Book - Introduction to Vertebrate Embryology 1935-5
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Shumway W. Introduction to Vertebrate Embryology. (1935) John Wiley & Sons, New York
- Shumway (1935): Preface - Contents | Part I. Introduction | Part II. Early Embryology | Part III. Organogeny | Part IV. Anatomy of Vertebrate Embryos | Part V. Embryological Technique
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Introduction to Vertebrate Embryology (1935)
Part V Microscopical Technique
Chapter XIV Preparation Of Embryological Material
A method much employed in the study of comparative embryology is that of cutting a preserved egg or embryo into a series of extremely thin slices, and arranging these in order upon a glass slide, so that they may be examined under the microscope. The older embryologists, however, were limited to the study of entire embryos and of minute dissections. These methods are still of great value in supplementing the study of serial sections, for it is a difficult mental exercise to translate sections into terms of the whole embryo. The single section, especially, is meaningless except when interpreted as a part of the complete series. It is very helpful, therefore, when facilities permit, for each student to prepare for himself a whole mount and a series of sections through one of the embryos he is to study.
A. COLLECTION AND REARING OF EMBRYOS
Although preserved embryos of the more important laboratory types may be obtained from the biological supply houses, it is often desirable to collect and rear live embryos.
THE FROG. — There are some sixty species of tailless Amphibia within the continental limits of the United States. Although the capture of adults in a pond where eggs are found is strong circumstantial evidence as to the species of the eggs, even this evidence is often lacking, so that the ability to identify the eggs or larvae from their own characteristics is highly desirable. A key to the eggs and larvae of some of the common Eastern frogs and toads is found in Wright’s ‘‘ Life History of the Anura of Ithaca, N. Y.” For the Pacific slope fauna, see Storer, “A Synopsis of the Amphibia of California.”’ The eggs of the salamander, Ambystoma, are laid at the same time and in the same localities as those of the early frogs, but may be distinguished from them by the greater proportion of jelly to the eggs in the
mass of spawn. 331 332 PREPARATION OF EMBRYOLOGICAL MATERIAL
Experiments dealing with the effect of pituitary hormones have led to the discovery that one of these hormones will induce ovulation in the female frog, and the drive to amplexus in the male, out of the breeding season. Rugh! (1934) has described in detail a technique for inducing ovulation and bringing about artificial fertilization which has been since used in several laboratories, including the author’s, with complete success.
The rate of development of the frog’s egg depends upon the temperature of the water. In the laboratory, the eggs will hatch in about one week after laying, at the ordinary room temperature. The egg masses should be kept in clean glass containers with at least ten times as much water. The water should not be changed until after hatching, when the larvae should be transferred to fresh water with aquatic plants. After the assumption of the tadpole form, they should be fed small pieces of finely ground meat. Metamorphosis may be hastened by feeding fresh or desiccated thyroid tissue.
Artificial fertilization is the best method of obtaining the earliest stages of development. The testes and vasa deferentia of the male are teased out in a watch glass of water. The eggs from the distal portions of the oviducts are placed in this water for five minutes and then removed to glass containers with not more than four inches of water.
THE CHICK. — In collecting hens’ eggs for incubation, it is a truism that they must be fresh and fertile. The best results are obtained from trap-nested eggs in the spring semester. The egg is normally laid in the gastrula stage (Chapter II), but in those cases where the egg does not reach the distal end of the oviduct by 4 P.M., it is retained till the following morning and undergoes further development. After laying, the egg cools and development ceases until incubation is commenced. The fertilized egg is viable for five weeks at a temperature of 8°-10° C. The time of hatching, as in the frog’s egg, is dependent upon the temperature. The minimum temperature at which development will take place is about 25° C.; the optimum is 37° C., at which temperature the egg will hatch in twenty-one days; the maximum temperature is about 41° C. In incubating eggs, care must be
1R. Rugh. Induced Ovulation and Artificial Fertilization in the Frog, Biol. Bull. 66, 22-29. PRESERVATION OF MATERIAL 333
taken to keep the air in the incubator moist and to rotate the eggs once a day.
