Book - The Elements of Embryology - Appendix
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- 1 Appendix - Practical Instructions for Studying the Development of the Chick
- 2 A. Incubators
- 3 B. On preparing sections of the embryo
- 4 2. Staining
- 5 3. IMBEDDING AND CUTTING SECTIONS
- 6 PRACTICAL DIRECTIONS FOR OBTAINING AND STUDYING CHICK EMBRYOS
- 7 =D. Surface view of the transparent embryo from above
- 8 APPENDIX - PRACTICAL DIRECTIONS FOR OBTAINING AND STUDYING MAMMALIAN EMBRYOS
- 9 Note A
- 10 Note B
- 11 Reference
- 12 Glossary Links
Appendix - Practical Instructions for Studying the Development of the Chick
OF all incubators, the natural one, i.e. the hen, is in some respects the best. The number of eggs which fail to develope is fewer than with an artificial incubator, and the development of monstrosities is rarer. A good sitter will continue to sit for thirty or more days at least, even though the eggs are daily being changed. She should never be allowed to want for water, and should be well supplied according to her appetite with soft food. It is best to place the food at some little distance from the eggs, in order that the hen may leave the eggs when feeding. She will sit most persistently in a warm, quiet, somewhat darkened spot. When an egg is placed under her, the date should be marked on it, in order that the duration of its incubation may be exactly known. When the egg is intended to remain for some time, e.g. for seven days or more, the mark should be bold and distinct, otherwise it will be rubbed off.
On the whole however we have found it more convenient to use a good artificial incubator. We have ourselves used with success two different incubators. One made by the Cambridge Scientific Instrument Company, and the other by Wiesnegg of 64, Rue Gay-Lussac, Paris (Fig. 65 in his catalogue for 1881). We have had the longest experience with the former, and have found it work exceedingly well : having been able to hatch chicks without more attention than now and then turning over the eggs.
Both these incubators consist essentially of a large water-bath fitted with a gas regulator. They are both perfectly automatic and when once regulated require no further attention.
The temperature within the incubator should be maintained at from 37 to 40C. A rise above 40 is fatal ; but it may be allowed to descend to 35 or in the young stages lower, without doing any further harm than to delay the development.
The products of the combustion of the gas should be kept as much as possible from the eggs, while ou supply of fresh air and of moisture is essential.
Tolerably satisfactory results may be obtained with an ordinary chemical double- jacketed drying water-bath, thoroughly covered in with a thick coat of cotton wool and flannel baize, and heated by a very small gas-jet. If the vessel be filled with hot water, and allowed to cool down to 40 or thereabouts, before the eggs are introduced, a very small gas flame will be sufficient to maintain the requisite temperature. A small pin-hole-nozzle, giving with ordinary pressure an exceeding narrow jet of flame about two inches high, is the most convenient. By turning the gas off or on, so as to reduce or increase the height of the jet as required, a very steady mean temperature may be maintained.
In the absence of gas, a patent night-light placed at a proper distance below the bath may be made to answer very well. When a body of water, once raised to the necessary temperature, is thoroughly surrounded with non-conducting material, a very slight constant amount of heat will supply all the loss.
B. On preparing sections of the embryo
a. Picric acid.
We find this reagent the most satisfactory for hardening the chick and in most instances mammalian embryos.
Klein enberg's solution of picric acid is the best.
With 100 parts of water, make a cold saturated solution of picric acid ; add to this two parts of concentrated sulphuric acid or nitric acid : filter and add to the filtrate three times its bulk of water.
In this solution of picric acid 1 the embryo must be placed and left for from 2 5 hours. It should then be washed in alcohol of 30 p.c. and placed in alcohol 50 p.c. for one hour. From this it must be removed into alcohol of 70 p.c. in which it should be left until all the picric acid is extracted ; to facilitate this the 70 p.c. alcohol should be frequently changed : when free from picric the embryo should be placed in 90 p.c. alcohol and kept there until required for further use.
1 It is sometimes advantageous to add to this solution of picric acid as much pure kreasote as it will dissolve (vide Kleinenberg, 'Development of Earthworm," Quarterly Journal of Mic. Sci. 1879).
2sT.B. Hardened embryos should always be kept in 90 p.c. spirit and only placed in absolute before imbedding, or staining with haematoxylin.
Some histologists prefer to keep hardened tissues in alcohol 70 p.c.
b. Corrosive sublimate.
Place the embryo in a large quantity of a saturated aqueous solution of corrosive sublimate to which a few drops of glacial acetic acid have been added, and allow it to remain for half-an-hour 1 . It is necessary thoroughly to extract the corrosive sublimate from the cells of the embryo ; to accomplish this, wash it thoroughly with water for from 10 minutes to 3 hours according to the size of the object. The washing may be limited to frequent changes of water or the embryo may be placed in a vessel through which a continuous stream of water is kept running. "When all the sublimate is removed, place it in 50 p.c. alcohol acidulated with nitric acid (half-a-dozen drops of acid to a 4 oz. bottle of spirit) for five minutes. The preservation of the embryo is completed by treating it with 70 p.c. alcohol for twenty-four hours and then keeping it in 90 p.c. alcohol. We have not found that corrosive sublimate gives such good results as picric acid in the case of chicks and mammalian embryos.
1 If there is only a small quantity of acetic acid mixed with the sublimate, a prolonged immersion will do the embryo no harm.
c. Osmic acid.
Osmic acid is a difficult reagent to use, but when properly applied it gives most excellent results.
It should be used as a weak solution ('I to 5 p.c.). The object should be left in it until it has acquired a light brown tint. The stronger the solution the less time is required for the production of this tint. It should then be removed and placed in picro- carmine, which arrests the action of the osmic and stains the embryo. The time required for the picro-carmine staining must be determined by practice. From the picro-carmine the object must be washed in 70 p.c. spirit; and then placed in 90, or may be preserved directly in glycerine.
If it is desired to use other staining agents (borax-carmine is good for some preparations), the object must be removed from osmic into water or weak spirit, thence through 50 into 70 p.c., stained, and passed through 70 to 90 p.c. spirit.
d. After using osmic it is well in some cases
(mammalian segmenting ova) to place the object in Miiller's fluid for 2 or 3 days, after which it may be preserved in glycerine or spirit. Miiller's fluid is made by dissolving 25 grms. of bichromate of potash and 10 grms. of sodic sulphate in 1000 cc. of water.
e. With chromic acid.
The embryo must be immersed in a solution of the strength of *1 p.c. for 24 hours. From this it should be removed and placed in a stronger solution (-3 p.c.) for another 24 hours. If it then appears sufficiently hard, it may be at once placed in alcohol of 70 p.c., in which it should remain for one day, and then be transferred to alcohol of 90 p.c.
f. Absolute alcohol has also been employed as
a hardening reagent, but is by no means so good as the reagents recommended above.
