various masses are prepared asfollows:—
Red injection mass(Woodhead’s formula) consists of gelatine softened by mixture with water and coloured by carmine.
(1)Carmine4grms.Liq. ammoniæ B.P.8grms.Distilled water150c.c.
Dissolve the carmine in the ammonia in a mortar. Pour on the water. Mix thoroughly and filter.
(2)Gelatine10grms.Distilled water50c.c.
Allow it to stand in the cold water until the water is absorbed and the gelatine has become soft.
Warm (1) almost to boiling point over a Bunsen burner, and add the gelatine slowly. Stir thoroughly and add a ten per cent. solution of acetic acid until the solution becomes slightly acid. This will be shewn by the mass assuming a darker and duller colour. A little salicylic acid may be added to preserve it.
Blue injection mass.—To the gelatine mass (2) prepared as above, and liquefied by heat, add instead of carmine
Soluble Prussian blue5grms.Distilled water60c.c.
Every trace of alkali must be kept away fromthe mass during and after the preparation. Sections of injected organs should be mounted in Farrant’s solution slightly acidulated with formic or acetic acid. With every care, however, the blue colour is apt to fade in the course of time.
Green injection mass.Robin’s formula (modified).
(1)Arseniate of soda (sat. sol.)80c.c.Glycerine50"(2)Sulphate of copper (sat. sol.)40"Glycerine50"
Mix and add one part to three parts of the gelatine mass made as for the red and blue injections.
Method of injection.—In injecting the vessels of tissues it is necessary that the organ or the entire animal, as the case may be, shall be kept during injection at a temperature well above that at which the gelatine mass will melt, otherwise the gelatine will “set” in the arteries and will never reach the capillaries. This warming is effected by immersing the animal in a water bath. The liquefied gelatine is forced into the artery by a syringe or by air pressure. It is essential that thepressure be uniform and steady. This is so much more easily managed with air pressure that this method is strongly recommended to the beginner. But, whatever method be adopted, perfect results can only be obtained with certainty after long practice. Sometimes too high pressure will be employed and the vessels give way, at others the injection may not reach the capillaries at all. The most scrupulous attention to details is essential.
By far the most effective apparatus for injecting is the modification of Ludwig’s constant pressure apparatus devised by Fearnley.2Although the apparatus appears complicated, the various parts are easily obtained and it would be easy to improvise a substitute for the water bath.
The apparatus which is shewn in figures 10 and 11 consists of a bath deep enough to contain the animal, and a vessel containing the injection fluid. The bath is kept at a temperature of about 110° by an ordinary Bunsen burner. A large Wolff’s bottle (20–40 oz.) with three necks, is fitted with three india-rubber stoppers perforated by glass tubes. Through the central stopper a glass tubeconnected by a rubber tube, with an ordinary Higginson’s syringe, passes almost to the bottom of the bottle. From one of the other necks a rubber tube passes to an ordinary mercurial manometer, while from the third a tube passes to the flask containing the liquefied injection mass, which isimmersed in the water bath. This flask is also firmly stoppered, and should be about half filled with injection material. The delivery tube from the large Wolff’s bottle should only just come through the cork. Another glass tube passesdown almost to the bottom of the flask, and is connected by a rubber tube with the cannula inserted into the artery. It will be evident from figure11that when water is pumped by the Higginson’s syringe into the Wolff’s bottle the pressure there will be raised (as indicated by the manometer). This increase of pressure will equally affect the air inside the bottle containing the injection fluid, and the fluid will be forced out along the tube and through the cannula into the artery.
Fig. 10.—Fearnley’s arrangement for injecting blood vessels. (Reproduced by permission of Messrs. Macmillan, from Fearnley’sPractical Histology).
Fig. 10.—Fearnley’s arrangement for injecting blood vessels. (Reproduced by permission of Messrs. Macmillan, from Fearnley’sPractical Histology).
Fig. 11.—Scheme shewing distribution of pressure in Fearnley’s Injection Apparatus (from Fearnley’sPractical Histology).
Fig. 11.—Scheme shewing distribution of pressure in Fearnley’s Injection Apparatus (from Fearnley’sPractical Histology).
Before using the apparatus a clamp should be placed on the exit tube of the vessel containing the injection fluid, and the pressure should be raised to see that the apparatus is everywhere air-tight. Any leaks should be sealed before the actual injection is commenced.
