Text-Book of Embryology 2-12 (1919)
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Kerr JG. Text-Book of Embryology II (1919) MacMillan and Co., London.
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Appendix
The General Methods of Embryological Research
Embryology is one of the youngest of the sciences and it offers a wide field for fascinating and important research. Regarded as a branch of morphology its main object is to gain information concerning the lines along which the structure of existing groups of animals has evolved. In the phylum Vertebrata there is an immense amount of work still to be done and it is important that the would-be researcher should be guided by certain general principles as to the technique of the subject, otherwise he is apt to achieve no more than the addition of relatively unimportant details to the vast accumulation of details which during the past few decades has tended to hide away general principles and incidentally to smother interest in the subject.
The incompetent or inexperienced investigator frequently betrays-himself by his choice of subject: he chooses a problem of relatively minor interest when there lie ready at his hand others which are of real importance, or he chooses a subject really important but of such difficulty that the probabilities are heavily against the feasibility of its solution under existing conditions. The beginner then should see that he has the aid of some competent adviser before he decides upon his line of research.
Having chosen his particular problem he has next to decide regarding the particular animals upon which his research is to be carried out. The earlier workers were guided mainly by the accessibility of the material. Fowls and Rabbits -—-of which embryos were easily obtained and easily investigated—--provided the material for the great pioneers of vertebrate embryology and the embryology of to-day suffers much from the difficulty of getting rid of general ideas founded on such narrow bases. N ow that embryology has taken its place as a branch of evolutionary science we recognize the importance of basing our general ideas upon the phenomena of development as displayed by the more primitive existing groups. In attempting any important problem of vertebrate morphology, evidence must be got from Elasmobranchs, Crossopterygians, Lung-fishes, Urodeles, before we can feel completely confident as to general principles: in other words we must go to groups which are admittedly archaic. Apart from directly adaptive features an animal which is archaic in its adult structure may be expected to show primitive features in its development. Naturally we should not look for this in cases where development takes place under peculiar conditions, for these necessarily involve adaptive modification. A pitfall into which investigators frequently stumble is that, starting from some particular group—-say Ampluloxus, or the Mammalia——with whose structure they'happen to be thoroughly familiar, they assume its general organization to be primitive. As a matter of fact it may be assumed with considerable probability that every existing vertebrate is to a certain extent a mixture of primitive features and specialized. It is only by careful comparative study that it can be decided which features are probably primitive and it is quite certain that these will not be found all within one group. Consequently speculations based upon the intensive study of one particular group are to be distrusted, though there is always less ground for distrust if the group is one which is recognized for reasons other than embryological, as being on the whole archaic. ‘ When minute histological details are concerned another qualification which should be possessed by the animal chosen for investigation is large size of its cell units.
The material should be abundant. Not only should there be a continuous series of stages but there should be numerous specimens of each stage. There is no such thing as an absolutely normal individual: the conception “normal” is an abstraction based upon the observation of numerous individuals. Only by observing numerous individuals can we therefore arrive at a knowledge of normal development. Work carried out on a? few specimens may of course provide isolated observations of much interest and value but it is inadequate to serve as a basis for general conclusions.
In all descriptive embryology it is necessary to have some method of specifying the stage of development of individual embryos. Unfortunately there has been a great lack of uniformity as to the particular method of doing this. One of the most frequently used is that of specifying the period of time during which development has been going on as for example a “chick embryo of 40 hours’ incubation.” This method is quite unsatisfactory, owing to the fact that the actual stage of development of any individual embryo is a function of other factors in addition to mere time, such as temperature and individual idiosyncrasy. Thus in many tropical freshwater animals a statement of the age of the embryo is practically worthless unless accompanied by a record of the temperature, and even then there remains the unknown element of individual peculiarity such as is for example illustrated by Fig. 251 where a number of sister eggs of a Frog are seen to have “lost step” with one another to a marked extent even at a comparatively early stage of development. In other words eggs or embryos of the same age are liable to vary greatly in their degree of development, and a statement of their age is not adequate as a precise indication of the stage of development. The want of precision varies in different cases: it is less for example in a Eutherian mammal where development takes place at a fairly definite temperature than it is in a Fish or Amphibian inhabiting a tropical pool or swamp where the temperature is liable to great variation.