The object of these so-called hardening reagents is to kill the tissues with the greatest possible rapidity without thereby destroying them. The subsequent treatment with alcohol completes the hardening which is only commenced by these reagents.
There is room for the exercise of considerable skill in the use of alcohol, and this skill can only be acquired by experience. A few general rules may however be laid down.
(1) Tissues should not, generally, be changed from water or an aqueous solution of the first hardening reagent into an alcoholic solution of too great strength, nor should the successive solutions of alcohol used differ too much in strength. The distortion produced by the violence and inequality of the diffusion currents is thus diminished. This general rule should be remembered in transferring tissues from alcohol to the staining agents and vice versa.
(2) The tissues should not be left too long (more than one or two hours) in alcoholic solutions containing less than 70 p.c. of alcohol.
(3) They should not be kept in absolute alcohol longer than is necessary to dehydrate them (see B. 1, p. 426). The alcoholic solutions we generally use contain 30, 50, 70, 90 p.c. of alcohol.
In most cases it will be found of advantage to stain the embryo. The best method of doing this is to stain the embryo as a whole, rather than to stain the individual sections after they have been cut.
We have found hsematoxylin and boraxcarmine the best reagents for staining embryos as a whole.
a. With hsematoxylin.
The best solution of hsematoxylin, one for which we are indebted to Kleinenberg, is made in the following way.
(1) Make a saturated solution of crystallized calcium chloride in 70 p. a alcohol, and add alum to saturation.
(2) Make also a saturated solution of alum in 70 p.c. alcohol, and add 1 to 2 in the proportion of 1 : 8.
(3) To the mixture of 1 and 2 add a few drops of a saturated solution of hsematoxylin in absolute alcohol.
(4) It is often the case that hsematoxylin solution prepared in this way has not the proper purple tint ; but a red tint. This is due to acidity of the materials used. The proper colour can be obtained by treating it with some alkaline solution. "We have found it convenient to use for this purpose a saturated solution of sodium bi-carbonate in 70 p.c. spirit. (The exact amount must be determined by experiment, as it depends upon the amount of acid present.)
The embryo should be placed for some hours in absolute alcohol, before staining with haematoxylin, and should be removed directly from absolute into the haematoxylin.
The time required for staining varies with the size of the object and the strength of the staining fluid. Hsematoxylin will not stain if the embryo is not quite free from acid.
If the embryo is stained too dark, it should be treated with a solution of 70 p.c. alcohol acidulated with nitric acid (*25 p.c. of acid) until the excess of staining is removed; and in all cases the hsematoxylin staining is improved by treating the embryo with acidulated 70 p.c. alcohol.
After staining the embryo must be well washed in 70 and placed in 90 p.c. spirit.
b. With borax-carmine.
Make an aqueous solution of 2 to 3 p.c. carmine and 4 p.c. borax, by heating: add an equal volume of 70 p.c. alcohol, and let the mixture stand for thirty-six hours; after which carefully filter.
Stain the object thoroughly by leaving it in this solution for one or even two days; it will attain a dull maroon colour : transfer it then to acidulated alcohol (see a) until it becomes a bright red, and afterwards keep it as before in 90 p.c. alcohol.
This staining solution permeates more thoroughly and uniformly a large object than does hsematoxylin : therefore when a four or five day chick is to be stained, borax-carmine is the best staining reagent to use. Embryos that have been preserved in corrosive sublimate will be found to stain more thoroughly in this than in the hsematoxylin solution.
c. With carmine.
Beale's carmine or some alcoholic solution is the best. Into this the embryo may be removed directly from 90 p.c. alcohol, left for 24 hours, and then placed again in alcohol until required.
d. With picro-carmine.
This reagent is useful as will be seen later for staining mammalian segmenting ova and very young blastoderms ; it is used with the greatest success after hardening in osmic acid.
There are several methods of making picrocarmiue, the following is the simplest, and we have found it answer our purpose fairly well.
To a solution made up of 1 grm. of carmine 4 cc. of liquor ammonia and 200 cc. of distilled water add 5 grms. of picric acid; agitate the mixture for some minutes, and then decant, leaving the excess of acid.
The decanted fluid must remain for several days, being stirred up from time to time; eventually evaporated to dryness in a shallow vessel, and to every 2 grms. of the residue add 100 cc. of distilled water.
e. With alum carmine.
To make it, boil a strong aqueous solution of ammonia-alum with excess of carmine for 10 to 20 minutes, filter, and dilute the filtrate until it contains from 1 to 5 p.c. of alum. Add a few drops of carbolic acid to prevent the growth of fungus.
Well hardened tissues may be left in this aqueous solution for 24 hours. It is especially good for staining nuclei ; as a rule the staining is not diffuse, but it is necessary after staining to treat with acid alcohol (see a).
3. IMBEDDING AND CUTTING SECTIONS
It is not possible to obtain satisfactory sections of embryos without employing some method of imbedding, and using a microtome. Many imbedding solutions and methods of cutting sections have been used, but we find the following far superior to any other. It combines several advantages \ in the first place it renders it comparatively easy to obtain, what is so essential, a complete consecutive series of sections of the embryo ; and secondly, all the sections when mounted are in the same relative position ; and the various parts of each section retain their normal position with regard to each other.
The substance we prefer for imbedding is paraffin. As will be seen below it is necessary to have at hand paraffins of various melting points, according to the temperature of the room at the time when the sections are cut.
It will be found most convenient to obtain paraffins of the highest and lowest melting points and to mix them together as experience dictates.
Place the stained embryo in absolute alcohol until completely dehydrated (two hours is sufficient for small embryos) : and when ready to imbed soak it in turpentine 1 until it is completely saturated : and transfer it thence with as little turpentine as possible to a dish of melted paraffin.
In cases of very delicate tissues, it is better to use chloroform instead of turpentine. The chloroform should be carefully added by means of a pipette to the absolute alcohol in which the tissue is placed. The chloroform sinks to the bottom of the bottle or tube and the embryo, which at first lies at the junction of the two liquids, gradually sinks into the chloroform. When this is accomplished, remove all the absolute with a pipette and add pieces of solid paraffin to the chloroform. Gently warm this on a water bath till all the chloroform is driven off ; then imbed in the usual way.
Care must be taken that no more heat is used than is necessary to melt the paraffin ; for this purpose the paraffin should be warmed over a water bath the temperature of which is kept constant (from 50 to 60C. but not more than 60C.).
A paraffin melting at 44C. is of the proper consistency for cutting when the temperature of the room is 15C.
With care a porcelain evaporating dish and a gas flame may be made to answer, but the student is advised not to imbed without a water bath.