If an isolated organ is to be injected, a cannula of glass or brass should first be inserted into the artery and securely tied in position. The organs, if cold, must be soaked in water at 120° F. for about half an hour and then transferred to a water bath.
In the case of injecting an entire animal, such as a rabbit, rat, or guinea pig, the injection is bestmade a few minutes after death. The animal may be chloroformed, and then bled to death by opening a large vein. As soon as death has occurred incise the skin over the thorax in the middle line. Cut through the costal cartilages to the right of the sternum, and through the junction of the manubrium and body of the sternum. These incisions being for most part through non-vascular parts will not lead to escape of fluid during injection. The sternum being forcibly raised towards the left, the pericardium will be exposed and must be carefully divided. An incision must be made into the left ventricle, and a cannula passed up into the aorta and firmly secured by a ligature passed round the aorta with the assistance of forceps or an aneurism needle. Any blood is cleaned away and the animal is then placed in the water bath for about ten minutes. The tube from the bottle containing the injection fluid is then filled by gentle pressure on the syringe, and clamped when full. Its end is then placed on the cannula and secured there by a ligature. The pressure should be raised by squeezing the syringe until the manometer registers one inch. The clamp should then be removed and the injection commenced. The pressure should be raised very gently and constantly by working the syringe, and the condition of the gums, lips, and eyes of the animal observed. The gums will soon shew a pink tinge. The best indications are obtained by watching the effect on the small vessels of the sclerotic. When these are completely filled, which will be in about five to ten minutes according to the rate at which the air pressure has been increased, the injection may be stopped. This result will be obtained, under good conditions, before the manometer indicates a pressure of five inches. The aorta should now be ligatured, and the animal placed in cold water frequently renewed until it is thoroughly cooled. The organs may then be removed and placed in methylated spirit and hardened. Sections are afterwards cut and mounted in the usual way.
Normal histology.—It cannot be too strongly impressed on the beginner that a thorough mastery of the normal appearances of tissues and organs is absolutely necessary before attempting to make an accurate study of morbid changes in them. He should not be satisfied with examining one specimen of an organ but as many as he conveniently can, in order to be fully acquainted with the many deviations from normal which may exist without actual disease. He should therefore obtain several animals, such as small dogs, cats, rabbits, frogs, &c., and remove their organs with all care, and harden them in the various appropriate fluids. He should also obtain specimens of normal human organs from the post-mortem room. Many normal tissues (skin, muscle, tendon, bone, &c.), can also be prepared from a limb amputated for an accident to a healthy patient. By preparing specimens in this way he will not only become the possessor of a set of slides illustrating normal histology, but will find also that he has acquired that proficiency in hardening and staining the specimens which practice alone can give.
The following account of the method of preparing different tissues is merely intended to indicate the lines on which the beginner should proceed. After some practice he will be quite able to select the modes of hardening and staining which special circumstances or cases may seem to demand.
The first part of these directions will refer to the preparation of normal tissues, the second part to morbid histology.
Blood.—For special methods of examination see ChapterVII.
Blood crystals—Hæmoglobin crystals, obtained from the blood of an animal, or enough may be collected at any operation. A little water or a little ether is added to the blood which is allowed to stand for half-an-hour after which a drop is allowed to evaporate slowly on a clean slide.
Hæmatin crystals.—The student should make himself thoroughly familiar with these, as their presence affords positive proof of the existence of blood colouring matter in a stain.
To obtain them a drop of blood should be allowed to dry on a slide. The dried blood is scraped into a little heap with a small piece of clean glass, and a drop of glacial acetic acid added. As it evaporates minute reddish-brown acicular crystals will appear.
Hæmatoidin crystals.—Obtained from the site of a bruise, or an old hæmorrhage,e.g., a cerebral apoplexy or a hæmatocele.
Simple squamous epithelium.—(Endothelium). Carefully strip off the lining of the parietal pericardium or parietal pleura, of a recently killed animal, or spread out its omentum on a piece of cork, and (1) stain the intercellular cement with nitrate of silver (p.82) so as to reveal the outlines of the cells. (2) Stain other specimens with hæmatoxyline or alum carmine to reveal the nuclei.
Stratified squamous epithelium.—Specimens from skin of various parts, finger, groin, lip,tongue should be prepared. Harden in Müller’s fluid.
Transitional epithelium.—Occurs in the pelvis of the kidney, ureter and bladder. It is very readily detached, especially if not hardened immediately after death. Remove as early as possible. If the bladder is taken it should be cut open and pinned out as flat as possible. Harden in osmic acid, or Müller’s fluid and spirit. Embed preferably in celloidin.