Instructive demonstrations may be made by opening the shell and shell membranes under aseptic conditions and removing a bit of the albumen. <A window of celloidin placed over the opening and carefully sealed will permit of observations on the development of the embryo for several days. An alternative method is that of opening the egg and placing the contents in a sterilized small stender dish. A glass ring is placed on the yolk to keep it beneath the surface of the albumen, and the dish is covered and placed in the incubator. If this operation is carried on under aseptic conditions, development will continue for two or three days.
THE PIG. — The early stages of development in any mammal are valuable. The larger embryos are visible as protuberances on the inner side of the uterine tubes. The tube should be slit open and the embryos exposed by cutting open the embryonic membranes which surround them. Smaller stages are obtained by washing out the contents of the tube with normal salt solution or preserving it entire.
Pig embryos may be obtained in quantities from any good-sized packing house. As many as eighteen may be found in a single female, but the average number is eight. The period of gestation in the pig is 121 days. Pig embryos of 10 mm. body length are the most useful in the elementary course. Later stages are of value in the detailed study of organogeny.
B. PRESERVATION OF MATERIAL
The preliminary preparation of material for microscopical work involves three distinct operations: killing, fixing, and preservation. In practice, two or three of these operations are performed by a single reagent known as a “ fixing fluid.” Such a reagent should kill the embryo so rapidly that it will undergo the minimum of post-mortem changes; it should preserve the structures of the embryo with as life-like an appearance as possible; and it should harden the soft parts so that they may undergo the later processes of technique without loss of form or structure. Some fixing fluids, such as alcohol or formalin, may be used indefinitely as preservatives, but the majority are used for a particular optimum period, and then washed out and replaced by alcohol. 334 PREPARATION OF EMBRYOLOGICAL MATERIAL
THE FROG. — The frog’s egg, before hatching, is best fixed by Smith’s fluid.
Potassium bichromate.......... cece eee eee eee 0.5 gram Glacial acetic aCid. . 6... ce cece cece cece eee eee 2.5 ce. Formalin. 2.0... ccc ccc cece cee cece cence eeaee 10.0 ce.
Distilled water... 2... . cece eee eee eee eee 75.0 cc.
1. Cut the egg masses into small pieces of about twenty-five eggs each, and submerge them in a dish of Smith’s fluid for twenty-four hours. A quantity equal to ten times the volume of the eggs should be used.
2. Rinse the eggs in water and wash with a 5 per cent aqueous solution of formalin until no more free color comes out. The eggs may be kept indefinitely in this fluid. If it is desired to remove the egg membranes, proceed as follows:
3. Wash in water for twenty-four hours, changing the water several times.
4. Place the eggs in eau de Javelle, diluted with three time its volume of water, and shake gently from time to time during a period of 15 to 30 minutes until the membranes are almost dissolved and will shake off.
5. Rinse in water and run through 50 per cent and 70 per cent alcohol, an hour to a day each, and preserve in 80 per cent alcohol.
After hatching, larvae are best fixed in Bouin’s fluid.
Picric acid, saturated aqueous solution................ 75 cc. Formalin. 2.0... cece cece eee een eeeeees 25 cc. Glacial acetic acid... .. ec cee cece eeeeees 5 ce.
1. Larvae are left in this fluid from one to eighteen hours, according to size.
2. After rinsing in 50 per cent alcohol, wash in 70 per cent alcohol, to which has been added a few drops of lithium carbonate, saturated aqueous solution, until the yellow color is extracted, and preserve in 80 per cent alcohol.
THE CHICK. — The chick embryo must be removed from the shell, albumen, and yolk before fixation. As the early stages are more difficult to handle, it is advisable to practice this operation on embryos of seventy-two hours’ incubation and then work backward toward the stages of the first day. THE CHICK 335
1. Place the egg in a dish 3 inches high and 6 inches in diameter, two-thirds full of normal saline solution, warmed to 40° C.
2. Crack the shell at the broad end with the flat of the scalpel, and pick away the pieces of shell until an opening slightly larger than a half dollar has been made. Remove the outer and inner shell membranes. Invert egg beneath the surface of the salt solution and allow the contents to flow out. The blastoderm, containing the embryo, will rotate until it is uppermost. With fine-pointed scissors, cut rapidly a circle of blastoderm, about the size of a quarter, with the embryo at the center. With blunted forceps, pull the blastoderm and adherent vitelline membrane away from the yolk and albumen, waving it gently beneath the surface of the salt solution to remove all yolk.