The embryo may be left in the paraffin two, three or more hours, after which it is imbedded by placing it along with the melted paraffin in either a box. made by bending up the sides and folding in the corners of a piece of stiff" paper, or what is better, a box formed by two L-shaped pieces of lead, placed on a glass slide in such a manner as to enclose a space, The latter is preferable because the object can be placed in any position required with great ease by moving it with a hot needle, and the whole can be cooled rapidly. It is advisable, at any rate at first, to arrange the embryo so as to cut it into transverse sections.
1 If the alcohol is not quite absolute kreasote should be used instead of turpentine.
When cool a block of paraffin is formed, in the midst of which is the embryo.
Other imbedding agents have been used. The best of these are, (1) pure cocoa butter ; (2) a mixture of spermaceti and castor oil or cocoa butter (4 parts of the former to one of the latter). With these imbedding substances, it is generally necessary to moisten the razor, either with olive oil or turpentine and ribbons of sections cannot be made (see b).
When the imbedding block is cold pare away the edges, then gradually slice it away until the end of the embryo is near the surface, and place it in a microtome.
The microtome we are most accustomed to is a ' sliding microtome' made by Jung of Heidelberg ; it gives excellent results. Recently however Messrs CaldweH and Threlfall have designed an automatic microtome which has been used with success at the Cambridge Morphological Laboratory and promises to effect a great saving of time and trouble in cutting sections (vide p. 471 and Proceedings of the Cambridge Phil. Soc. 1883). A convenient small microtome is one made by Zeiss of Jena (also by the Cambridge Scientific Instrument Company), in which the object is fixed and by means of a finely divided screw raised through a hole in a glass plate, across which a razor held in the hand is pushed. We will briefly describe the method of manipulation for the small microtome, it will be found easily applicable to Jung's sliding microtome.
The paraffin block is pared in such a manner that the edge nearest to the operator and that opposite to him are parallel. A dry razor is then pushed upon the glass plate over the hole through which the block of paraffin projects upwards, and a section cut which remains upon the razor. Care must be taken that the edge of the razor is parallel to the parallel edges of the paraffin block. The block having been raised by the screw, a second section is made in the same way and on the same part of the razor as the first ; in consequence of which, the first section will be pushed backwards by the second. Similarly each new section pushes backwards those already made ; and a ribbon of sections formed which, if the paraffin is of the right consistency, will adhere firmly together.
Experience must teach the manipulator how to mix the paraffin in such a manner that it is neither too hard nor too soft ; if it is too hard, the sections will not adhere together and will curl up on the razor, if too soft they will stick to the razor and be found to be creased. When it is not possible to keep the temperature of the room constant it will be found convenient to use a hard paraffin, and when necessary to raise the temperature by means of a lamp.
The paraffin should completely surround the embryo and fill up all the spaces within it.
c. Mounting sections.
When the sections are cut, place them in rows on a slide prepared in the following manner. Make a solution of white shellac in kreasote by heating, and let it be of the consistency of glycerine, or slightly more fluid. With a camel's hair-brush paint a very thin and uniform layer of this gum over the slide which must be clean and dry, and while the gum is wet place the sections in rows upon it. Now place the slide on a water bath which is heated up to the melting point of the paraffin. The sections sink down into the thin layer of shellac and kreasote, the kreasote slowly evaporates and the shellac becoming hard fixes the section in the position in which it was placed on the slide. When the kreasote has been evaporated, pour turpentine carefully upon the slide, this dissolves the paraffin and clears the sections which may at once be mounted in Canada balsam.
A turpentine or chloroform solution of Canada balsam should be used.
This method of cutting ribbons of sections was first introduced by Mr Caldwell, to whom we are also indebted for the account given above for mounting sections (vide Note B, p. 471). The latter however is a modification and improvement of Dr Giesbrecht's method. (Zoologischer Anzeiger No. 92, 1881.)
C. Preservation of the embryo as a whole.
Chick embryos of the first or second day may be easily preserved whole as microscopic objects. For this purpose, the embryo, which has been preserved in the ordinary way (B, a) should be stained slightly, dehydrated, soaked in oil of cloves until transparent and mounted in balsam.
Whole embryos of a later date cannot be satisfactorily preserved as microscopic objects.
PRACTICAL DIRECTIONS FOR OBTAINING AND STUDYING CHICK EMBRYOS
II. Examination of a 36 to 48 hours 1 embryo.
The student will find it by far the best plan to begin with the study of an embryo of this date. The manipulation is not difficult ; and the details of structure are sufficiently simple to allow them to be readily grasped. Earlier embryos are troublesome to manage until some experience has been gained; and the details of later ones are so many as to render it undesirable to begin with them.
A. Opening tlie Egg
Take the egg warm from the hen or the incubator, and place it (it does not matter in what position, since the blastoderm will at this stage always be found at the uppermost part of the egg) in a small basin large enough to allow the egg to be covered with fluid. It is of advantage, but not necessary, to place at the bottom of the basin a mould, e.g. a flat piece of lead with a concavity on the upper surface, in which the egg may rest securely without rolling. Pour into the basin so much of a '75 per cent, solution of sodium chloride warmed to 38C. as will cover the egg completely. With a sharp tap break through the shell at the broad end over the air-chamber, and let out as much air as has already been gathered there. Unless this is done, the presence of air in the air-chamber will cause the broad end to tilt up. At this date there will be very little air, but in eggs of longer incubation, inconvenience will be felt unless this plan be adopted.
Instead of being broken with a blow, the shell may be filed through at one point, and the opening enlarged with the forceps; but a little practice will enable the student to use the former and easiermethod without doing damage.
With a blunt pair of forceps, remove the shell carefully bit by bit, leaving the shell-membrane behind; begin at the hole made at the broad end, and work over the upper part until about a third or half of the shell has been removed.
Then with a finer pair of forceps remove the shell-membrane; it will readily come away in strips, torn across the long axis of the egg in a somewhat spiral fashion. The yolk and embryo will now come into view.
It is the practice of some simply to break the egg across and pour the yolk and white together into a basin, very much as the housewife does. We feel sure, however, that the extra trouble of the method we have given will be more than repaid by the results.
During this time, and indeed during the whole period of the examination of the embryo in situ, the basin and its contents must be maintained, either by renewal of the salt solution, or by the basin being placed on a sand-bath, at about 38C.
B. Examination of the blastoderm in situ
This may be done with the naked eye, or with a simple lens of low power. Observe :
- Lying across the long axis of the egg, the pellucid area, in the middle of which the embryo may be obscurely seen as a white streak.
- The mottled vascular area, with the blood-vessels just beginning to be formed.
- The opaque area spreading over the yolk with the changes in the yolk around its periphery.
- (With a simple lens), the contractions of the heart; perhaps the outlines of the head of the embryo may be detected.