It is necessary then in referring to particular stages of development to define them by structural features. Here however a new difficulty presents itself in the fact that the relative rate of development of different organsystems is not the same in different individuals. It follows that if a. number of individuals be grouped together as being at the same stage of development as judged by a particular organ A it Wlll be found that other APP. METHODS OF EMBRYOLOGICAL RESEARCH 575
organs B, C, etc. are not exactly at the same stage of development—some are less developed some more in the various individuals. Still for practical purposes this is a useful way of indicating roughly the stage of development. For example early stages in the development of Vertebrates may be defined by giving the number of mesoderm segments which have developed——these being fairly conspicuous structures and definable by a number. A much better system, however, is to use numbered stages defined by the general external form——the first structural feature met with in the examination of an embryo. Keibel has published “normal plates” of the development of various Vertebrate types in which standard stages in development are defined by accurate figures. Unfortunately some of the normal plates are incomplete as regards the earlier stages during segmentation and gastrulation, but wherever the plates extend over the whole period of development they should be made use of by the working embryologist as his standard stages. Where no normal plates exist the embryologist should m_ake_it his first business to construct one by carefully working over the external features of development and defining by careful drawing and description a series of stages which he judges to be roughly equidistant. The embryology of any animal is an account of the observable changes which take place in its structure from the zygote stage up to the adult. Logically the investigation of its embryology should proceed similarly from zygote to adult but in actual practice it is better to work in the opposite direction—-—to commence by getting a clear idea of the adult organization and then to work back from the known to the unknown of earlier stages. An embryological investigation should commence with a careful study of the entire embryos or larvae at the various stages. Each stage should be examined first alive by transmitted and reflected light, careful note being taken of any movements due to muscular contraction, ciliary action etc. Particular attention should be paid to the arrangement of the blood-vessels, the time of commencement of heart movements, of circulation of the blood and of the appearance of haemoglobin in the corpuscles. The appearance of chromatophores should be noted: the seat of their first appearance and their reactions-——whether by changes of form, movement of pigment granules in their protoplasm, or by actual migration-—in response to changes in direction or intensity of light. During this phase of the work constant use should be made of the binocular microscope and rough sketches should be made.
Embryos of each stage should be submitted to the action of various fixing agents and it is important to watch the embryo during the process of fixing, for the fluid as it gradually penetrates the tissues often makes special structures stand out distinctly for a short space of time——to disappear again with further penetration. The fully fixed embryo should be subjected to further careful scrutiny by reflected light under the Greenough binocular. To detect small inequalities of the surface it will be found necessary to arrange the lighting carefully. The light from an mcandescent gas-mantle may be concentrated by a large condenser and caused to illuminate the embryonic surface in a tangential direction. It is often well to cover the specimen with a little house of opaque cardboard or metal resting on the stage of the microscope and possessing two apertures one in its roof through which the observation is made and one at the side through which light is admitted. The embryo must of course be completely submerged in fluid and is preferably contained in a round glass dish with a layer of pitch or black wax on the bottom in which, if necessary, small excavations can be made in which the embryo can rest securely in the desired position. The glass vessel should be rotated slowly during the observations so as to allow of the incidence of the light from different directions. It is important to observe a number, preferably a considerable number, of embryos of the same stage, as owing to individual variation particular features may be much more distinct in some than in others.
A number of thoroughly typical specimens of each stage should be picked out for further investigation and these should be carefully drawn under the camera lucida, a piece of millimeter scale being placed by the side of the embryo and drawn at the same time so as to form a reliable record as to dimensions.