3. Submerge a syracuse watch glass in the salt solution and float the embryo into this. Remove the watch glass carefully from the large dish and examine the embryo with a dissecting lens. If the vitelline membrane has not yet separated from the blastoderm, it should be removed at this time with fine-pointed forceps and needles. Make sure that the embryo lies dorsal side up, as it did when the egg was opened.
4, Slide a cover glass under the embryo, and remove all salt solution: with a pipette, taking care that the embryo lies in the center of the cover glass. Lift the cover glass by one corner so that the overhanging edges of the blastoderm fold under, and place it in a dry watch glass on a piece of thin absorbent tissue paper and add fixing fluid at once. While the embryo is becoming attached to the cover glass, remove the yolk, albumen, and pieces of shell from the dish of salt solution to a slop jar, reheat the salt solution to 40° C., and prepare another embryo. Three embryos of each stage are to be prepared.
5. After five minutes, drop the cover glass, embryo side up, into a small stender dish of Bouin’s fluid and leave from two to four hours.
6. Rinse in 50 per cent alcohol, wash for two days in 70 per cent alcohol to which lithium carbonate has been added or until the yellow color is extracted from the embryo, and preserve in 80 per cent alcohol. 336° PREPARATION OF EMBRYOLOGICAL MATERIAL
THE PIG. — Embryos of 6 mm. body length and over are easily located in the uterine wall. Slit open the uterus and remove the embryo with fine-pointed forceps and a horn spoon, taking pains not to rupture the membranes. Place at once in Bouin’s fluid. Embryos of 10 mm. body length should be fixed for four hours. Rinsing and preserving are done as for the frog or chick. Larger embryos should have the body cavity slit open to admit the fixing fluid. Fetal pigs of 6 inches or more should be injected through the umbilical artery with formalin (20 per cent aqueous solution). This solution is also injected into the body cavity and cranium, after which the fetus is submerged in the same medium for a week and preserved in 6 per cent formalin.
“°C. WHOLE MOUNTS
It is very helpful to have some embryos mounted entire for comparison with the serial sections. In making these whole mounts, the embryos are stained, cléared, and mounted, i.e., transferred to a final medium for preservation and examination on the slide beneath a cover glass.
THE FROG. — Frog eggs and embryos may be mounted as opaque objects with the natural pigmentation, or they may be cleared and stained as transparent mounts.
Opaque mounts. —
1. Prepare a saturated aqueous solution of thymol. Filter the solution, and add gelatin until saturated. Remove the supernatant liquid.
2. Liquefy the gelatin by immersing a small quantity, in a test tube, in a dish of hot water. Fill a hollow-ground depression slide with gelatin and allow to cool.
3. With a hot needle, melt a small hole in the gelatin, sufficiently large to hold the embryo. Place the embryo in the desired position and hold it in place until the gelatin has cooled.
4, Add a drop of gelatin just warm enough to be liquid and cover with a cover glass which has been slightly warmed. When the gelatin has cooled, any surplus may be removed from the edges of the cover glass with a toothpick wrapped in moist cotton. In order to prevent the later formation of bubbles, the edges of the cover glass should be painted with gold size or Valspar.
Free-hand sections and dissections are admirably mounted by THE CHICK 337
this method, but great care must be exercised to prevent the formation of air bubbles through cracks in the gold size.
Transparent stained mounts. —
1. Bleach the embryo, until white, in hydrogen peroxide. About one week is required for this purpose. Embryos that have been preserved in 80 per cent alcohol should first be passed through 70 and 50 per cent alcohol to water, an hour or more in each fluid. Embryos in formalin must be rinsed in water for one hour.
2. Stain in dilute borax carmine four days or more.
Borax, 4 per cent aqueous solution. .................. 100 ce. Carmine. . 0.0.2... ccc cece eee eeneee 1 gr. Boil until dissolved and add alcohol, 70 per cent....... 100 ce.