C. Removal of the embryo
Plunge one blade of a sharp fine pair of scissors through the blastoderm, just outside the outer margin of the vascular area, and rapidly carry the incision completely round until the circle is complete, avoid as much as possible any agitation of the liquid in the basin.
With a little trouble, the excised blastoderm may now be floated into a watch-glass, care being taken to keep it as flat as possible. With a pair of forceps or with a needle, aided by gentle shaking, remove the piece of vitelline membrane covering the blastoderm.
If any yolk adheres to the blastoderm, it may with a little gentle agitation easily be washed off. Sometimes it is of advantage to suck up the yolk with a glass syringe, replacing the fluid removed with clean ('75 p.c.) salt solution.
The blastoderm should now be removed from the watch-glass to a microscopic glass slide ; since it is difficult in the former to prevent the edges of the blastoderm from curling up.
The transference may easily be effected, if both the watch-glass and slide are plunged into a basin of clean warm salt solution. With a little care, the blastoderm can then be floated from the one to the other, and the glass slide, having the blastoderm with its upper surface uppermost spread flat upon it, very gently raised out of the liquid.
A thin ring of putty may now be placed round the blastoderm, a small quantity of salt solution gently poured within the ring, and the whole covered with a glass slide, which may be pressed down until it is sufficiently close to the embryo. The presence of any air-bubbles must of course be avoided.
Provided care be otherwise taken to keep the embryo well covered with liquid, the putty ring and the coverslip may be dispensed with. They are often inconvenient, as when the embryo has to be turned upside down.
The object is now re&dy for examination with a simple lens or with a compound microscope of low objective. It is by far the best for the student to begin at least with the simple lens. In order that everything may be seen at its best, the slide should be kept warmed to about 38, by being placed on a hot stage.
=D. Surface view of the transparent embryo from above
The chief points to be observed are :
- The head-fold.
- The indications of the amnion; especially the false amnion, or outer amniotic fold.
- The neural tube : the line of coalescence of the medullary folds, the first cerebral vesicle, the commencing optic vesicles, the indications of the second and third cerebral vesicles, the as yet open medullary folds at the tail end.
- The heart seen dimly through the neural tube; note its pulsation if present.
- The fold of the somatopleure anterior to the heart (generally very faintly shewn).
- The fold of the splanchnopleurt (more distinctly seen) : the vitelline veins.
- The mesoblastic somites.
- Indications of the vitelline arteries.
- The as yet barely formed tail-fold.
- The commencing blood-vessels in the pellucid and vascular areas.
E. Surface view of the transparent embryo from below
The coverslip must now be removed and the glass slide again immersed in a vessel of clean salt solution. By gently seizing the extreme edge of the opaque area with a pair of forceps, no difficulty will be found in so floating the blastoderm, as to turn it upside down, and thus to replace it on the slide with the under surface uppermost.
The points which most deserve attention in this view, are :
- The heart : its position, its union with the vitelline veins, its arterial end.
- The fold of the splanchnopleure marking the hind limit of the gut ; the vitelline veins running along its wings.
- The mesoblastic somites on each side of the neural canal behind the heart; farther back still, the vertebral plates not divided into somites.
F. The examination of the embryo as an opaque object
This should never be omitted. Many points in the transparent embryo only become intelligible after the examination of it as an opaque object.
Having removed the putty ring and coverslip, if previously used, allow the blastoderm so far to become dry, that its edge adheres to the glass slide. Care must of course be taken that the embryo itself does not become at all dry. Place the glass slide with the blastoderm extended flat on it, in a shallow vessel containing a solution of picric acid (I. B.).
If the blastoderm be simply immersed by itself in the picric acid solution, the edges of the opaque area will curl up and hide much of the embryo. The method suggested above prevents these inconveniences.
The embryo thus hardened and rendered opaque by immersion in the acid (a stay of 2 to 3 hours in the solution will be sufficient) may be removed to a watch-glass, containing either some of the solution, or plain water, and examined with a simple lens, imder a strong direct light. The compound microscope will be found not nearly so advantageous for this purpose as the simple lens. A piece of black paper placed under the watch-glass, will throw up the lights and shadows of the embryo, with benefit. The watchglass should have a flat bottom; or a shallow flat glass cell should be used instead.
a. Looking at the embryo from above, observe :
- The head-fold ; the head distinctly projecting from the plane of the blastoderm, and formed chiefly by the forebrain and optic vesicles.
- The elevation of the medullary canal, and the indications of the side walls of the embryo.
- The indications of the tail.
- The Amnion partly covering the head. Tear it open with needles. Observe its two folds.
b. Having turned the blastoderm upside down, observe the following points, looking at the embryo from below.
- The hinder limit of the splanchnopleure in the head-fold, marking the hind limits of the foregut. The opaque folds now conceal the head almost entirely from view.
- The commencing tail-fold, and the shallow boatshaped cavity (of the alimentary canal) between it and the head-fold.
The student should not fail to make sketches of the embryo, both as a transparent, and as an opaque object, seen from below as well as from above. These sketches will be of great service to him when he comes to study the sections of the same embryo.
G. The following transverse sections will perhaps be the most instructive
Manipulation as in I. B. 3.
- Through the optic vesicles, shewing the optic stalks.
- Through the hind-brain, shewing the auditory sacs.
- Through the middle of the heart, shewing its relations to the splanchnopleure and alimentary canal.
- Through the point of divergence of the splanchnopleure folds, shewing the venous roots of the heart.
- Through the dorsal region, shewing the medullary canal, mesoblastic somites and commencing cleavage of the mesoblast.
- Through a point where the medullary canal is still open, shewing the mode in which its closing takes place.
Longitudinal sections should also be made and compared with the transverse sections.
III. Examination of an Embryo of about 48-50 hours.
A. Opening the egg as in II. A.
B. Examination of the blastoderm in situ. Observe
- Thejform of the embryo, which is much more distinct than at the earlier stage.
- The beating of the heart.
- The general features of the circulation.
C. Removal of the Embryo from the yolk, as in II. C.
D. Surface view of the transparent embryo from
above. Notice :
- General form of the embryo. a. Commencing cranial flexure. b. The tail and side folds.
- Amnion. Notice the inner and outer (false amnion) limbs and remove them with a needle. When the amnion has been removed the features of the embryo will be much more clearly visible.
- The organs of sense. a. Eye. Formation of the lens already nearly completed. b. Auditory involution, now a deep sac with a narrow opening to the exterior.
- The brain. a. The vesicles of the fore-, mid-, and hind-brsiui. b. The cerebral vesicle. c. The cranial flexure taking place at the midbrain.
E. Transparent embryo from below. Manipulation as in II. E.
- The increase of the head-folds of the somatopleure and splanchnopleure, especially the latter, and the commencement of these folds at the tail.
- The now as-shaped heart ; for further particulars vide Chap. iv.