Simple columnar epithelium.—Occurs in many parts. It may be studied in the salivary ducts, the intestine, kidney, &c., of any mammal.
Goblet-cells.—Seen abundantly among the columnar cells of the intestinal glands, and in the mucous glands of the mouth and of the cervix uteri.
Stratified columnar epithelium.—Occurs only in the urethra. Harden the penis of a cat in Müller’s fluid, and cut transverse sections.
Ciliated epithelium.—Harden the trachea of a recently killed cat in osmic acid or Müller’s fluid. Beautiful specimens may also be obtained from an ordinary nasal polypus, which should beput into hardening fluid immediately after removal.
Stain all sections of epithelium in picrocarmine, and in eosine and hæmatoxyline.
Ordinary areolar tissue.—Difficult to obtain free from fat. It may be studied in the subcutaneous tissue of the section of the cat’s penis already made. A fragment of the tissue should also be removed and carefully teased in a drop of picrocarmine. Areolar tissue may also be studied in sections of skin, and in the capsules of the different internal organs.
Elastic tissue.—May also be studied in most sections of skin. If the ligamentum nuchæ of a large quadruped (horse, bullock), &c., is available it yields the best specimens, or the human ligamenta subflava may be examined. Pin a piece out on a piece of wood or wax. Harden in Müller’s fluid. Stain in picrocarmine. Both sections and teased specimens should be prepared.
Tendon.—Readily obtained from an amputated limb. Harden in Müller’s fluid. Make transverse and longitudinal sections. Stain with eosine and hæmatoxyline.
A preparation should also be made by teasing a little of the fresh tendon in normal salt solution, and staining with picrocarmine.
Retiformorlymphadenoid tissue.—Seen in lymphatic glands and in the lymphoid follicles scattered along the sub-mucous coat of the alimentary canal.
Prepare sections in the ordinary way. Stain in eosine and hæmatoxyline or in picrocarmine.
Some sections should also be prepared by pencilling (i.e., dabbing with a camel’s hair brush) or by shaking sections up in a test tube with water or normal salt solution. By this means the leucocytes are removed, and the structure of the adenoid tissue itself becomes more evident.
Fat.—Best studied in sections of skin and subcutaneous tissue, or in the mesentery of the cat. One specimen should be stained with osmic acid and picrocarmine and mounted in Farrant’s medium, and another in eosine and hæmatoxyline and mounted in Canada balsam.
Pigment cells.—Branched cells are best studied in the living foot of the frog, where amœboid movements may be seen in them when thelight falling on the retina is made to vary in intensity. Permanent preparations are most conveniently made from the pallium of the common snail. The shell is removed, and the pallium snipped out with the scissors. It is then pinned out flat, hardened for a day in methylated spirit, and mounted unstained in Farrant’s medium. They are also well seen in sections of the choroid coat of the eye.
Hyaline cartilage.—Specimens may be obtained from any joint, from the costal cartilages of young animals, or from the thyroid cartilage and tracheal rings. It may be hardened in spirit. Stain with picrocarmine, eosine and hæmatoxyline, and with methyl violet.
Elastic cartilage.—Prepared from the epiglottis, or from the cartilages of the ear,e.g., of a cat. Harden in spirit. Stain in picrocarmine or in dilute fuchsin.
White fibro-cartilage.—Obtained from intervertebral disc. Prepare and stain as for hyaline cartilage.
Bone:—
Unsoftened Bone.—Cut as thin a section aspossible with a fine saw. Rub the section with the hand on a dry oil stone until it is as thin as possible. Then cement it by Canada balsam (liquefied by warming) to a piece of plate glass and continue the rubbing process with this, examining it now and then with the low power to see if it is thin enough. As soon as it is thin enough it is washed off the slide with methylated spirit, and washed to get rid of the fine bone dust. It should then be transferred to turpentine and may be mounted in balsam.
Softened bone.—Specimens may be obtained from an amputated limb or from the femur of a cat.
Specimens should be decalcified in chromic and nitric fluid, and the hardening completed in spirit. In studying the process of ossification,e.g., in the head of the humerus of a kitten, it is best to embed the specimen in celloidin before cutting sections, as the trabeculæ of bone are very delicate, and easily detached.
Very beautiful double staining effects may be obtained with either picrocarmine, or eosine and hæmatoxyline, and with eosine and methyl violet.
