Outlines of practical histology

Front matter

i i'

Private Library

OF

Preface

TO

THE SECOND EDITION.

No subject more than Practical Histology requires for its successful pursuit that guidance which oral instruction alone can give. If the beginner attempts to study it without a teacher, he is baffled in learning how to use the microscope, how to manipulate the tissues, and how to apprehend rightly the phenomena that come under his observation. Much valuable time is thus lost, and the final result is generally failure and disappointment.

These Outlines are therefore primarily intended to assist beginners who are working under the guidance of a teacher in a laboratory furnished with the usual appliances, together with a full set of those microscopical specimens that cannot be readily made. They are at the same time, however, designed to assist those who may already have learned the rudiments of vi PREFACE.

the subject at a time when its details were not taught as they now are. Care has been taken to render all instructions as definite as possible, and the advanced student will find in Part I. such directions for the preliminary preparation of the tissues as will enable him to work through the Histological Demonstrations in Part III. without embarrassment. The paragraphs in small type are especially intended for the advanced student. ,

These Outlines are essentially the Notes of the course of Practical Histology given by me in King's College, London, and in the University of Edinburgh. That course is conducted on the principle that, inasmuch as the time at the disposal of most students of medicine is very limited, the only practicable method of teaching large numbers is what may be termed the regimental system, whereby the students all work at the same moment, at one subject, and are addressed collectively. In this way classes, under thirty in number, can be conducted through the leading points of Practical Histology in about thirty-six lessons, extending from an hour to an hour and a half. As it is impossible in this short time to prepare every desirable microscopical specimen, special demonstrations are made by the teacher in order that the introduction to the subject may be as comprehensive as possible. The special

.prepare. :vu

working- out; qf rfiany complicated processes; cannot be attempted in a general cour.se of this sort, bu$ must be done by the advariced student working in the laboratory for several hours daily. The exceptionally high .magnifying powers sometimes mentioned in the following pages, may be used by him, but in the case of the beginner it is understood that they are intended to be used for special demonstration by his teacher.

In the course of the demonstrations (Part III.), the student is desired to read the general considerations regarding methods, in Part IV. ; and when he finds no statement regarding the preservation of a specimen, it is to be inferred that it is needless to preserve it.

In this edition, the book has been considerably extended by an account of the microscope, and fuller descriptions of the tissues. All figures of the latter have been omitted, however, for they are to be found in the systematic text books already in the hands of everyone. Their repetition here would therefore have entailed needless addition to the expense of the work. It is hoped that the demonstrations of the tissues, if made as directed, will supply the place of pictures, and that the descriptions will, together with hints from the teacher, prove a sufficient guide to them.

via PREFACE.

The blank paper at the end is intended for drawings and noteS4

The author is glad of the indication of the usefulness of the book, afforded by the rapidity with which the first edition was exhausted.

University of Edinburgh, September 1876.

Apparatus required .

Preliminary Preparation of Tissues

PAGE I

Part II. The Microscope

Fundamental Principles ..... 8

Theory of the Simple Microscope . . . .16

Theory of the Compound Microscope . . .17

Faults of a Lens ...... 19

Optical Parts of the Compound Microscope . . 22

Mechanical Parts of the Compound Microscope . . 30

Different Forms of Microscopes .... 33

Accessory Apparatus for Illumination . . .36

Method of Testing the Microscope . . .39

Method of Working with the Microscope . . 40

Preparation of a Simple Object . . . 41

Description of the Object ..... 41

Delineation of the Object ..... 43

Estimation of the Magnifying Power of the Microscope 46 Estimation of the Size of an Object . .48

Part III. Histological Demonstrations

Brownian Movement. Tradescantia

Si

Penicillium. Starch. Cotton Fibres

Linen Fibres. Disc-Bearing Tissue. Bacteria .

Blood of Newt

Blood of Bird, Fish, and Man

Mucus. Epithelium . .

Connective Tissues-

Cartilage ....

Bone ....

Tooth ....

Muscle . . .

Nerve Tissue .

Blood-vessels .

.

Circulation of the Blood .

Lymphatics

Blood Glands .

Lung ....

no

Tongue ....

Stomach . . .

Intestine

"S

Liver . . ...

Salivary Glands and Pancreas

Kidney .

Urinary Deposits

Skin ....