- The commencing 1st and 2nd visceral clefts arid the aortic arches.
- The circulation of the yolk sac, vide Fig. 36. Make out all the points there shewn and ascertain by examination that what have been called the veins and arteries in that figure, are truly such.
F. The embryo as an opaque object, Treatment as in. II. F.
FROM ABOVE :
Observe the amnion, which is a very conspicuous object, and remove it with needles if not done previously. The external form of the brain and the auditory sac appear very distinctly. FROM BELOW :
Observe the nature of the head- and tail-folds, which are much more easily understood from the opaque than from the transparent embryos.
Observe also the alimentary canal, the widely open hind end of the fore-gut, and the front end of the as yet very short hind-gut.
G. Sections. Manipulation as in I. B. 3.
The more important sections to be observed, are 1 . Through optic lobes, shewing :
a. The formation of the lens.
b. The involution of the primary optic vesicle.
c. The constriction, especially from above, of the optic stalk.
2. Through auditory sac, shewing :
a. Auditory sac still open.
b. The thin roof and thick sides of the hind-brain.
e. Closed alimentary canal.
3. Through dorsal region, shewing the general appearance of a section of an embryo at this stage, which should be compared with a similar section of the earlier stage.
It shews :
a. The commencement of the side folds; the alimentary canal still however open below.
b. The "Wolffian duct lying close under the epiblast on the outside of the mesoblastic somites.
c. The notochord with the aortse on each side.
IY. Examination of an Embryo at the end of the third
A. Opening the egg, as in II. A.
B. Examination of the blastoderm in situ. Observe :
- The great increase of the vascular area both in size and distinctness. The circulation is now better seen in situ than after the blastoderm has been removed.
- That the embryo now lies completely on its left side and that it is only connected with the yolk-sac by a somewhat broad stalk.
C. Removal of the embryo. See II. C.
It is now unnecessary to remove the whole of the blastoderm with the embryo ; indeed it is better to cut away the vascular area unless it is wanted for examination.
D. Surface view of the transparent embryo.
Since the embryo now lies on its side we shall not have to speak of the view from above and below. The views from the two sides differ chiefly as to the appearance of the heart.
The embryo (freed from the blastoderm and the amnion) is to be floated on to a glass slide in the usual way. It is necessary to protect it while under examination, with a coverslip, which must not be allowed to compress it. To avoid this, we have found it a good plan to support the coverslip at one end only, since by moving it about when thus supported, a greater or less amount of pressure can be applied at will to the object.
The details which can at this stage be seen in a transparent embryo are very numerous and we recommend the student to try and verify everything shewn in Fig.* 37. Amongst the more important and obvious points to be noticed are
- The increase of the cranial flexure and the bodyflexure.
- The condition of the brain. The mid-brain now forms the most anterior point of the head.
The fore-brain consists of the inconspicuous vesicle of the third ventricle and the two large cerebral lobes.
The hind-brain consists of a front portion, the cerebellum with a thickened roof; and a hinder portion, the fourth ventricle with a very thin and delicate roof.
3. Organs of sense.
The eye especially is now in a very good state to observe. The student may refer to Fig. 51, and the description there given.
The ear-vesicle will be seen either just closing or completely closed.
4. In the region of the heart attention must also be paid to :
a. The visceral clefts.
b. The investing-mass, Le. the growth of mesoblast taking place around the end of the notochord.
c. The condition of the heart.
5. In the region of the body the chief points to be observed are :
a. The increase in the number of the somites.
b. The Wolfflan duct, which can be seen as a streak along the outer side of the hinder somites.
c. The attantois, which is now a small vesicle lying between the folds of the somatopleure and splanchnopleure at the hind end of the body, but as yet hardly projects beyond the body cavity.
E. The embryo as an opaque object. Preparation as in II. F.
The general form of the embryo can be very satisfactorily seen when it is hardened and examined as an opaque object; but the most important points to be made out at this stage in the hardened specimens are those connected with the visceral clefts and folds and the mouth.
If the amnion has not been removed it will be necessary to pick it completely away with needles. Without further preparation a view of the visceral folds and clefts may be obtained from the side ; but a far more instructive view is that from below, in order to gain which the following method may be adopted.
Pour a small quantity of melted black wax (made by mixing together lampblack and melted wax) into a watch-glass, using just enough to cover the bottom of the glass. While still soft make a small depression in the wax with the rounded end of a pen-holder or handle of a paint-brush and allow the wax to cool. In the meantime cut off the head of the hardened embryo by a sharp clean transverse incision carried just behind the visceral clefts, transfer it to the watch-glass and cover it with water or spirit. By a little manipulation the head of the embryo may now be shifted into the small depression in the wax, and thus be made to assume any required position. It should then be examined with a simple lens under a strong reflected light, and a drawing made of it.
When the head is placed in the proper position, the following points may easily be seen.
- The opening of the mouth bounded below by the first pair of visceral folds, and commencing to be enclosed above by the now very small buds which are the rudiments of the superior maxillary processes. Compare Fig. 56.
- The second and third visceral arches and clefts.
- The nasal pits.
F. Sections. Manipulation as in I. B. 3. The most important sections are :
- Through the eyes in the three planes, vide Fig. 50, A. B. C.
- Through the auditory sac.
- Through the dorsal region, shewing the general changes which have taken place.
Amongst these, notice
a. The changes of the mesoblastic somites: the commencing formation of the muscle -plates.
b. The position of the Wolffian duct and the formation of the germinal epithelium.
c. The aortce and the cardinal veins.
d. The great increase in depth and relative diminution in breadth of the section.
V. Examination of an Embryo of the Fourth Day.
A. Opening the egg, as in II. A.
Great care will be required not to injure the embryo, which now lies close to the shell-membrane.
B. Examination in situ. Observe:
- The now conspicuous amnion.
- The allantois, a small, and as yet hardly vascular vesicle, beginning to project from the embryo into the space between the true and the false anmion.
- The rapidly narrowing somatic stalk.
C. Removal of the embryo, as in II. C. and IV. C.
The remarks made in the latter place apply with still greater force to an embryo of the fourth and succeeding days.
D. Surface mew of the transparent embryo. For manipulation, vide IV. D.
The points to be observed are :
- The formation of the fifth, seventh, and ninth cranial nerves. To observe these, a small amount of pressure is advantageous.
- The formation of the fourth visceral cleft, and the increase in size of the superior maxillary process.
- The formation of the nasal pits and grooves.
- The great relative growth of the cerebral lobes and the formation of the pineal gland from the roof of the vesicle of the third ventricle.
- The great increase in the investing mass.
- The formation and growth of the muscle-plates, which can now be easily seen from the exterior.
- The allantois. Make out its position and mode of opening into the alimentary canal.
