The frog; an introduction to anatomy, histology, and embryology

Preface To The First Edition

The Owens College Course of Elementary Biology, which forms part of the scheme of study prescribed by the Victoria University, is of a rather more extended and comprehensive nature than the courses held elsewhere under the same name ; and experience has shown me that there is want of a book that will guide and direct the student through the practical part of his work, the whole ground of which is covered by no one of the existing manuals. It is to meet this want that the present little work has been prepared.

This first instalment of the work consists of an Introduction, containing practical instruction in the methods employed in biological investigation ; followed by the application of these methods to the examination, both anatomical and histological, of an actual animal. For this purpose the frog has been selected as being convenient to dissect, easy to obtain, and a fairly typical example of the great group of Vertebrate animals. Where, from its small size or for other reason, the frog proved unsuitable, other animals have been substituted for it.

For convenience of reference, and in order to definitely stamp the practical character of the work, directions for dissection, etc., have throughout been printed in italics.

It is not expected that the student should do the whole of

IV the work here given the first time he goes over it. The dissection of the muscles and of the cranial nerves should only be attempted if time remain after the other work is completed.

In preparing this first part I have received very valuable assistance from Dr. Hartog, Demonstrator of Biology in the College, and from my friend and pupil Mr. C. H. Hurst. I am also much indebted to Prof. Gamgee and to Mr. Waters for the important help they have given me in the Histological portions.

Owens College,

August, 1882.

V

Preface To The Second Edition

In preparing the present edition I have again received valuable help from Mr. C. H. Hurst, Assistant Lecturer in Zoology in the College.

The illustrations are, with the exception of Figs. 1, 11, 12, and 1 4, original ; and will, I hope, add to the usefulness of the book. They have been carefully selected in order to aid the student in his work without in any way replacing the drawings he must make from his own dissections.

The second instalment of the work, containing directions for the examination and dissection of a number of animals chosen as types of the principal zoological groups, is in active preparation, and will be published shortly.

Owens College, vi

Preface To The Third Edition

Since the appearance of the former editions, the publication of the Junior Course of Practical Zoology by Mr. Hurst and myself has completed, so far as Animal Morphology is concerned, the Owens College Course of Elementary Biology.

I have retained The Frog in its original form, as the mode of treatment is more elementary, and in some ways different from that of the larger book. The present edition has been carefully revised, and an account of the development of the Frog has been added. In preparing this account, I have had the advantage of examining some excellent series of sections mounted by my friends and former pupils, Mr. H. Sidebotham, and Mr. A. E. Giles.

Owens College,

The Nervous System of the Frog.

The Central Nervous System. The Peripheral Nervous System.

Histology of Nerves 73-91

Introduction

I- LABORATORY RULES.

1. The Laboratory is open to members of the Elementary Biology Class from 10-30 to 5 on Tuesdays and Thursdays. Students are required to attend at least three hours on each of these days.

2. Each student has a definite seat assigned him in the Laboratory, which he is not allowed to change without permission.

3. Each student has the free use of the drawer and locker belonging to his seat : the key may be obtained on payment of a deposit of half a crown, which will be returned if the key is given up before the end of the term, but otherwise will be forfeited.

4. All necessary reagents and specimens for dissection are provided by the Laboratory, but each student is required to furnish himself with dissecting instruments, note book, and pencil, as explained in the next section. Microscopes are provided by the College at a charge of five shillings per term.

II— LIST OF APPARATUS REQUIRED.

Each student 'is required to provide himself with the following

1. Two or three scalpels or dissecting knives of different sizes.

2. Two pairs of forceps, one large and one small. Both pairs should be straight, and should have the tips roughened in order to secure a firmer hold.

3. Two pairs of scissors; one pair large and strong, for cutting bone and other hard tissues ; the other pair small, for fine dissections. The latter pair should have the blades either bent at an angle (elbow scissors), or else curved. In selecting scissors be careful to see that they cut quite up to the points of the blades.