At this stage the normal plates should be constructed if not already in existence and the embryos classified in accordance with them,
For the study of internal structure the great method is that of cutting the embryo into serial sections but a much older method, that of dissection, should by no means be ignored. Careful dissections made under the Greenough binocular are often extraordinarily instructive. It is advisable to experiment with embryos fixed according to various methods as diffcrrent methods give difl‘erent degrees of consistency, opacity etc. Van Beneden and N eyt’s fluid will be found in many cases to give very good results.
In section - cutting a fetish to beware of is excessive thinness of sections. The expert section cutter is liable to become so interested in his feats in accomplishing the preparation of sections of an extraordinary degree of thinness that he is apt to forget that the criterion of good sections is not simply their degree of tenuity but the relation which their thickness bears to the size of the cell-elements of the particular embryo. Thus while in some cases it is of advantage to have sections so thin as 1 [L2 or even '5 ii, in other cases, such as segmentation and gastrulation stages of some of the large heavily-yolked holoblastic eggs, the sections should reach as much as 80 p. or 100 ,u in thickness.
Before an embryo is cut into sections its soft protoplasm has to be supported by infiltration with some suitable embedding mass. For this purpose the two substances used at the present time are parafiin of high melting-point and celloidin. Of these the first is used frequently alone but the student should realize from the beginning that if he is to obtain reliable results, especially yvhere yolk is present in the embryonic tissues, he must use both methods and control and check the results obtained from one by those obtained from the other.
The process of infiltrating the embryo with paraflin is usually carried out in a hot-water oven heated by oil, gas or electricity and kept at a temperature just above the melting-point of the paraffin by a thermostat. The melted paraflin may be contained in small copper pans preferably plated inside with silver or nickel. An essential preliminary is a very thorough dehydration followed by a very thorough soaking in the clearing agent. To get the best results it is well to take the embryo through three changes each of 90% alcohol, absolute alcohol, and xylol or other clearing fluid. The actual process of infiltration with paraffin should last for the minimum time (which will have to be determined by experiment 1) and be carried out at the minimum temperature.
- 1 A useful guide for beginners is.Sect7}on- Cutting by P. Jemieson in preparation. For those who already possess an elementary knowledge of the subject an .xcellent work of reference is Bolles Lee's Miicrotomicfs V ads-nwcum.
It may be remembered that the complicated and bulky water-bath with its thermostat is in no way necessary for the embedding process. A very simple apparatus which is perfectly eflicient consists of a small metal trough (copper, or tinplate) resting upon a metal table kept heated at one end by a small flame. By sliding the trough lengthwise along the table a position can be found such that the entire thickness of paraflin is fluid at the end next the flame and solid towards the other end. Between these two points stretches an inclined plane of solid paraffin upon the surface of which the embryo rests without any risk of the temperature rising appreciably above melting-point. A simple embedding trough of the kind indicated is of great use in the field as there is no method of storing and transporting embryos so free from danger of accident or of histological deterioration as having them embedded in solid paraffin.
To get a block of parafiin in good condition for section~cutting the embryo should be transferred to a bath of fresh paraffiu as soon as it is infiltrated. With certain clearing agents, e.g. cedar oil, it is well to give two or three changes of paraffin. The vessel containing the embryo in"a considerable volume of paraflin should now he floated on cold water so as to give a homogeneoustranslucent block of solid paraffin. On no account should the vessel be actually submerged in the cold water for in this event the contraction of the inner paraffin as it cools within the already rigid outer layers will lead to the formation of cavities into which the water penetrates.
For the actual process of section-cutting it is necessary to use a mechanical microtome. The Cambridge Rocking microtome is one of the most convenient for ordinary enibryological work while the ReinholdGiltay ‘microtome is a most excellent instrument both as regards accuracy and rapidity of working.
The paraflin block containing the embryo is trimmed down so as to be rectangular in section and is then fixed by, the interposition of a hot spatula to the parafiined surface of the microtoine carrier in such a position as may be necessary to give the required direction of sections.
Where the object is a “diflicult” one, e.g. containing much yolk, it is advisable to have it surrounded by a paraffin block of considerable size. A considerable mass of paraffin above the specimen makes it out better, while a considerable mass to the side causes successive sections, with their long edges, to adhere better together and form a continuous ribbon. The embryo should be near one of the lower corners of the block to facilitate exact orientation.