E. The embryo as an opaque object. Manipulation as II. F. For mode of examination vide IV. E.
The view of the mouth from underneath, shewing the nasal pit and grooves, the superior and inferior maxillary processes and the other visceral folds and clefts, is very instructive at this stage. Compare Fig. 69.
F. Sections. Manipulation as in I. B. 3. The most important sections are,
- Through the eyes.
- Transverse section immediately behind the visceral arches, shewing the origin of the lungs.
- Transverse section just in front of the umbilical stalk, shewing the origin of the liver.
- Transverse section at about the centre of the dorsal region, to shew the general features of the fourth day. Compare Fig. 68.
Amongst the points to be noticed in this section, are
b. Spinal nerves and ganglia.
c. Wolffian duct and bodies.
d. Miiller's duct.
f. Commencing changes in the spinal cord.
5. Section passing through the opening of the allantois into the alimentary canal.
For the points to be observed in embryos of the fifth and sixth days, the student must consult the chapters devoted to those days.
In the hardened specimens, especial attention should be paid to the changes which take place in the parts forming the boundaries of the mouth.
VI. Examination of a Blastoderm of 20 hours.
A. Opening the egg, as in II. A.
B. Examination in situ.
It will not be found possible to make out anything very satisfactory from the examination of a blastoderm in situ at this age. The student will however not fail to notice the halones, which can be seen forming concentric rings round the blastoderm.
C. Removal of the embryo.
Two methods of hardening can be adopted at this age. One of these involves the removal of the blastoderm from the yolk, as in II. C. In the other case, the yolk is hardened as a whole. If the latter method be employed, the embryo cannot be viewed as a transparent object.
In the cases where the blastoderm is removed from the yolk, the manipulation is similar to that described under II. C, with the exception of more care being required in freeing the blastoderm from the vitelline membrane.
D. Surface view transparent, from above. Observe :
- The medullary groove between the two medullary folds, whose hind ends diverge to enclose between them the end of the primitive groove.
- The head-fold at the end of the medullary groove.
- The one or two pairs of mesoblastic somites flanking the medullary groove.
- The notochord as an opaque streak along the floor of the medullary groove.
E. Surface view transparent \ from below.
Same points to be seen as from above, but less clearly.
F. Embryo as an opaque object.
As an opaque object, whether the embryo is hardened in situ or after being removed from the yolk, the same points are to be seen as when it is viewed as a transparent object, with the exception of the notochord and mesoblastic somites (vide D). The various grooves and folds are however seen with far greater clearness.
Two methods of hardening may be employed ; (1) with the embryo in situ, (2) after it has been removed.
To harden the blastoderm in situ the yolk must be hardened as a whole. After opening the egg either leave the yolk in the egg-shell or pour it out into a Berlin capsule ; in any case freeing it as much as possible from the white, and taking especial care to remove the more adherent layer of white which immediately surrounds the yolk.
Place it in picric acid or a weak solution of chromic acid (first of '1 p.c. and then of '5 p.c.) with the blastoderm uppermost and leave it in that position for two or three days.
Care must be taken that the yolk does not roll about ; the blastoderm must not be allowed to alter its position : otherwise it may be hard to find it when everything has become opaque. If at the end of the second day the blastoderm is not sufficiently hard, the strength of the solution, if chromic acid be used, should be increased and the specimen left in it for another day.
After it has become hardened by the acid, the yolk should be washed with water and treated suecessively with weak and strong spirit, vide I. B. After it has been in the strong spirit (90 p.c.) for two days, the vitelline membrane may be safely peeled off and the blastoderm and embryo will be found in situ. The portion of the yolk containing them must then be sliced off with a sharp razor, and placed in absolute alcohol.
The staining, <fec. may be effected in the ordinary way.
If osmic acid, which we believe will be found serviceable for these ear]y stages, is employed, it will be necessary to remove the blastoderm from the yolk before treating it with the reagent.
The following transverse sections are the most important at this stage :
1. Through the medullary groove, shewing
a. The medullary folds with the thickened mesoblast.
b. The notochord under the medullary groove.
c. The commencing cleavage of the mesoblast.
2. Through the region where the medullary folds diverge, to enclose the end of the primitive groove, shewing the greatly increased width of the medullary groove, but otherwise no real alteration in the arrangement of the parts.
3. Through the front end of the primitive groove with the so-called axis cord underneath it, while on each side of it are still to be seen the medullary folds.
4. Through the primitive groove behind this point, shewing the typical characters of the primitive groove.
VII. Examination of an unincubated Blastoderm.
A. Opening the egg. Vide II. A.
B. Examination of the blastoderm in situ.
Observe the central white spot and the peripheral more transparent portion of the blastoderm and the halones around it.
C. Removal of the blastoderm. Vide VI. C.
With the unincubated blastoderm still greater care is required in removal than with the 20 hours' blastoderm, and there is no special advantage in doing so unless it is intended to harden it with osmic acid.
D. Surface view transparent from above. Observe the absence of the central opacity.
E. Surface view transparent from underneath. Nothing further to be observed than from above.
F. As an opaque object.
There is nothing to be learnt from this.
G. Sections. Manipulation as in VI. G. The sections shew
a. The distinct epiblast.
b. The lower layer cells not as yet differentiated into mesoblast and hypoblast.
c. The thickened edge of the blastoderm.
d. The segmentation cavity and formative cells.
VIII. Examination of the process of Segmentation.
To observe the process of segmentation it will be found necessary to kill a number of hens which are laying regularly. The best hens lay once every 24 hours, and by observing the time they usually lay (and they generally lay pretty regularly about the same time), a fair guess may be made beforehand as to the time the egg has been in the oviduct. By this means a series of eggs at the various stages of segmentation may usually be obtained without a great unnecessary sacrifice of hens. For making sections, the yolk must in all cases be hardened as a whole, which may be done as recommended in VI. G. Chromic acid is an excellent reagent for this and it will be found very easy to make good sections.
In the sections especial attention should be paid,
- To the first appearance of nuclei in the segments, and their character.
- To the appearance of the horizontal furrows.
- As to whether new segments continue to be formed outside the limits of the germinal disc, or whether the fresh segmentation merely concerns the already formed segments.
- In the later stages, to the smaller central and larger peripheral segments, both containing nuclei.
For surface views, the germinal disc, either fresh or after it has been hardened, can be used. In both cases it should be examined by a strong reflected light. The chief point to be noticed is the more rapid segmentation of the central than of the peripheral spheres.
IX. Examination of the later changes of the Embryo.
For the later stages, and especially for the development of the skull and the vascular system of the body of the chick, it will be found necessary to dissect the embryo. This can be done either with the fresh embryo or more advantageously with embryos which have been preserved in spirit.
If the embryos are placed while still living into spirit a natural injection may be obtained. And such an injection is the best for following out the arrangement of the blood-vessels.