B

ON DISSECTION

4. A pair of stout needles, firmly mounted in handles.

5. A pair of the finest sewing needles, mounted in handles : only about a quarter of an inch of the needle should project. They are used for teasing histological preparations.

G. A seeker, i.e. a blunt needle mounted in a handle, and bent at an angle half an inch from the end.

7. A pocket lens, containing two or three lenses mounted in a handle, and giving when combined a magnifying power of at least six diameters.

8. Slides and coverslips, for mounting microscopical specimens.

9. A blank note book, for drawing in ; an HB pencil, and a piece of india-rubber.

Ill— ON DISSECTION.

The object of dissection is to separate the several parts and organs from one another, so as to define their boundaries and display clearly their mutual relations. Dissection consists mainly in removing the “connective tissue” which binds the several parts together.

The following rules should be carefully attended to : —

1. Always pin down the animal you are dissecting firmly to the dissecting board. Never attempt to dissect a specimen that is not so fixed.

2. In pinning out a dissection stick the pins in, not vertically, but obliquely, so that their heads do not get in the way or obscure the dissection.

3. Never cut away anything until you are quite certain what it is you are removing.

4. Put the part you are dissecting slightly on the stretch ; e.g. when dissecting the bloodvessels or nerves of the throat, distend it by passing a small roll of paper or the handle of a seeker down the oesophagus ; or when dissecting the muscles of the leg, pin out the leg in such a position as to stretch the muscles you are cleaning.

5. In cleaning bloodvessels or nerves always dissect along them and not across them ; and avoid layiug hold of them with the forceps. Similarly when cleaning muscles, dissect along their fibres and not across them.

6. Fine dissections should always be done under water, which

ON DRAWING.

supports the parts and greatly facilitates the operation. A stream of water allowed to play gently on the dissection from time to time is often a valuable aid.

7. The dissection of muscles, and still more of nerves, is greatly aided by placing the specimens in spirit for a day before dissecting.

8. Always keep your instruments clean and sharp. Be careful not to blunt your fine scissors or scalpel by using them for cutting hard parts.

9. If you get in a muddle, stop and wash the dissection thoroughly under the tap before proceeding further.

IV— ON DRAWING.

It is absolutely essential to draw your dissections, and this must on no account be omitted. Keep a separate book for your drawings, and draw every dissection you make. Do not be discouraged if you find it difficult at first : you will never regret time spent on it.

The following rules will be useful to those who have not learnt drawing systematically : —

1. Always make your drawing to scale, i.e., either the exact size of the natural object, or half or double or treble that size as the case may be.

2. In commencing a drawing, first determine by careful measurement the positions of the principal points, and sketch in lightly the whole outline before finishing any one part.

3. If the object you are drawing is bilaterally symmetrical, draw a faint line down the middle of your paper, and sketch in the left hand half first ; by measuring from your median line it will be very easy to make the two halves symmetrical.

4. Always name the several parts shown in your drawing, and mark also the scale adopted. Thus, if your drawing be of the natural size mark it thus — x 1 ; if it be double the size of the object mark it x 2 ; if half the size, x and so on.

5. Coloured pencils are very useful, and water-colour paints still better. Always keep certain colours for particular organs or tissues; e.g., when drawing the skeleton colour the cartilage blue, the cartilage bones yellow, and the membrane bones either red or white ; when drawing the bloodvessels colour the arteries red and the veins blue.

THE USE OF THE MICROSCOPE.

V.-THE USE OF THE MICROSCOPE.

The microscope consists essentially of a stand and a body, the latter of which bears at its ends the lenses by which the magnifying power is obtained.