For thorough investigation of the structure of embryos it is advisable to have specimens cut into sections in the three sets of planes-—transverse, psagittal or longitudinal vertical, and coronal or longitudinal horizontal. To obtain these it is tiecessary to have the embryo orientated exactly on the microtome. In most cases this can be accomplished with a sufficieutly close approximation to accuracy when fixing the paraflin block on to the carrier, especially if care has been taken to trim the surfaces of the block parallel to the three chief planes of the embryo.
E.g. for a Chick at about the middle of the second day about 20 minutes will be found to be suflicient.
Where greater accuracy is needed, as in the case of very small embryos, they should be arranged in position in the melted paraffin with warm needles under the prism binocular microscope. This may be done by placing the watch-glass or other vessel on the top of a small flat copper cistern full of water, provided with inlet and outflow tubes, and heated up by contact with the top of the water-bath or hot stage. In the bottom of the embedding vessel is placed a small plate of glass on the upper surface of which are engraved parallel lines intersecting one another at right angles. When the embryos have been accurately orientated with regard to the engraved lines a stream of cold water is allowed to run through the cistern and this causes the paraliin rapidly to solidify. When the block is quite llard the glass plate is picked off and the ridges formed by its engraved lines serve as accurate guides to the position of the embryo.
Still greater accuracy is obtainable by arranging that the melted paraffin in which the embryo is being orientated is already in its definitive position on the holder of the microtome, the paraffin being kept melted as long as necessary by an electric current passing through a loop of high resistance wire.‘
' For the actual cutting care must be taken that the razor (solid ground) or other knife has a very fine edge which does not show irregularities when examined under the low power of the microscope. The blade should be thoroughly cleaned with pure spirit before commencing work. If very thin sections, e.g. of l [L in thickness, are required it is well to commence with sections of 5 pt, then without stopping to change to 4 p., then to 3 p., then to 2 p, then to l p.——cutting a continuous ribbon throughout and going ahead rapidly when the 1 /1. sections are cutting properly.
The celloidin method should be constantly used as a check on the paraffin method. Where yolk.y eggs or embryos are being cut the celloidin method gives the only trustworthy sections as by it the yolk granules are held in position and prevented from sticking on the edge of the knife, ploughing through the tissues and destroying much of the fine detail, as is always liable to happen if paraffin alone is used under such circumstances.
In cases where there is no need for specially thin sections (say under 25 pi.) a convenient method is that in which the celloidin block is hardened by exposure to: chloroform vapour and then cleared by immersion in cedar-wood oil.
The block of celloidin is usually fixed to a block of wood which is gripped by the holder of the microtome. Care should be taken that such wooden blocks are baked for several days so as to ensure their being
absolutely dry. Otherwise moisture will diffuse out and produce a milky opacity in the celloidin which ought to be absolutely clear and transparent. Sometimes it will be found that the block becomes too hard and will
not cut properly, its edges frilling or breaking. This is sometimes due to the presence of a trace of chloroform in the cedar oil used for clearing.
When this is the case the cut surface of the block should have perfectly pure cedar oil applied to it with a brush just before each section is ‘cut.
‘ A special apparatus for this purpose is made by the Cambridge Scientific Instrument Company. APP. METHODS OF EMBRYOLOGICAL RESEARCH 579
To obtain thinner sections it is necessary to embed the celloidin block containing the object in parafiin. This may be done simply by transferring the block saturated with cedar oil to melted paraffin. A better method is to use a solution of celloidin in clove oil of about the consistency of treacle. The object, thoroughly permeated by this and surrounded by a small quantity of the celloidin, is hardened and cleared in chloroform. The block is then carefully trinnned with one face accurately parallel to the plane of the required sections. It is now immersed in melted paraiiin for a minimum time (ten minutes suilices for a small object). After cutting and mounting the sections the slide is immersed in xylol ,until the parafiin is dissolved out, then in absolute alcohol, then in a mixture of equal parts of absolute alcohol and ether until the celloidin is removed. The slide is now taken down through the series of alcohols and the sections stained and mounted in the ordinary way.