Bone marrow.—To obtain good sections ofred bone marrow, take a piece of the clavicle or a rib, or of one of the carpal or tarsal bones. Decalcify in chromic and nitric fluid. Embed in celloidin. Stain with eosine and logwood, eosine and alum carmine, or alum carmine and picric acid. Mount in Canada balsam. The various cells present in bone marrow may also be studied by squeezing some fresh marrow from a rib, and making a cover-glass film, and preparing in exactly the same way as is directed in the case of blood films on page116.
Tooth.—Best cutin situfrom the jaw of a cat. Decalcify in chromic and nitric fluid, and cut both vertical and transverse sections. Stain in picrocarmine, or eosine and hæmatoxyline.
Developing tooth.—Extremely good specimens may be obtained from the jaw of a newly-born kitten or puppy. Sections can easily be made shewing a milk tooth and a developing permanent tooth by its side.
The enamel is dissolved by decalcifying fluids. To study it a specimen of unsoftened tooth should be made, according to the directions given for bone.
Striped muscle.—Should be studied in various animals.
The leg of an insect such as a cockroach may be hardened in osmic acid. One leg should be hardened in a straight position so as to fix the fibrils in the fully extended position, another should be bent up so as to get specimens of relaxed fibrils.
Portions of muscle should be removed, and teased on a glass slide in some staining fluid such as picrocarmine, a tenth per cent. solution of eosine or quarter per cent. of safranine.
Sections of amphibian and mammalian muscle should be prepared to show their differences in structure. The most convenient part to select is the tongue, as a view of the fibres is obtained both in longitudinal and transverse sections. Sections should be stained in eosine and hæmatoxyline which gives a beautiful effect. For special stains for intra-muscular nerve endings see page92.
Heart muscle.—A portion should be teased fresh in picrocarmine or eosine, another portion hardened in Müller’s fluid, and sections made and stained with eosine and hæmatoxyline.
Unstriped musclemay be obtained by teasing a fresh portion of the muscular coat of the small intestine of an animal, or by sections of the hardened intestine, bladder or uterus. Stain in picrocarmine or preferably eosine and hæmatoxyline.
Nerves.—The special methods for staining nerve tissues are detailed in ChapterVI. The student must remember that the ordinary staining methods are also applicable to nervous tissues.
Nerve terminations:—
Meissner’s corpuscles.—Take the tip of an index finger immediately after amputation. Place part of it at once in chloride of gold solution, and the rest in Müller’s fluid until it is hardened.
Sections stained with chloride of gold should be mounted in Farrant’s medium. The other sections may be stained in picrocarmine or eosine and hæmatoxyline.
Pacini’s corpuscles.—May be dissected out on the smaller branches of the digital nerves, or may be found in the mesentery of the cat. The latter should be spread out on wood, hardened in Müller’s fluid, stained in hæmatoxyline, and mounted in balsam.
Other forms of tactile corpuscles may be studied in the tongues of frogs, ducks, or geese. A network of nervous fibrils should be studied in the cornea. Take the cornea of a newly killed frog or cat and stain with chloride of gold (p.82).
The end plates in which the nerves terminate in muscle may be studied by placing specimens of living muscle of some cold blooded animal into chloride of gold solution, and staining rather deeply.
Arteries.—Take a piece of the aorta, a piece of some medium artery, as the renal or radial, and harden in Müller’s fluid. Stain in picrocarmine and always in eosine and hæmatoxyline. Arterioles are best studied in sections of the various organs. Thus they are seen in each Malpighian body of the spleen, in the boundary zone of the kidney, and so on. A longitudinal surface view can also be obtained by staining and examining the pia mater.
Veins.—Remove, harden, and stain in the same way.
Capillaries.—May be very well seen in the foot of the frog.
Stun a frog by striking its head, or by chloroforming it. Fix it on a piece of card with a V shaped notch at one end. Tie one of the hind feet by means of threads attached to its toes so that the web of the foot is gently stretched over the V. The foot can then be readily examined under a1/2inch objective. The foot must be brushed from time to time with normal salt solution to keep it moist. The movement of blood in the capillaries, &c., can then be studied for an hour or two. After death the mesentery should be spread out on a piece of wood, and hardened for a few days in Müller’s fluid.
Stain with eosine and hæmatoxyline.
Lymphatics.—Commencement of lymphatics in serous membrane. Stain a piece of cat’s omentum in nitrate of silver (p.82) for some minutes. After washing keep in glycerine for about a week and then stain in hæmatoxyline and mount in Farrant’s medium.