The stand is an upright pillar, the lower end of which is attached to a heavy foot to ensure steadiness. A little way above the foot the stand supports a horizontal plate — the stage — on which the object to be examined is placed. The stage is perforated in the middle by a hole, the size of w'hich can be varied by means of diaphragms. Through this hole light is directed on the object to be examined by means of a mirror attached to the stand below the stage. Above the stage the stand supports a vertical tube, in which the body of the microscope slides up and down.

The body is a tube, in the upper end of which is placed a combination of lenses, known as the eyepiece , while to the lower end is screwed another combination of lenses — the objective.

A microscope is usually provided with a couple of eyepieces and a couple of objectives of different magnifying power. An objective magnifying only a small number of times is called a low power ; one magnifying many times (200 diameters or more), a high power. Similarly eyepieces are spoken of as high or low according to their magnifying power.

In order that an object may be seen clearly the objective must be at a certain definite distance from the object, this distance varying with different objectives, and to a slight extent with different observers. The higher the power employed the closer must the objective be bi’ought to the object. As the position of the object on the stage of the microscope is a fixed one, this distance is regulated by moving the body of the microscope up and down in the tube in which it slides.

This process of focussing is effected in twro ways: (1) by simply sliding the body up and down by hand : this is known as the coarse adjustment ; it should always be performed with a slight screwing motion, and can only be used when low powers are being employed.

(2) With high powers the distance between the objective and the object examined is so small that a more delicate method of adjustment is necessary. This fine adjustment is

THE USE OF THE MICROSCOPE.

effected by a screw with a milled head placed at the top of the vertical pillar forming the stand. By turning the head from right to left, in the direction of the hands of a watch, the body of the microscope is lowered and the objective brought nearer to the object : by turning in the reverse direction the objective is raised.

In using the microscope attend to the following rules : —

1. Always examine an object first with the low power. Having adjusted the eyepiece and objective, direct the light up the tube of the microscope by means of the mirror, and then place the object on the stage. Twist down the body until the objective is about a quarter of an inch from the cover-glass ; look down the microscope, and gradually twist the body up until the object becomes visible. Focus accurately by means of the fine adjustment.

2. When using a high power begin with the objective close to the cover-glass, and then proceed as before. It will facilitate the process if, while focussing with the right hand, you move about the object slightly with the left hand.

3. Take extreme care never to let the objective touch the cover-glass ; and never to touch or allow any dirt to get on the face of the objective. The face of an objective cannot be cleaned without doing harm to it.

4. Should by any chance a drop of glycerine get on the face of the objective, wash it carefully with water from a waslibottle, and wipe it gently with a silk handkerchief or piece of chamois leather. Should Canada balsam be allowed to get on the objective, do not attempt to clean it yourself, but give it at once to the assistant.

5. See that the body of the microscope slides smoothly in its tube. If it does not, remove it, and clean it by rubbing with a few drops of olive oil : wipe off the oil before replacing in the tube.

6. Keep both eyes open when looking through the microscope : a very little practice will enable you to do this, and it will save you much fatigue. Also get into the habit of using either eye.

7. With a high power, use a small diaphragm: the amount of light will be somewhat diminished, but the clearness and definition of the object much increased.

8. When examining an object, keep one hand on the fine

THE PREPARATION OP MICROSCOPICAL OBJECTS.

adjustment, and keep screwing it up and down slightly : in this way parts of the object at different depths are brought into focus, and a much clearer idea of the object is obtained.

9. If the object appears dim or dirty, find out where the fault lies, in this way : —

While looking down the microscope, turn round the eyepiece with your right hand : if the dirt turns round too, remove and clean the eyepiece. If the fault is not in the eyepiece, move the slide about gently ; if the dirt move with the slide remove the slide and clean it. If the dirt does not move with either the eyepiece or the slide the fault is almost certainly in the objective, which should be removed and examined ; if dirty, it must be cleaned very carefully with a piece of silk or chamois leather.

VI.— THE PREPARATION OF MICROSCOPICAL OBJECTS.