. 125

. 128

Cochlea. ...

• 134

CONTENTS.

xi schneiderian membrane . <

Spinal Cord and Medulla Oblongata Cerebellum and Cerebrum . .

Male Generative Organs , ,

Female Generative Organs , , tAGE

136 137 139 141 142

Part IV. General Considerations Regarding Histo- Logical Methods

Methods op Applying Re-agents to the Tissues Application of Fluids and Gases . .

Application of Heat ....

Application of Electricity .

Methods of Studying Living Tissues Methods of Hardening the Tissues .

Methods of Softening the Tissues*.

Mechanical Dissociation of Tissue Elements Methods of Making Sections Methods of Increasing the Transparency of ti. Methods of Staining the Tissues .

Methods of Injection. . . , .

Methods of Preserving the Tissues Method of Keeping Preparations .

Index .....

• 144 . 144

• 145 148

• 157 . 159

• 159 Tissues 169

. 171

• 179 . 185

. 191

ABBREVIATIONS.

H. means high power ; a combination of lenses such as the \ inch objective of an English microscope, with an eyepiece of medium power, or the No. 7 objective and No. 3 eye-piece of Hartnack's microscope — with the tube of the microscope drawn out — magnifying about 300 diameters linear,

L. means low power ; a combination of lenses such as the 1 inch objective of an English microscope, with an eyepiece of medium power, or the No. 3 objective and No. 3 eye-piece of Hartnack's microscope, magnifying about 50 diameters.

V. S., L. S., and T. S., respectively mean vertical, longitudinal, and transverse section.

iMP

Fig. i. — Hartnack's No. IIIa Microscope. A, Eyepiece. D, Coarse adjustment C, Condenser. H, g^,lgiy^<# cr ^®ectiv e . L - Sta S e - M > Diaphragm, N 3 Mirror.

OUTLINES

OF

PRACTICAL HISTOLOGY.

Part I. Introduction

APPARATUS REQUIRED.

i. The student who is entering upon the study of Practical Histology must be provided with a compound microscope capable of giving a low magnifying power of about 50 diameters, and a high power of about 300 diameters ; a thin-bladed razor of good steel (§ 294); a pair of strong needles in handles (§ 290); scalpels, forceps, and scissors, such as are found in an ordinary dissecting case, the bent scissors shown in Fig. 55 are, however, to be preferred; three or four camel-hair brushes of medium size ; five or six dozen ground glass slides, 3x1 inch ; about a hundred extra thin circular cover-glasses, f inch in diameter ; a box to hold about fifty preparations. The student will thus be enabled to preserve the principal specimens made during the course of study indicated in the following pages.

2. He should also be provided with the following set of the ordinary reagents and mounting fluids. These may be conveniently placed in one-ounce bottles, with ordinary corks (Fig. 2).*

PRACTICAL HISTOLOGY.

i. f per cent Salt Solution (§ 269)

2. Magenta Solution (§ 324 a)

3. Picrocarmine Solution (§ 322)

4. Logwood Solution (§ 323)

5. Dilute Acetic Acid (§ 313)

6. Dilute Alcohol (§ 284)

7. Glycerine

8. Farrants' Solution (§ 349)

9. Dammar mounting fluid (§ 347c) 10. Clove Oil. In a bottle, with a

brush fixed to a stick (Fig. 2).

Gold Size Cement ) botUfig> ^ «„„£.

11. 12.

White Zinc Cement

In bottles, with a glass rod fixed in the cork.

It is convenient to place these bottles in a wooden framework similar to that used for test-tubes, and to cover

the whole with a paper or glass shade, to protect it from dust.

The advanced student will find it advantageous to place the first eight of the above-mentioned reagents in the bottles with capillarystoppers (Fig. 3). In these the reagent is completely protected from the entrance of dust by a glass cap. These bottles, however, though excellent for very fine work, are not to be recommended for ordinary purposes, because of the serious length of time required to obtain a

"^fiSSr^P of ±e reagent — the bottle requiring to be inverted, and held in the hand until its heat expands the air and expels the fluid.