To dilute, take 5 cc. of the borax carmine and 95 ce. of 35 per cent alcohol and add a crystal of thymol.
3. If overstained, remove the surplus color with hydrochloric acid (1 per cent solution in 70 per cent alcohol) after passing through water and 50 per cent alcohol, an hour each.
4, Run up through 80, 95, and 100 per cent alcohol, an hour each, and place in xylene (xylol) until transparent.
5. Prepare a mounting diagram by drawing an outline of a slide on a piece of cardboard and in this laying off an outline of the cover glass to be used. Place a clean slide on the diagram, and, just’ inside the right and left margins of the cover-glass outline, attach a thin strip of celluloid, 15/1000 of an inch in thickness, by means of a drop of acetone. Greater thicknesses may be obtained by attaching other strips as necessary. When these supports are dry, place a few drops of Canada balsam, dissolved in xylene, between the supports, place the embryo in position, and lower a clean cover glass gently. Try to avoid the formation of air bubbles. If these appear later they may be removed by a needle which has been heated or dipped in xylene. A little fresh balsam may be run into the cavity.
THE CHICK. — Total mounts may be stained either with the borax carmine or with Conklin’s modification of Delafield’s hematoxylin. Delafield’s hematoxylin, which gives a blue color to the embryo, is made as follows: 338 PREPARATION OF EMBRYOLOGICAL MATERIAL
Hematoxylin (16 per cent solution in 100 per cent al COMO]... cc ee een eee nes 25 ce. Ammonia alum (saturated aqueous solution).......... 400 ce. Hydrogen peroxide, neutralized. . 0... 6. cee eee ee eee 25 cc. Glycerin. 20... cc cece eet ee eee eens 100 ce. Alcohol methyl... 0.0.0... ccc cence nens 100 ce.
Conklin’s modification consists of diluting the stain with four times the volume of distilled water and adding to each 100 ce. of the dilute stain 1 ec. of picrosulphuric acid, prepared by adding 2 cc. of sulphuric acid to 98 cc. of picric acid (saturated aqueous solution).
1. Run the embryo from the 80 per cent alcohol down to water through changes of 70 and 50 per cent alcohol, an hour each.
2. Stain in borax carmine, undiluted, over night, or in hematoxylin from one to three hours. Either stain may be diluted still further and the staining period prolonged. In the author’s laboratory the schedule demands a four-day staining period and the borax carmine is diluted 5 x, the hematoxylin 20 x.
3. Destain, if necessary, in acid alcohol until the desired color is obtained. Embryos stained with hematoxylin will turn red in the acid alcohol, and the blue color must be restored by washing them in running water or, after washing in neutral 70 per cent alcohol, placing them in alkaline alcohol (1 per cent ammonia in 80 per cent alcohol).
4. Run up the alcohols, 80, 95, and 100 per cent, half an hour each. Pour off half the 100 per cent alcohol and add an equal amount of xylene. When the diffusion currents disappear, transfer to pure xylene and leave until the embryo is transparent. In rainy weather, or when 100 per cent alcohol cannot be obtained, phenol-xylene (phenol crystals, 25 gr. and xylene 75 cc.) may be substituted.
5. Remove the embryo from the cover glass (if it has not already detached itself) and trim the surrounding blastoderm to the form of an oblong or circle. Arrange a clean slide on the mounting diagram, as described for the frog, attach celluloid support, and mount the embryo in Canada balsam with the same side uppermost as when the egg was opened. Put the slide away where it may lie flat and free from dust until the balsam has hardened. This will take at least a week, after which the slide EMBEDDING IN PARAFFIN 339
may be cautiously cleaned and studied. The process may be hastened by drying the slide in the paraffin oven.
THE PIG. — Embryos up to 10 mm. body length may be prepared as whole mounts by staining in dilute borax carmine, destaining until only a trace of color persists, and mounting in Canada balsam. The time spent in each alcohol should be at least an hour for the larger embryos.
D. SERIAL SECTIONS
In the preparation of serial sections of an embryo, the fixed material is (1) embedded in a suitable matrix and (2) sliced into extremely thin sections, which are (3) mounted in serial order upon slides. The embryo may be stained before or after sectioning.