In mounting microscopical objects be careful that your slides and coverslips are thoroughly clean. Slides should be labelled as soon as they are prepared, and should be kept in a box or cabinet in which they lie flat.

A. Methods of Mounting.

There are various media in which objects may be mounted. The method of procedure is much the same with all. Put a small drop of the fluid in the middle of the slide, place the object in the middle of the drop, and arrange it with needles in any position that may be desired. Then place the cover-glass carefully on the top, letting it rest by one edge on the slide and supporting the opposite edge by a needle : withdraw the needle gradually so as to let the cover-glass down slowly, and drive out any air-bubbles there may be in the fluid. If any air-bubbles still remain, leave them alone, as they will probably work out by themselves. Be careful not to use too large a drop of your mounting medium, and above all things be careful not to let any of it get on the top of the cover-glass ; should this happen, the cover-glass must be removed at once and the specimen mounted again with a clean one.

The most important mounting media are the following.

1. Normal Salt Solution ; a 0-75 per gent, solution of common salt in water. This is very useful in the examination of

THE PREPARATION OP MICROSCOPICAL OBJECTS. 7

fresh specimens of animal tissues, as, unlike water, it has practically no action whatever on them. It cannot be used, however, for making permanent preparations.

2. Glycerine can be used either pure or diluted with its own bulk of water. If the preparations are intended to be permanent, a narrow ring of cement must be painted round the edge of the cover-glass to fix it to the slide.

3. Canada Balsam is the most generally useful, requiring no cement. Specimens that are to be mounted in balsam must first be deprived of all water they may .contain by placing for an hour or so in absolute alcohol, and should then, before mounting, be soaked for a few minutes in a mixture of creosote and turpentine in order to clear them, i.e., render them permeable by the balsam. Canada balsam, if too thick, may be diluted with chloroform, turpentine, or benzole.

B. Teasing.

The object of teasing is to separate the several parts of a tissue or organ from one another as completely as possible, in order to show their minute structure.

The fragment to be teased should be placed on a slide in a drop of the medium in which it is to be mounted, and then torn up into as minute particles as possible by means of a couple of needles held one in each hand. The process is often greatly facilitated by placing the slide on a piece of black paper, which renders the particles easier to see. When torn up as finely as possible, a cover-glass is placed on as before. The two rules to be borne in mind in teasing are the following.

1. Take a vexy small fragment to commence with.

2. Tease it as finely as you possibly can. Your object is to separate the component cells from one another.

C. Maceration.

The process of teasing is in many cases very greatly facilitated by previously macerating the specimen, i.e., soaking it in some fluid, which, while preserving the individual cells, tends to loosen their connections with one another. The most important macerating fluids are as follows.

1. Ranvier’s Alcohol : a mixture of one part of strong spirit with two parts of water. The specimens should be put fresh into the mixture and allowed to remain twenty-four hours.

THE PREPARATION’ OF MICROSCOPICAL OBJECTS.

2. Baryta Water is very useful for isolating the individual cells of tendons.

3. Muller’s Fluid : a solution of bichromate of potash with a little sodic sulphate in water.

D. Staining.

Various reagents are employed for the purpose of staining preparations ; some of these merely colour the whole preparation more or less uniformly, but the most useful ones are those which stain certain parts of the cells only, or at any rate stain these much more strongly than the other parts. The most important are the following.

1. Haematoxylin. There are various preparations of hsematoxyliu, or logwood, used in microscopical work : the best is that proposed by Kleinenberg and called by his name. It is prepared thus : —

(a) Make a saturated solution of crystallised calcium chloride

in 70 per cent, alcohol, and add alum to saturation.

( b ) Make a saturated solution of alum in 70 per cent, alcohol,

and add (a) to (b) in the proportion of 1 to 8.

(c) To the mixture of (a) and ( b ) add a few drops of a

saturated solution of hsematoxylin in absolute alcohol.