Fig. 2.— Corked reagent bottle^

INTRODUCTION. 3

3. The advanced student who may be desirous of working through the following course of instruction privately — without the resources of a laboratory— ^requires, in addition to the foregoing apparatus and reagents, a magnifying power of about 800 or 1000 diam. ; a stage and an eye-piece micrometer (§§ 59, 60) ; a freezing microtome (§ 3 02 ) > a camera lucida (§ 58, a or b) ; a lamp with a chimney of pale blue glass for night work (§ 51) ; a warm stage (§ 261); a small and a large injection syringe (Fig. 60), and perhaps a pressure bottle injection apparatus (Fig. 61) ; a turn-table (Fig. 63). A polarising apparatus may be added, but other accessories are of very little use. Various other reagents are required, but these need not be particularised here.

PREPARATION OF TISSUES PREVIOUS TO THEIR EXAMINATION.*

4. The following fluids require to be prepared : —

I. f- per cent Salt Solution. Dissolve 7^5 grammes of dried ordinary NaQ.in 1000CC distilled water.

II. One per cent Chromic Acid Solution. Dissolve 10 grammes chromic acid in 1000CC water. This can be

. readily diluted when required:

III. Chromic Acid and Spirit Solution. Dissolve 1 gramme chromic acid .in 20CC water, and slowly add it to 180CC rectified spirit.

IV. Chromic and Bichromate Solution. Dissolve 1 gramme chromic acid, 2 grammes potassium bichromate in 1200CC water.

V. Chromic and Nitric Fluid. Chromic acid 1 gramme, water 200CC, then add 2CC nitric acid.

VI. Mailer's Fluid. Dissolve 25 grammes potass, bichrom. and 10 grammes sodium sulphate in 1000CC water.

VII. One per cent Bichromate Solution. Dissolve 10 grammes potass, bichromate in 1000CC water.

* This section is designed to assist the advanced student when he undertakes histological work in private,

5. Most of the requisite tissues and organs may be obtained from the cat and guinea-pig. Feed the cat, and an hour or so afterwards place it in a bag ; drop chloroform over its nose until insensibility is produced. Open the chest by a linear incision through the sternum ; speedily open the right ventricle, and allow the animal to bleed to death.

6. Divide the trachea immediately below the cricoid cartilage, and inject it with £ per cent chromic acid fluid ; tie it to prevent the escape of the fluid, and place the distended lungs in the same fluid, and cover them with cotton wool. Change the fluid at the end of eighteen hours. Allow them to remain in this fluid for a month, then cut into small pieces and transfer to rectified spirit* until required.

7. Open by a linear incision the stomach, small and large intestine, and wash their inner surface with f per cent salt solution. Place the tongue, divided transversely into a number of pieces, and a portion of the small intestine in chromic and bichromate fluid (Solution 4). Change the fluid at the end of eighteen hours ; and at the end of a fortnight transfer them to rectified spirit until required. Place the tongue, divided transversely into five or six pieces, together with a portion of the stomach and large intestine, in £ per cent chromic acid solution. Change the fluid at the end of eighteen hours ; and at the end of a month transfer them to rectified spirit till required.

8. Open the bladder, wash it with salt solution, and place it in a 1 per cent solution of bichromate of potash (Solution 7) for two or three days.

9. Remove the kidneys ; divide one transversely, the other longitudinally, and place them in Miiller's fluid. Change the fluid at the end of eighteen hours ; and at the end of four weeks transfer them to rectified spirit until required. They will be ready after having been a fortnight in the spirit. It is also advantageous to have the kidneys

* In this edition rectified spirit has been substituted for methylated spirit in the preparation of the tissues, for undoubtedly the tissue elements are better prepared in it. (Its sp. gr. is 0-838.) Digitized by Microsoft®

INTRODUCTION. 5

of a rabbit prepared as follows : — Divide the kidneys longitudinally ; place them for a fortnight in a 1 per cent solution of chromic acid, then in rectified spirit till required.

1 o. Cut half of the liver into small pieces, and place them in Miiller's fluid. Change the fluid at the end of eighteen hours; and at the end of four weeks transfer them to rectified spirit till required.

11. Place the spleen, uterus, some thin muscles from the limbs or abdomen, in £ per cent chromic acid. Change the fluid at the end of eighteen hours ; and at the end of a month transfer them to rectified spirit. .

12. Through several points in the tunica albuginea of testis of cat, inject a 1 per cent solution of osmic acid, and then place it in rectified spirit ; and finally — a day or two before making sections — in absolute alcohol.