Lymphatic glands.—The lymphatic glands of the neck of the cat may be used. Harden in Müller’s fluid. Stain in picrocarmine, eosine and hæmatoxyline.
Skin and sweat glands.—Sections should be made from pieces taken (a) from the sole, (b) from the skin of the body, (c) from the axilla of an adult to study the pigment. Harden in Müller’s fluid. Stain in picrocarmine or eosine and hæmatoxyline.
Hairs and sebaceous glands.—Take a portion of the scalp, or of the skin of a puppy. Harden in Müller’s fluid. Stain in eosine and hæmatoxyline, and mount others unstained.
Hairs from various parts of the body should also be soaked for some hours in liq. potassæ and mounted unstained in Farrant’s medium. They may be bleached subsequently by treatment with eau de Javelle (p.27).
Brain and spinal cord.—Must be removed from the body with extreme care, all stretching or squeezing being avoided. Harden slowly in Müller’s fluid to which a fourth of its bulk of water may be added.
The best staining reagents to employ are eosine and hæmatoxyline, alum carmine or borax carmine, aniline blue-black, &c. Staining methods, see ChapterVI.
Eye.—Harden the eye of a recently killed bullock, cat, or other animal in formal (p.23), puncturing the sclerotic in places to allow the hardening fluid to penetrate. In about a week make a horizontal section through the eye. The anterior half (the lens having been removed) may be satisfactorily cut in gum. Sections of the crystalline lens are not very satisfactory. The best way to get specimens of the fibres is to tease a piece of the fresh lens of a fish (e.g., a cod) in a1/40per cent. aqueous solution of eosine. Wash the eosine off the slide with1/2per cent. acetic acid, and mount in Farrant’s solution.
The posterior half of the eye should be embedded in celloidin, as otherwise it is extremely difficult to get sections of the retina in its proper relation to the other coats.
Mount some specimens unstained. Stain others with the ordinary stains.
Internal ear.—Decalcifying the temporal bone of a cat, dog, guinea pig, &c., in chromic and nitric fluid. As soon as the bone is decalcified complete the hardening of the soft parts in methylated spirit, embed in celloidin, and cutsections in the longitudinal axis of the cochlea. Owing to the extreme hardness of the bone in adults it will be found best to use the petrous bone of newly born animals.
The semi-circular canals will be most readily studied in the temporal bone of fishes, or of birds,e.g., the common fowl. They also must be cut in celloidin, and stained in the ordinary way.
Nose and olfactory epithelium.—It is difficult to obtain specimens from the human subject, but very satisfactory preparations may be made from the dog, or more conveniently in a new born puppy where the bones are still cartilaginous. Harden the latter in Müller’s fluid, decalcify adult specimens in chromic and nitric fluid. Specimens of ciliated epithelium, &c., will be obtained from the lower part, and of the special olfactory epithelium from the upper part. Stain in eosine and hæmatoxyline.
Lungs.—Carefully remove the lungs of a cat without injuring the bronchi or trachea. Introduce a cannula into the trachea and gently inflate the trachea with air. Ligature the trachea and place the lung in Müller’s fluid, a weight beingattached to keep the organ submerged. Harden for about six weeks, and then make sections of the various parts.
To demonstrate the endothelium of the alveoli, inject instead of air, nitrate of silver. Allow it to remain in for half an hour, then remove it by washing, and harden in Müller’s fluid.
Beautiful casts of the alveoli, &c., may be obtained by placing a cat’s or human lung under the receiver of an air-pump, and when the air is completely exhausted, injecting fusible metal into the bronchus. The lung tissue is then removed by corrosion or by maceration. Portions of the casts should be removed, fixed in a glass cell with a spot of Canada balsam, and examined by reflected light.
Thyroid gland.—Best obtained from a young subject either human or an animal.
Harden in Müller’s fluid. Stain in picrocarmine or eosine and hæmatoxyline. Also stain sections in safranine, which stains the colloid material, and also picks out any colloid formation in the cells themselves.
Thymus.—Remove from a fœtus or a very young animal, and prepare in the usual way.
Tongue.—That of the cat or rabbit serves very well.
Ordinary transverse sections should be made, and also sections through the circumvallate papillæ in order to study the “taste buds.”
Salivary glands.—Those of a cat or dog do very well.
Sections should be made from each of the three glands.