The specimens, which must be perfectly free from all trace of acid, should be left in the haematoxylin in a covered vessel or stoppered bottle for from one to twenty-four hours, according to the size of the specimen and the depth of staining desired, and then placed in strong spirit for some hours before mounting. Haematoxylin stains the nuclei of cells much more strongly than the other parts.

2. Borax-carmine. This, which is perhaps the most generally useful of all the stains in ordinary use, is prepared as follows. Dissolve 2 parts of carmine and 4 parts of borax in 100 parts of water: add an equal volume of 70 per cent, alcohol; let the mixture stand for a couple of days, and then filter.

Specimens may be left in borax-carmine for from one to twenty-four hours, or even for two or three days : on removal they should be placed in acid-alcohol — i.e., 70 per cent, alcohol to which a few drops of hydrochloric acid have been added — until they become a bright scarlet colour, when they should be transferred to 70, and then to 90 per cent, alcohol, in which

TlfE PREPARATION OF MICROSCOPICAL OBJECTS.

latter they may be left till required. The time of immersion in acid alcohol will vary, according to the nature and size of the specimen, from a quarter of an hour up to a day or more.

3. Piero- carmine is a very useful, and to a certain extent a differential stain, as it colours the several tissues different tints. It may be prepared thus. Dissolve 1 gramme of carmine in 4 cc. of liquor ammonia and 200 cc. of distilled water. Add 5 grammes of picric acid ; shake the mixture well for some minutes, and then decant from the excess of acid. Leave the decanted liquid for some days, stirring it occasionally : then evaporate it to dryness, and to every 2 grammes of the dried residue add 100 cc. of distilled watei\

Picro-cannine answers best with specimens preserved in 70 per cent, alcohol. They should be left in the stain for a day, and then placed in 70, and afterwards in 90 per cent, alcohol. Some specimens give better results by washing freely with water on removal from the picro-carmine, and then placing in 1 per cent, acetic acid for an hour before transferring to alcohol.

4. Magenta stains very rapidly, but diffusely : the colour also is not permanent.

5. Silver Nitrate. A 1 per cent, solution in water stains the intercellular substance, which binds together the several cells of a tissue, much more strongly than the cells themselves, and is therefore chiefly used when we wish to render prominent the outlines of the individual cells. The specimens should be placed fresh in the silver solution for from two minutes to a quarter of an hour, then washed thoroughly with distilled water and exposed to the light until stained sufficiently deeply, when they may be mounted in glycerine. Such preparations are rarely permanent, as the reduction of the silver, to which the staining is due, continues until the specimens ultimately become too dark to be of any use.

6. Osmic Acid. A 1 per cent, solution of osmic acid in water forms an extremely useful staining reagent. It is especially useful for the detection of fat, which is stained by it a dark brown or black colour. Specimens, which must be quite fresh, should only be left in it a few minutes, and may then be mounted in glycerine, or else dehydrated and mounted in balsam.

7. Acetic Acid. Although not strictly a staining agent inasmuch as it does not colour the specimens, acetic acid majr

ON SECTION CUTTING.

conveniently be mentioned here as it is used for the same purpose as the true stains, i.e., for the sake of rendering certain parts of the cells especially distinct. Acetic acid, of which a 1 per cent, solution is employed, causes the protoplasm of cells to swell up and become transparent, and brings the nuclei into special prominence. It is used with fresh specimens.

VII— ON SECTION CUTTING.

Many tissues and organs can only be studied satisfactorily by cutting them into thin sections, and this method of investigation is of such importance as to require special notice. There are three chief stages : Hardening, Imbedding, and Cutting, which will be noticed in succession.

A. Hardening.

Before the object can be cut into sections it is necessary to harden it : this may be effected by freezing, but the more usual plan is by means of reagents. The general action of these hardening reagents is to coagulate the protoplasm of the tissues ; and the objects to be attained are to effect this coagulation quickly, before the tissues can undergo any alteration ; and thoroughly, i.e., throughout the whole thickness of the object to be hardened. To ensure the latter result it is always advisable to use small pieces of the substance to be cut.