12A. Place the ovaries of cat or dog in Miiller's fluid. Change the fluid after the first, day, and after three weeks transfer to a mixture of equal parts of absolute alcohol and water for thirty-six or forty-eight hours, then in absolute alcohol for a day, after which they will be ready for section.

13. Remove both eyes. Divide them transversely behind the crystalline lens. Remove the vitreous. Place the posterior halves in the chromic and spirit (Solution 3). Change the fluid at the end of eighteen hours. Transfer to rectified spirit at the end of ten days. Place the crystalline lens in chromic acid and spirit for two weeks, and then in rectified spirit. Place the cornea in J per cent chromic acid for a month, and then in rectified spirit.

14. Cautiously open the cranial and spinal cavities* Remove brain and spinal cord, and strip off arachnoid. Partially divide spinal cord into pieces half an inch long., Partially divide the brain transversely into a number of pieces. Place them in a cool place in rectified spirit for eighteen hours. Transfer the spinal cord to \ per cent chromic acid for six or seven weeks. Change the fluid at the end of eighteen hours. Prepare, the sciatic nerve in the same manner. Place the brain in chromic and bichromate fluid. Change at the end of eighteen hours, and then once a week, until the brain is hard and tough. If it be not tough and leathery at the end of six weeks, place it in £ per cent chromic acid for a fortnight, and then in rectified spirit. If the brain be placed in a too strong chromic acid solution, or if it be too long exposed to the action of even a moderately strong solution, it becomes friable and useless. Procure a portion of human cerebrum {parietal lobe), as fresh as possible, and treat it in the same manner as the cat's brain. A portion of human cerebellum may be prepared in the same manner. At the end of six or seven weeks transfer all the tissues to rectified spirit to complete the hardening. The brain and spinal cord should always be placed on cotton wool in their hardening fluid, in order that the fluid may reach every part equally.

1 5. Remove the muscles, but not the periosteum, from the bones of the upper or lower limb, and remove both from the lower jaw. Divide the jaw and the long bones transversely in two or three places, and put them into the chromic and nitric fluid (Solution 5). Change the fluid repeatedly until the bone is just sufficiently softened, and then transfer it to rectified spirit. If the softening is not completed in a month, double the quantity of nitric acid in the fluid, or place them simply in dilute nitric acid (2 per cent). The softening is most rapidly accomplished by suspending the bones in the fluid. For this purpose they may be placed in a muslin bag. The bone must not, however, remain in the softening fluid after all calcareous matter has been removed.

16. Place a piece of human scalp, skin from palmar surface of finger, in chromic and spirit fluid (Solution 3). Change the fluid at the end of eighteen hours. At the end of a month transfer to rectified spirit.

17. Remove the petrous portion of the temporal bone, open the tympanum, pull the stapes from the oval fenestra, and place the cochlea in chromic and spirit fluid. Change it at the end of eighteen hours, and again on the sixth or seventh day. But if a copious brown precipitate fall, change the fluid every third day. On the tenth or twelfth day transfer to chromic and nitric fluid. Change the fluid frequently until the bone is soft. Then plage

INTRODUCTION. 7

it in rectified spirit. In the guinea-pig the cochlea forms a very evident projection into the tympanum, and is therefore very convenient for enabling the student to see how the cone is to be sliced when sections are to be made. Kill a young guinea-pig, sever the head from the body, disarticulate the lower jaw, open the tympanic bulla, remove the stapes, and prepare the cochlea as above. In a young animal the chromic and spirit fluid alone may suffice to soften the bone, and the chromic and nitric fluid may therefore not be required, or at any rate only for a short time.

It is very important to place all the above tissues in a cool place. A cellar is best in summer. Protoplasm rapidly undergoes change in even a medium temperature.

ERRATA.

Page 8, line 4, for behind, read within.

Part II. The Microscope

FUNDAMENTAL PRINCIPLES.

: 18. The Visual Angle. — We form an opinion of the magnitude of an object from the size of the image which it produces on the retina. The lateral rays from any object intersect behind the crystalline lens. The size of the retinal imageaepends on the size of the visual angle — that is, the angle formed by the intersection of the rays

II Mfi

Fig. 4. — Visual angle.

from the extreme points of the object. All objects which have the same visual angle appear of the same size. Thus the objects ABC (Fig. 4), although actually of different sizes, have — when placed at proper relative distances from the eye — the same apparent size ; because the visual angle is the same for all, and the magnitude of the image on the retina (N) consequently the same.