Sections of course will be available for study, especially when combined with dissections.
X. Study of the development of the Blood-vessels.
Observations on this subject must be made with blastoderms of between 30 40 hours. These are to be removed from the egg, in the usual way (vide II. A. and C.), spread out over a glass slip and examined from below, vide II. E.
The blastoderm when under examination must be protected by a coverslip with the usual precautions against pressure and evaporation, and a hot stage must also be employed.
Fresh objects so prepared require to be examined with a considerable magnifying power (400 to 800 diameters). From a series of specimens between 30 and 40 hours old all the points we have mentioned in Chapter iv. p. 92, can without much difficulty be observed.
Especial attention should be paid in the earlier specimens to the masses of nuclei enveloped in protoplasm and connected with each other by protoplasmic processes; and in the later stages to the breaking up of these masses into blood corpuscles and the conversion of the protoplasmic processes into capillaries, with cellular walls.
Blastoderms treated in the following ways may be used to corroborate the observations made on the fresh ones.
With gold chloride.
Immerse the blastoderm in gold chloride (-5 p.c.) for one minute and then wash with distilled water and mount in glycerine and examine.
By this method of preparation, the nuclei and protoplasmic processes are rendered more distinct, without the whole being rendered too opaque for observation.
The blastoderm after the application of the gold chloride should become a pale straw colour; if it becomes in the least purple, the reagent has been applied for too long a time.
With potassium bichromate.
Immerse in a 1 p.c. solution for one day and then mount in glycerine.
With osmic acid.
Immerse in a *5 p.c. solution for half an hour and then in absolute alcohol for a day, and finally mount in glycerine.
APPENDIX - PRACTICAL DIRECTIONS FOR OBTAINING AND STUDYING MAMMALIAN EMBRYOS
XI. Animals and breeding.
For class work the Rabbit is the most convenient animal from which to obtain embryos, it will breed freely in the early spring months of the year and will give ample opportunity for the student to observe the exact time when the female is covered. A number of does should be kept together in a large pen, and two or three bucks in separate small cages also placed within the pen ; at the period of heat, the doe should be temporarily placed with the buck and the exact time of copulation noted, the age of the embryo being calculated from that hour.
XII. Examination of segmenting ova.
It will be well to mention here that although a doe may have been satisfactorily covered, embryos are not always obtained from her. A superficial examination of the ovaries will determine whether or no fertilized ova are present. If ova have been recently dehisced from the ovary, the Graafian follicles from which they were discharged will be found to be of a distinctly red colour. In case no such ' corpora lutea ' as they are called are present further search is useless.
A. To obtain ova from i to 60 hours old.
Cut open the abdomen from pubis to sternum, and from the pubis round the thigh of each side, and turn back the flaps of the body wall so formed. Remove the viscera and observe below (dorsal) the single median vagina, from the anterior end of which the uterine horns diverge.
Observe at the anterior end of each uterine horn a small much coiled tube, the oviduct (Fallopian tube) near the anterior end of which a little below the kidney lies the ovary. Cut out the uterus and oviduct together and lay them in a small dissecting dish. Carefully stretch out the oviduct by cutting the tissue which binds it, and separating it from the uterus, taking care to obtain its whole length, lay it upon a glass slide.
With the aid of a lens it is frequently possible to distinguish the ovum or ova, through the wall of the oviduct. In this case cut a transverse slit into the lumen of the duct with a fine pair of scissors a little to one side of an ovum ; press with a needle upon the oviduct on the other side of the ovum, which will glide out through the slit, and can be with ease transported upon the point of a small scalpel, or what is better spear-headed needle. In case the ovum cannot be distinguished in the oviduct by superficial observation, the latter must be slit up with a fine pair of scissors, when it will easily be seen with the aid of an ordinary dissecting lens.
B. Treatment of the ovum.
The ovum may be examined fresh in salt solution, it is however more instructive when preserved and stained in the following manner.
a. Immerse it in a \ p.c. solution of osmic acid for 5 or even 10 minutes, transfer it thence to the picrocarmine solution described above (I). After staining the ovum should then be washed in distilled water and placed in a weak solution of glycerine in a watch-glass half glycerine, half water. It should be allowed to remain thus under a bell jar for several days (7 to 14 or longer) in a warm room until the water has evaporated. By this means shrinkage and distortion are avoided, the glycerine becoming very gradually more and more dense. It should be mounted in glycerine in which 1 p.c. formic acid has been mixed to prevent fungoid growths. Care must be taken that there is no pressure upon the ovum this being insured by the insertion of a couple of slips of paper one on each side of the ovum under the cover glass. b. Another method of preservation is used, but does not appear to us so successful as the one already described. It consists of an immersion of the ovum for 5 minutes in i to J p.c. osmic acid, subsequent treatment with Mtiller's fluid for two or three days, and finally mounting in glycerine.
C. Examination of the ovum.
The most instructive stages to observe are ova of
a. 18 hours old, when four segmentation spheres will be observed.
b. 36 hours old when the segmentation is more advanced and the spheres numerous.
The chief points to be noted are :
- The number and size of the segmentation spheres; in each of which, when treated as described in B. a., a large deeply stained nucleus will be visible. The spheres themselves are also stained slightly.
- The presence of one or two polar bodies on the outer side of the segments in ova of not more than 48 hours old: these also are slightly stained.
- The zona radiata immediately surrounding the segments, and
- The thick albuminous coat, marked with concentric rings.
D. The fully segmented ovum. 70 hours old.
The fully segmented ovum is found in the uterus at its anterior end close to the place where the oviduct opens into the uterus.
To obtain this stage the uterus must be slit open and examined carefully with a dissecting lens : the ovum will be seen as a somewhat opaque spot on the glistening moist mucous epithelium of the uterus.
It may be treated in the manner described under B. a., but the segments being closely pressed together their outlines are not rendered distinct by this method. A more advantageous mode of treatment is the following : wash the ovum rapidly in distilled water, and place it in a 1 p.c. solution of silver nitrate for about 3 minutes : then expose it to the light in a dish of distilled water until it be tinged a brown colour.
The brown colour is due to the reduction of the silver, which takes place chiefly in the cement substance between the cells and thus defines very exactly their size and shape. The ovum may now be treated with glycerine and mounted as described in B.
The points to be observed are :
- The division of the segmentation spheres into the layers an outer layer of cubical hyaline cells, and an inner of rounded granular cells.
- The blastopore of van Beneden.
- The presence of a thin layer of mucous outside the concentrically ringed albuminous coat of the ovum.
XIII. Examination of the blastodermic vesicle, 72 90 hours.
A. To obtain tJie embryo see XII. D.
B. Prepare the ovum either as in XII. B. or D.
or in picric acid see I. B. i.