The hardening reagents in most common use are as follows.

1. Osmic Acid. For this purpose a 1 per cent, solution in water is used : it acts almost instantaneously, and so allows no change to occur in the tissues ; it has also the merit of staining the tissues as well as hardening them. It can, however, only be employed when the specimens are very small, as it hardens the surface layers so rapidly that it is unable to penetrate beyond a very slight depth. A few minutes immersion is usually sufficient.

2. Corrosive Sublimate. A saturated solution in water is employed, in which the object is placed for half an hour or more. After removal it is thoroughly washed with water or weak alcohol, and then transferred to 70 per cent, alcohol before staining.

3. Chromic Acid. A 0"25 to 0'5 per cent, solution of chromic acid in water is a useful hardening reagent ; it acts much more

ON SECTION CUTTING.

slowly than osmic acid, but is thereby enabled to penetrate to greater depths. Specimens should usually be left in the solution for 24 hours.

4. A Mixture of chromic acid with a few drops of osmic acid is often very useful, as it combines the advantages of both reagents.

5. Picric Acid is a very valuable hardening reagent, of which the best preparation is Kleinenberg’s. Specimens should be left in it from 12 to 24 hours. It is prepared thus : with 100 cc. of water make a cold saturated solution of picric acid : add 2 cc. of concentrated sulphuric acid : filter, and add to the filtrate three times its volume of water.

6. Absolute Alcohol is often a useful hardening reagent.

B. Dehydration.

Specimens that have been hardened in any of the preceding reagents, with the exception of the last, should, on removal be placed for a few hours in 30 per cent, alcohol, and then transferred to 50 per cent, alcohol : on the following day they should be transferred to 70 per cent, alcohol, which should be changed daily until the specimens are colourless : they may then be left in 90 per cent, alcohol until required.

C. Staining.

The hardened specimens, if not too large, may now be stained with either hmmatoxylin, borax-carmine, or picro-carmine ; they should then be replaced in 90 per cent, alcohol and transferred from that to absolute alcohol immediately before imbedding. If the specimen is'too large to stain whole, the sections must be stained after cutting.

D. Imbedding.

The preparation of sections is greatly facilitated by imbedding the specimen in some waxy substance. For this purpose various materials have been employed, but by far the most useful is paraffin, which is used in the following manner.

The stained specimen is placed in absolute alcohol for an hour or two in order to completely dehydrate it. It is then transferred to turpentine, in which it is left for half an hour or more until completely saturated. From the turpentine it is transferred to paraffin, which is kept by means of a water bath

ON SECTION CUTTING.

at a temperature just above its melting point. In this it isleft for several hours, or even for a whole day, in order that it may be thoroughly permeated. It is then placed in a small box of paper, or other material, filled with melted paraffin. By means of hot needles it can readily be arranged in any desired position ; and the paraffin should then be cooled quickly.

E. Section-Cutting.

When thoroughly set the block is removed from the box, and the paraffin pared away with a knife until the specimen just comes into view.

The block is then placed in a microtome, and cut into thin sections. These may be transferred one by one to a slide, but a great saving of time is effected by the method of cutting continuous ribbons, devised by Mr. Caldwell.

This depends on the fact that if the paraffin is of proper consistency the successive sections, as they are cut, will stick together at their edges, so as to form a ribbon. To ensure this the razor should be placed at right angles to the direction of stroke, and the edges of the block of paraffin cut parallel to one another, and to the edge of the razor. If for any reason it is desirable to imbed the specimens in a paraffin too hard to form ribbons, the block should, before cutting, be coated with a layer of soft paraffin, by dipping it for a moment in a dish of melted soft paraffin. This outer coating should be left on the sides of the block parallel to the edge of the razor, but cut away from the sides at right angles to it.