Embedding. — There are two principal methods of embedding, in paraffin or in celloidin. For especially delicate objects, the best results are obtained by a combination of these methods, the embryo being first impregnated with celloidin in order to avoid the shrinkage (about 10 per cent) caused by paraffin embedding, and the block of celloidin then immersed in paraffin so that ribbons of serial sections may be cut.
Embedding in paraffin. — In preparing the first few embryos for sectioning, it is advisable to stain, dehydrate, dealcoholize, and clear as if for a total mount. Later, the staining may be omitted until after the sections are affixed to the slide.
1. After clearing in xylene, which should be done in a warm place, for example, the low-temperature oven at about 40° C., pour off half the xylene and add an equal amount of paraffin chips. In the author’s laboratory a paraffin of about 55° melting point, obtained by mixing commercial paraffin with parawax, is used. The parawax, unfortunately, varies in melting point, so that the formula is empirical. The embryo may be left in this xylene paraffin for two days.
2. If the mixture has hardened it should again be melted in the low-temperature oven. Fill a clean stender dish with melted paraffin, transfer the embryo to this, and place in the high-temperature oven at about 56° C. (or one degree above the melting point of the paraffin used) for not more than two hours. The xylene paraffin should be thrown in the slop jar. Take care not 340 PREPARATION OF EMBRYOLOGICAL MATERIAL
to get any xylene in the high-temperature oven or paraffin used for the final embedding.
3. Smear the interior of a small watch glass with a 10 per cent aqueous solution of glycerin (or vaseline), and fill with fresh melted paraffin. Transfer the embryo to this, making any necessary adjustments in position with a heated needle. Place the embryo dorsal side up, and note the position of the head. Cool the surface of the paraffin by blowing on it gently until it is congealed. Then plunge it immediately into a dish of cold water or waste alcohol and leave it there for five minutes. Mark the block for identification. Objects may be left in paraffin indefinitely.
4. On removing the block of paraffin from its container, examine for the following flaws:
a. Air bubbles, if they are not near the embryo, may be removed with a hot needle. Otherwise it is better to trim the block close to the embryo, put it into melted paraffin, and re-embed.
b. Milky streaks are due to the presence of xylene. These will crumble during sectioning, so that it is best to re-embed if they occur near the embryo.
c. If the paraffin has “ fallen ” in the center, it is because the surface was cooled too long before the block was immersed in the water. If any part of the embryo is exposed, it must be reembedded.
Sectioning after paraffin embedding. — Before sectioning your first embryo, be sure you understand the mechanism of the microtome (there are many varieties, of which the rotary type is best adapted to beginning students), and have practised the technique on a block of paraffin. There are three standard planes of sectioning corresponding to the axes of the body (Fig. 238). Transverse sections are obtained by cutting the cephalic end of the body first, with the knife entering the left side. Sagittal sections are made by cutting the right side first, with the knife entering the ventral surface. Frontal sections are made by commencing at the ventral surface, the knife entering the left side. It is best to begin with transverse sections.
1. Attach the paraffin block to the object-carrier of the microtome in the proper manner to obtain the type of section desired. This is done by heating the surface of the carrier until it will just SECTIONING AFTER PARAFFIN EMBEDDING 341
melt paraffin, pressing the block against it in the desired orientation, and lowering into a dish of cold water. A little melted paraffin may be poured around the base of the block and this again cooled to secure additional support.
2. Place the object-carrier in the microtome and, after orienting the block with respect to the knife, trim it so that the end of the block is a perfect rectangle with one of the longer sides parallel to the knife edge. If one of the angles is cut off slightly there will be a series of indentations in the ribbon which will assist in orienting the sections on the slide.
3. If microtome knives are not available, place a new safetyrazor blade (Autostrop type) in the holder provided, allowing the
Fig. 238. — Diagram to show method of orienting embryo with reference to microtome knife according to type of section desired.
edge to project between a sixteenth and an eighth of an inch. Screw the holder in the knife-carrier so that the edge of the blade is tilted inward about 10° from the perpendicular.