The arriving at a clear idea of the structure of an embryo from the study of a series of sections involves fitting the successive sections together into a continuous whole. To a great extent this reconstruction of the whole from the successive sections can be done mentally but where complicated structures are being investigated, some aid- is either absolutely necessary or at least desirable for the sake of accuracy. The preseht writer finds the most reliable as well as the most convenient of such aids in the method of reconstruction by means of glass plates.‘ Successive sections are drawn with a hard (9 H) lead pencil by means of a camera lucida upon finely ground sheets of glass such as is used for photographic focusing screens and then the successive drawings are fitted together, a fluid of as nearly as possible the refractive index of the glass being interposed between them so that the ground surfaces disappear and the heap of plates appears as a clear block with the structures drawn running through it and appearing as a kind of solid model.
The following details may be noted. Sections are cut to a standard thickness of 10 ,u (z'.e. T55 mm.): the glass plates are 1 mm. thick: the drawings are made at a magnification of 100 diameters. But it will be found in practice that much use can be made of the method even if these three dimensions are not so exactly correlated. The outlines made with pencil of the particular organ that is being studied are filled in with water colour. Vermilion is the most generally useful colour for it retains its opacity and light-reflecting properties to an unusually high degree when submerged in fluid of high refractive index. When the plates are dry N o. 1 is laid, ground side up, on a flat surface——_preferably a glass stage with a_ mirror beneath so that light may be refleeted up through it—a few drops of the fluid used, e.g. clove oil or cedar oil or a mixture of fennel oil (two parts) and cedar oil (one part) as recommended by Budgett 2 are placed by a pipette on the centre of the ground surface and then plate N o. 2 is lowered gently into position and fitted into its place over plate N 0. 1. The outlines of the drawings should be made to coincide exactly, and the two plates should be pressed firmly into contact care being taken to avoid interposed air bubbles which act as elastic cushions and prevent the upper plate from settling down into contact with the other. Successive
1 Quart. Jowm. Micr. Sea, xlv, 1902. 2 Trans. Zool. Soc. Landon, xvi, Pt. 7. 1902. 580 EMBRYOLOGY OF THE LOWER VERTEBRATES APP.
plates are fitted on in a similar manner until the particular organ stands out like a solid model in the mass of plates. _
The same set of drawings may be used for different organs : the clove oil is removed by treating with strong spirit, and the water colour by holding under the tap, and then, after drying, a new organ can be coloured in. By colouring merely the cavity of an organ the relations of the cavity can be displayed as by an injection. When finally done with the drawings are removed by scrubbing with “ Monkey brand” soap.
By this method, after a little practice, reconstructions can he made with great rapidity and accuracy.
Though less accurate and much more tedious the older method of reconstructing with plates of wax is useful for building up a permanent model. Its use is also indicated where only a single specimen is available. Instead of wax plasticine may be used 1 which allows of a kind of dissection being made, in as much as particular parts of the model may be bent out of the way to display structures which would otherwise be hidden.
1n investigating the development of the skeleton the cartilage is often found to pass by imperceptible gradations into unmodified mesenchyme. The absence of sharply defined surfaces in such cases makes the reconstruction method unreliable and it is advisable to supplement it by subjecting the embryo to treatment with a specific stain which picks out the cartilage while leaving the other tissues uncoloured so that the cleared and transparent specimen may be studied as a whole under the binocular microscope.
An excellent stain for this purpose is v. Wijhe’s Methylene Blue.” The embryo is fixed preferably in '5% watery solution of corrosive sublimate, with 10% formalin added just before use, and preserved in alcohol. When about to be stained it should be treated for a day or two with alcohol containing :1-% hydrochloric a.cid——care being taken_ to renew this so long as it develops any yellowness due to traces of iodine. The stain consists of a solution of 1% methylene blue in 70% alcohol to which 1% hydrochloric acid has been added some time before use. The embryo is stained for a week and is then treated with 70% alcohol containing {/0 hydrochloric acid and renewed several times the first day and thereafter once daily until no more colour comes away. The embryo is now dehydrated, cleared gradually in xylol, passed through stronger and stronger olutions of canada balsam in xylol, and preserved eventually in balsam so thick as to be solid at ordinary temperatures though liquid at 60° C.