Stomach.—That of the cat or dog should be studied. The organ must be removed immediately after death before any post-mortem digestion of the coats has occurred. The stomach should be opened, washed gently and pinned out flat, with as little stretching as possible on a piece of wood, and hardened in Müller’s fluid.
Sections should be made (a) longitudinally through the cardiac end to show the transition from the œsophageal to the gastric mucous membrane, (b) from a portion of the greater curvature, (c) longitudinally through the pyloric valve.
Eosine and hæmatoxyline form the best stain for the alimentary canal.
Intestine.—Prepare in the same way as thestomach. Make sections from (a) the upper part of the duodenum to show Brunner’s glands, (b) the ileum, (c) a Peyer’s patch, (d) the vermiform appendix, (e) the colon.
Liver.—Make an injection of one specimen with carmine and gelatin (p.120). Harden in methylated spirit. Others should be hardened in Müller’s fluid and stained in the usual way.
Kidney, supra-renal, and pancreas.—Same preparation as for liver.
Spleen.—Harden in Müller’s fluid.
Mount one section unstained. Shake another up with water in a test tube to shew the structure of the pulp. Stain others in eosine and hæmatoxyline.
Bladder.—Must be removed and pinned out immediately after death, as otherwise the epithelium will be macerated off. Consequently it must be taken from an animal, as a cat. Harden in osmic acid. Cut in celloidin as the coats are very apt to become detached.
Penisandtestis.—Readily obtained from dog, cat, or rat.
Stain with eosine and hæmatoxyline.
Uterus, ovaries, and Fallopian tubes.—May be obtained from the post-mortem room or from the lower animals. Harden in Müller’s fluid, and make sections from the cervix, the body of the uterus, the Fallopian tube, and the ovary.
Stain with eosine and hæmatoxyline.
Embryological specimens.—For systematic work special manuals should be consulted.
Specimens should be hardened in osmic acid or in Müller’s fluid, and cut in celloidin, or paraffin.
Cloudy swelling.—Specimens are obtained from organs of subjects who have died in the early stage of some fever. They should be always hardened in Müller’s fluid, as the appearances alter if the tissue is kept in spirit for any length of time.
Fatty degeneration.—Prepare from patients who have died of exhausting diseases, phosphorus poisoning, &c.
Stain in osmic acid. Mount in Farrant’s medium and keep in the dark.
Mucoid degeneration.—Study in goblet cells of normal intestine or of ovarian cysts. There are no satisfactory selective stains for mucin.
Colloid degeneration.—Occurs in the thyroid gland, in the tubules of the kidney in many diseases, and the prostate of the old.
Stain in safranine.
Waxy or lardaceous degeneration.—Best studied in liver, spleen, or kidneys. It should be searched for in persons who have died from a long illness, accompanied by suppuration,e.g., phthisis or bone disease. Mount one section unstained, stain another in methyl violet, a third in a weak solution of iodine, and examine the latter at once both by transmitted and reflected light. The iodine stain is not permanent. Another section should be stained in osmic acid, followed by methyl violet, as waxy and fatty degeneration frequently co-exist.
Hyaline degeneration.—Seen in the arterioles of the spleen in some cases of typhoid and diphtheria. The ordinary staining methods must be used.
Calcareous degeneration.—Occurs after fatty degeneration in gummata and in atheromatous arteries. It also occurs in the matrix of the costal cartilages after middle life. Mount onesection unstained and examine if possible with the polariscope. Stain others in safranine.
Pigmentary degeneration.—May be studied in brown atrophy of heart, nutmeg liver, &c. It is also seen well in spinal and cerebral nerve cells of the aged. Harden in Müller’s fluid and mount sections unstained.
It will be unnecessary to recapitulate the methods for hardening the various diseased organs as the directions for the normal organs hold good. If the presence of micro-organisms be suspected, harden in methylated spirit or absolute alcohol, but as a rule both for diseased organs and tumours Müller’s fluid will be found the most satisfactory reagent for general use.
It sometimes happens, however, that it is inconvenient to wait several weeks, until the Müller’s fluid has hardened the specimen sufficiently, before making sections. In this case the best plan is to make fresh sections, or else to cut a slice about one-eighth of an inch thick, and harden for about three days in plenty of methylated spirit, or in formal (p.23).
Tumours.—Müller’s fluid should be employed, unless a more rapid agent is required.
Methylated spirit may be used in the case of epithelioma, adenoma, &c., but for sarcoma, myxoma, tumours containing cysts or much blood, Müller’s fluid yields by far the best results.