4. Set the regulator for 20 microns (thousandths of a millimeter). .
5. Run the feed screw as far back as it runs freely; do not force it.
6. Advance the knife-carrier until the edge of the blade just clears the block.
7. Release safety catch and turn the wheel steadily until the knife begins to cut the block. Cut slowly, making necessary adjustments to the block and knife until you are cutting a perfectly straight ribbon without wrinkles or splits. The principal causes of trouble and their remedies are as follows: 342 PREPARATION OF EMBRYOLOGICAL MATERIAL
a. The ribbon curls to right or left. This happens because (1) the block is thicker on the side away from which the ribbon curls, or (2) the knife is duller on the side toward which the ribbon curls. Remedy: (1) trim the sides of the block parallel; (2) shift the knife to one side.
b. The sections curl and the ribbon is not continuous. This is due to (1) too much tilt of the knife, (2) too hard a grade of paraffin, or (3) too cold a room. Remedy: (1) lessen tilt of knife; (2) re-embed in softer paraffin; (3) move microtome to warmer place, light an electric light or micro-bunsen burner near microtome, or cut thinner sections.
c. The ribbon wrinkles badly. This is caused by (1) too little tilt to the knife, (2) too soft a grade of paraffin, (3) too warm a room, or (4) a dull or dirty knife. Remedy: (1) increase the tilt of the knife; (2) re-embed in harder paraffin; (8) move to a cooler room, or cool the knife and block by dropping alcohol on them and blowing vigorously, or cut thicker sections; (4) clean knife edge with cloth moistened in xylene or shift to a new place on the knife.
d. The ribbon splits lengthwise. This is due to (1) a nick in the knife, (2) a bubble in the paraffin, or (3) dirt on the knife edge or side of the block. Remedy: (1) shift to new cutting edge; (2) paint surface with thin celloidin; (3) clean knife edge and block.
e. The sections refuse to ribbon; they fly apart or cling to the knife or the block. This is due to the electrification of the sections caused by unfavorable atmospheric conditions. Many remedies have been suggested; the best is to ground the microtome to a water pipe. Usually it is advisable to wait for more favorable conditions.
8. Remove the ribbon in 6 inch lengths with a camel’s hair brush and arrange these in order, shiny side down, in a cardboard box cover. Avoid air currents of all kinds. The ribbons may be put away in a dust-free place if the room is not too warm. It is better to affix them to slides as soon as possible.
Affixing paraffin sections to the slide. — 1. Prepare a mounting diagram by laying off the outline of a slide as before, but enclose in this the outline of a long cover glass (25 by 50 mm. approximately) and leave space for a label on the right-hand side.
2. Clean a slide thoroughly by washing with acid alcohol EMBEDDING IN CELLOIDIN 343
followed by distilled water. Place this over the mounting diagram and brush over the surface above the outline of the cover glass with the following dilute solution of egg albumen:
Egg albumen, beaten and skimmed.................. 50 ce. Glycerin... 0... cece cece eee eee neeeeeeees 50 ce. Filter and add Thymol.............. 0. ccc cee ee ee ees a crystal Dilute 2 drops of this to distilled water............... 25 ce.
3. Cut the ribbon into lengths about 2 per cent shorter than the length of the cover glass. Using the wet brush from which most of the albumen solution has been squeezed, pick up these lengths and arrange them on the albumenized slide so that the sections will follow each other like the words on a printed page. The shiny side of the ribbon should be next to the slide. Great care should be taken to lower the ribbon slowly so as to prevent the formation of air bubbles beneath it.
4. Carefully warm the slides on a warming plate or a piece of plate glass, previously heated in the paraffin oven, until the sections are expanded and perfectly smooth. If bubbles appear beneath the ribbon, prick them with a hot needle while the ribbon is still soft and hot. Drain off the surplus water, carefully realign the sections, mark the slides with a glass-marking crayon, and set them away in the low-temperature oven to dry, at least two days. They may be kept indefinitely in this condition if not exposed to dust. .
Embedding in celloidin. — This method is preferred by some technicians as no heat is used in the process and the shrinkage is less than that resulting from the paraffin method. However, thin sections are not so easy to obtain and the sections must be handled individually.