Figs. 4 and S , the crossing of the^ial^ys oflhe peripheral „«,,-•> . _.__oagfatto_b e within the crystalline lens. P ^^

Moreover, an object of the same actual size appears of different sizes at different distances from the eye,— the magnitude appearing less the greater the distance. This depends on the size of the visual angle being inversely as the distance of the object from the eye. Thus, if :we look at a line in the position A (Fig. 5), it produces the

-Visual angle.

image a a on the retina: in the position B it appears larger ; because its image b b excites a greater retinal area ; in the latter the visual angle is evidently greater than in the former.

19. Theory of the Microscope.— The eye can see the same object clearly when it is successively placed at various distances, because of an alteration of the curvature of the crystalline lens, whereby parallel rays from an object at a great distance, or divergent rays from a near object, may be successively focalised accurately upon the retina. The more minute the object, the nearer one brings it to the eye in order to increase the visual angle, and get as large an image as possible. But there is a limit to this. Within eight or ten inches from a normal eye of an adult, the object cannot be seen without an unpleasant effort to render the crystalline lens sufficiently convex to focalise the divergent rays on the retina ; and within two inches or so the eye completely fails to accomplish the task ; the image is therefore ill denned. But if a convex lens (Fig. 19) be interposed between the near object and the eye, it acts in the same sense as the crystalline lens, and assists it focussing the divergent rays, and thus the object apparently magnified can be- clearly seen.

PRACTICAL HISTOLOGY.

20. Refraction and Reflection of Light. — The

action of a lens depends on the following principles. As long as a luminous ray travels in a uniform medium it pursues a straight line. If it pass at a right angle from one medium to another, it does not deviate from its course, however different the refractive powers of the two media may be. But if the ray traverse the plane separating two media of different refractive powers at any

other than a right angle, it is bent. Thus, the ray R (Fig. 6), passing through air and striking the surface of a glass plate obliquely at B, is bent in two directions ; a portion (r) is reflected, while the- remainder is refracted towards the perpendicular (J>). The refracted ray again strikes the plane between the two media obliquely at C ; a portion (r ) is reflected, while the remainder is refracted ' from the perpendicular as it Te-enters the air. The emergent ray is of less intensity after traversing the glass, in consequence of its partial reflection at the two surfaces ; there is therefore a loss of light at every refraction ; a very important fact in microscopical optics.

21. Index of Refraction. — The refractive power of any medium is expressed by its index of refraction. This is ascertained by measuring the amount of refraction which a ray undergoes in passing from air into another medium ; thus, let r (Fig. 7) be a ray passing through air, incident upon water at ;", and refracted in the direction r. The sines of the angles of incidence r i p, and of refraction r' i p', are obtained by describing a circle with the point

Fig. 6. — Refraction and reflection by parallel plate.

Fig. 7. — Law of refraction.

{Lcmmtl.)

Fage 1 1, line 4, after are, insert in the ratio of.

THE MICROSCOPE.

Diamond

2-44

Flint glass

1*642

Crown glass

Canada balsam

Clove oil

Oil of turpentine

1-478

Glycerine

1 '47 5

Alcohol

1-372

Aqueous humour

1 '33 7

Pure water

Air

1-000294 i as the centre, and drawing lines from the perpendicular at right angles with it to the points at which the circle intersects the incident and refracted rays. Thus a b and c d are^he sines of the angles of incidence and refraction ^_ respectively. In the case of air and water the sines are as 4:3. The refractive index of air is so small, that for practical purposes its refractive power is discarded, and therefore -i^i'333 the refractive index of water. The following table shows the indices of refraction for various substances, many of which are used in practical histology : ; —

Indices of Refraction.

As will afterwards be seen, such substances as Canada balsam, clove oil, turpentine, and glycerine, having a comparatively high refractive index, are constantly employed in histology, for the purpose of increasing the transparency of tissues.