An excellent method of cleaning small cartilaginous skeletons is to place. them amongst Frog tadpoles which remove the muscle etc. from the surface of the cartilage by means of their oral combs.
In regard to the general principles of embryological research it need hardly be said that, as in other branches of science, accuracy of observation occupies the first place. And yet, curiously, accuracy may become a fault. In those branches of science which are more effectively under the control of mathematics it is well recognized that in any type of investigation there is a limit of- probable error of observation-—due to instrumental or sensory imperfections or to disturbing factors of one kind or another— ‘ Harmer, Pterobranclyia of Seiboga Expedition, 1905. 9 Proceedings Akad. Wetensch. Amsterdam, J une 1902.
beyond which it is mere waste of time to push observation. In all biological observation the limit of probable error is particularly high yet this fact is peculiarly apt to be ignored and it is no unusual thing to find dimensions or other numerical data stated to three or four places of decimals when anything beyond the first place is worthless for the reason indicated. _
To secure accuracy of observation not merely training and experience in the art of observing is needed but also a proper psychological outlook: the observer must be able to take a completely detached point of view and must ever be on the watch to guard against some particular hypothesis or preconceived idea causing actual error instead of fulfilling its proper function of keeping the powers of observation tuned up to the highest pitch of alertness. '
The whole spirit and aim of scientific investigation is directed toward the seriation of facts and the devising of general expressions or formulae _ which unite them together. In this it contrasts with the more primitive
state of mental development which observes isolated phenomena, noting the differences between them but blind to the common features which link them together. In embryology as in other departments of knowledge the able investigator sees the general principles which run through and organize the masses of detail: he interests himself in discovering the likeness which is hidden under superficial difference; he is constructive not destructive.
In this volume embryology is treated as a branch of morphology but it must be borne in mind that morphology and physiology are inseparably intertwined. The living body whether of an embryo or an adult is above all a piece of exquisite mechanism fitted to live and move and have its being, and to ignore this is to make morphology as sterile and as misleading as would he the study of machinery apart from the movements and functions of its various parts. More particularly in attempting to delineate the evolutionary past of an organ, or set of organs, speculation must always be rigidly controlled by the reflexion that at each phase in evolution it nmst have been able to function.
When at length the stage is reached of putting results into form for publication the first thing to aim at is absolute clearness of expression. It must be remembered that clearness of language and clearness of thought are closely interdependent. Sloppy obscure language means sloppy obscure thought. The greatest care should be taken in the correct and precise use of technical terms. Argumentation in regard to scientific and other matters is, when the disputants are equally well informed, due as a rule to some word or expression being used in slightly different senses. Elegant literary style, however desirable, must always be subordinate to clarity and precision of language. Indeed actual harm is sometimes done to scientific progress by the writer whose literary skill carries away not merely himself but others of uncritical and impressionable mind. Scientific problems are eventually settled not by skill in dialectic but by increase of knowledge.
As a rule the proper presentment of an embryological thesis involves pictorial illustration. In this the elaborate coloured lithographs of former days may conveniently be replaced to a great extent by simple line or half-tone drawings in India ink ‘or process black which can be reproduced photographically and inserted in the text in contiguity with the passage which they illustrate. Their function is to render more clear the statements of the author: they represent as accurately as possible phenomena as observed by the skilled and trained eye with a brain behind it. Actual photographs, which repr'(:sent merely details lying in one particular plane and as seen by the untrained photographic lens, should be avoided. Apart from the imperfections indicated they are so blurred by the ordinary processes of reproduction as to be liable to misinterpretation and in these days of skilful manipulation they are of course useless as guarantees of truth.