C. Surface view, or in section see I. B. 3. Observe :
- The great increase in size of the ovum and the reduction in the thickness of the membranes.
- The flattened layer of outer cells enclosing a cavity.
- The rounded cells of the inner mass attached as a
lens-shaped mass to one side of the vesicle.
XIV. Examination of a blastodermic vesicle of 7 days, in which the embryonic area and primitive streak are present.
A. To obtain the embryo.
On opening the body cavity the uterus will be found to be uniformly swollen and very vascular.
Remove the uterus and open it carefully with fine scissors along the free, non-mesometric edge, taking care to keep the point of the scissors within the uterus close against its wall.
- The oval thin-walled vesicles lying at intervals on the walls of the uterus.
- The presence of the pyriform embryonic area, at the posterior end of which is seen the primitive streak.
- The commencement of the area vasculosa around the hind end of the area. This is seen better after treatment with picric acid.
- Treatment and Examination of the embryo.
- Preserve the vesicle in picric see I. B. 1. Stain in haematoxylin, cut out the embryonic area, leaving a considerable margin, imbed and cut into sections.
b. In transverse sections observe :
- At the anterior end of the area the single row of columnar epiblast and the single row of flattened hypoblast cells.
- Immediately in front of the primitive streak between these two layers a few irregularly shaped mesoblast cells.
- Through the middle of the primitive streak,
a. Several ] ay ers of rounded mesoblast cells attached to, and continuous with, the epiblast in the middle line, and stretching out laterally beyond the edge of the area.
b. A single layer of flattened hypoblast.
4. The epiblast outside the embryonic area in the form of flattened cells and, except in the region around the primitive streak, overlying a layer of flattened hypoblast.
XV. Examination of an eight days 7 embryo.
A. To obtain the embryo.
The uterus will be found here and there to be swollen. In these swellings the embryos lie; and owing to the fact that the wall of the embryonic vesicle is exceedingly thin, and attached to the uterine wall, they are very difficult to obtain whole.
Cut the uterus transversely on each side of the swellings and pin the pieces so obtained slightly stretched out in small dissecting dishes. Cover the tissue with picric acid solution and allow it to remain untouched for an hour. Then with two pairs of fine pointed forceps carefully tear the uterus longitudinally, slightly to one side of the median line of the free side. This operation will necessarily take some time, for but a small portion should be done at once, the picric acid being allowed time to penetrate into that part of the uterus which has been most recently torn open.
With care, however, the student will be able to open completely the swelling and will observe within the thin walled vesicle. Great care must also be exercised in freeing the vesicle from the uterus.
This dissection should be performed with the aid of a dissecting lens. In case the embryonic vesicle is burst it will still be possible to extract the embryonic area which lies on the mesometric side of the uterus ; the area itself is not attached to the uterine walls.
B. Examination of surf ace view. Observe :
- The increased size of the embryonic area.
- In the anterior region the medullary folds; diverging behind and enclosing between them,
- The primitive streak.
- The area opaca now completely surrounding the embryo.
C. Examination of sections.
Prepare and cut into transverse sections as advised in XIY. B.
1. In the sections of the anterior region, (L The lateral epiblast composed of several layers of columnar cells.
b. The epiblast in the median line one layer thick and in the form of a groove (medullary groove).
c. The lateral plates of mesoblast.
d. The flattened lateral hypoblast, and columnar hypoblast underlying medullary groove (notochord).
2. In sections through the anterior end of the primitive streak.
Note the continuation of the epiblast, mesoblast and hypoblast in the middle line.
3. In sections through the posterior end of the area the same points to be seen as in XIV. B. b. 3.
XVI. Examination of an embryo about 8 days 12 hours.
A. Manipulation as in XV. A.
B. In surface view observe (cf. Fig. 106) :
- Area pellucida surrounding embryo, outside which is the well marked area vasculosa.
- Widely open neural canal, at anterior end dilated, and partially divided into the three primary vesicles of the brain : note the optic vesicles. At the posterior end, the sinus rhomboidalis.
- The mesoblastic somites, 4 to 8.
- The two lateral tubes of the heart, and the commencement of the two vitelline veins.
- The rudiment of the primitive streak.
- The commencing head and tail folds.
- The commencing folds of the amnion. Compare Fig. 106.
XVII. Examination of the foetal membranes of an embryo of 14 days.
A. To obtain t/ic embryo, with its membranes.
Manipulate as in XV. A. only dissect under salt solution instead of picric acid.
B. Observe before removing tJie embryo from the
1. The attachment of the vesicle to the mesometric side of the uterus over a discoidal area, the placental area.
2. The position and form of the placenta.
C. Remove the embryo with its membranes intact, and observe :
1. the vascular yolk sac, extending completely round the chorion with the exception of a comparatively small area where
2. the allantois is situated. The vascularity of the allantois. The foetal villi projecting into the maternal placental tissue.
D. Separate the membranes from one another without tearing them,
and notice :
- The embryo surrounded by the amnion.
- The allantois; its position dorsal to the embryo; its attachment to the chorion ; its circulation.
- The flattened yolk sac, ventral to the embryo ; its long stalk; its circulation.
- The heart.
E. The embryo in surface view. The points to be observed are
- The cranial and body flexure, the spiral curvature of the hinder portion of the body.
- The vesicles of the brain : cerebral hemispheres, fore-brain, mid-brain and hind-brain.
- The eye, and the ear.
- The heart.
- The visceral arches and clefts.
- The fore and hind limbs, and the tail.
Since writing the account of section-cutting on p. 434, we have obtained more experience as to the practical working of Messrs. Caldwell and Threlfall's microtome there mentioned. We find that it cuts more accurately and better than any other microtome with which we are acquainted, and can confidently recommend it to investigators and teachers with large classes. In the Cambridge Laboratory, it is driven by a small water engine and will cut at a rate of 500 a minute, without detriment to the sections.
Mr Threlfall, of Caius College, has recently elaborated a method of mounting sections which in our opinion has many important advantages over the shellac method. It is as follows. Make a solution of pure india-rubber in benzine or chloroform. Spread a thin film of this on a clean glass slide, and allow it to dry. Arrange the sections on the film y melt the paraffin ; allow the slide to cool, then immerse the slide for a moment in benzoline (liquid paraffin), which dissolves the paraffin, and mount in balsam. The chief advantages of this method are that the sections do not adhere to the india-rubber until warmed, and they can be stained after they are fixed on the slide if necessary. For the latter purpose, wash the benzoline away with absolute alcohol ; treat with weaker alcohol ; stain ; return to absolute ; clear with oil of cloves or kreasote, and mount in balsam (vide Zoologischer Anzeiger, 1883).
Foster, M., Balfour, F. M., Sedgwick, A., & Heape, W. (1883). The Elements of Embryology. (2nd ed.). London: Macmillan and Co.
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