22. Lenses. — If a ray pass obliquely through a medium with parallel faces (Fig. 6), the excident assumes the same direction as the incident ray, but, if the faces be not parallel, the excident ray assumes a new direction. Thus, let r (Fig. 8) be a ray impinging on a triangular prism at a, it bends towards the perpendicular /, and on emerging at b, bends from it. The excident ray r thus takes up a direction entirely different from r. If two prisms be joined by their bases (Fig. 9), the rays A B, being refracted as indicated in Fig. 8, are rendered convergent and brought to a focus (C). On the other hand, if the angles of the

■5 Fig. 8.— Refraction by prism.

Fig. q. —Convergence of rays by prisms.

prisms be turned towards each other (Fig. 10), the rays A B, are rendered divergent. The converging lenses A B and C in Fig. 11 act after the manner of the prisms in Fig. 9, while the diverging lenses D E and F (Fig. 11) act like the prisms arranged as in Fig. 10.

Fig. 10.— .Divergence of rays by prisms.

Fig. 11. — Lenses.

23. Focal Length. — Take the simple case of a piano: convex lens (Fig. 1 2). It may be regarded as composed of two rectangular prisms joined by their bases, with the oblique surfaces curved so as to form the segment of a sphere whose radius is C S. The rays A R R all strike the plane surface of the lens at right angles, and therefore undergo no refraction until the spherical surface is reached. The ray AS passing through the centres of both surfaces of the lens' is termed the axial ray. Being perpendicular to both surfaces, it undergoes no refraction. The rays K. S, on emerging from the lens, are bent from the perpendicular C S, and meet in the focus F. In a plano-convex lens the

Fig. 12. — Effect of plano-convex lens. (Hannover.)

optical centre is placed at the point where the axial ray meets the convex surface ; in a bi-convex lens it is placed within the lens, at its very centre, if the convexity of the surfaces be equal. The distance J of the focal point F from the optical centre is the focal distance of the lens. In a plano-convex lens of crown glass the focal distance is almost exactly twice the radius of curvature (C S), while in a biconvex lens of the same substance having equally curved surfaces, the focal length is very nearly the same as the radius of curvature. But the focal length depends not only upon the degree of curvature of the lens, it also depends on the refractive power of its material ; thus, if the focal lengths of two lenses — the one of flint, the other of crown glass — of equal curvatures be compared, the flint lens is found to have a shorter focus than the other, because of its higher refractive power (§ 21).

24. Principal Focus and Conjugate Foci — The principal focus of a convex lens (A, Fig. 13) is the point (F) to which rays that were parallel before entering the lens are brought. (The same is shown in Fig. 14, where P P are the parallel rays, and F the principal focus.) If the rays be divergent (D D, Fig. 14) before entering the lens, they are focalised outside the principal focus (D') ; while, if they be convergent (C C), they are focalised within

H

PRACTICAL HISTOLOGY.

it {€'). It is evident that if a cone of rays proceeded from a luminous point at F, the divergent pencil would be converted into a cylinder of parallel rays. If the luminous

Fig. 13.— Principal focus. (Deschanei.

.point were placed within the principal focus— say at C', the more divergent cone would not be rendered parallel by the lens, but would still continue to diverge, though to a smaller degree, after passing through it ; but if the luminous pencil

Fig.=I4.— Principal focus (F) and secondary foci (C C). (Hannover.

prpceeded from a point outside the principal focus, say at D', the less divergent rays would be rendered convergent (D D) by the lens, and would come to a focus somewhere on the other side of it. In Fig. 15a cone of rays is seen proceeding from a point S', outside the principal focus F, and brought by a lens to a real focus at S. At S a real image of the point S' would be produced. Conversely, if the rays proceeded from S, they would come to a real focus at S', and if S were placed nearer the lens, but outside the

THE MICROSCOPE.

IS principal focus, S' would recede from the lens. The points S and S' are therefore termed real conjugate foci ; that is,

Fig. 15. — Conjugate foci, both real. (Deschanel.)

points so correlated that rays diverging from the one converge to the other. In Fig. 15 the luminous point is placed in the principal axis of the lens ; if it were placed above or below the axis, the rays would be focalised on the opposite side of the axis (Fig. 17).

25. "Virtual Focus. — Let Fig. 16 represent a luminous point T placed within the principal focus F of the lens A B, the rays are never brought to a focus, but are only rendered less divergent by the lens.