1. Embryos are dehydrated as for the paraffin method. Leave in absolute alcohol one day.
2. Absolute alcohol and ether, equal parts, one day.
3. Thin celloidin, three days to one week.
Alcohol, 100 per cent. ... 0... eee eee eee eee eee 100 ce
Ether... 0.2... cece ccc eee cence eee eeeeee 100 ce
Celloidin. £0... ccc ccc cece eee e eee e eee eaes 5 gr. 4, Thick celloidin, two days to two weeks.
Alcohol, 100 per cent... 1... 0.0... ccc cece eee 100 ce
BO 6) a 100 ce 344 PREPARATION OF EMBRYOLOGICAL MATERIAL
5. Remove the embryo to a small watch glass and pour thick celloidin over it. Cover lightly, or place under a bell jar until the celloidin is hard enough to cut with a scalpel.
6. Dip a block of vulcanized fiber in thick celloidin. Cut out a block of celloidin containing the embryo from the watch glass and, after moistening the end by which it is to be attached in ether alcohol, press it firmly against the prepared fiber block.
7. Pour a little chloroform into a stender dish, add the block and embryo, cover tightly, and allow the celloidin to harden in the fumes for thirty minutes.
8. Fill the stender dish with chloroform and cover. Leave for thirty minutes.
9. Pour off half the chloroform and add an equal amount of cedar oil. Leave for one hour.
10. Transfer to pure cedar oil where it may remain indefinitely.
Sectioning after celloidin embedding. — Celloidin sections are usually cut with some form of sliding microtome. Be sure to study the mechanism and cut a piece of hardened celloidin before proceeding further.
1. Set the knife with a little more tilt than would be used for paraffin, and obliquely to the object so that at least half the cutting edge will be drawn through the block.
2. Orient the block upon the object-holder so that the desired type of sections may be obtained. The long side of the block should be parallel to the edge of the knife.
3. Cut sections 20 » or more in thickness, using a steady drawing cut. Mount sections as they are cut.
Affixing celloidin sections to the slide. — This is best done as the sections are cut.
1. Using the mounting diagram as before, rub on a thin film of undiluted albumen solution to cover the areas of the cover glass. Rub in well with the ball of the finger.
2. Arrange the sections in order on this area. When this is filled, lay a cigarette paper over the sections and press gently with another slide. The slides may be kept in a dust-free container.
Double embedding in celloidin and paraffin. — This process, although tedious, combines the best points of the two methods already given. AFTER STAINING IN BULK 345
1. Embed in celloidin according to the method above, omitting step 6.
2. Trim the celloidin block close to the embryo and wash out the cedar oil with xylene, three changes in two hours.
3. Embed in paraffin as described above, commencing at step 2.
4. Section according to the method given for paraffin.
5. Affix to the slide according to the method given for paraffin sections.
Staining serial sections. — When the embryo has been stained before sectioning, it is only necessary to remove the paraffin (or celloidin), replace with Canada balsam, and cover, if the stain proves to be satisfactory. Sometimes, however, it is advisable to strengthen or weaken the stain or to add a contrasting dye.
After staining in bulk. —
1. Paraffin sections on the slide should be put in a Coplin staining jar of xylene and left until the paraffin is dissolved, up to fifteen minutes.
2. Transfer to a mixture of xylene and 100 per cent alcohol, equal parts, five minutes.
3. Transfer to 100 per cent alcohol, five minutes.
4, Examine slide rapidly under microscope after wiping the back of the slide.
a. If the stain is satisfactory:
5a. Absolute alcohol and xylene, five minutes. 6a. Xylene, ten minutes. 7a. Mount in balsam under cover glass.
b. If the stain is too intense: 5b. Ninety-five and 85 per cent alcohol, one minute each. 6b. Acid 70 per cent alcohol, until stain is correct. 7b. Sections stained in hematoxylin should have the blue color restored in alkaline 85 per cent alcohol.
8b. Eighty-five, 95, and 100 per cent, one minute each. 9b. Absolute alcohol and xylene, five minutes.
10b. Xylene, ten minutes.
11b. Mount in balsam.
c. If the stain is too light: 5c. Ninety-five, 85, 70, and 50 per cent alcohol, one minute each, 346 PREPARATION OF EMBRYOLOGICAL MATERIAL
6c. Stain until desired effect is secured. 7c. Distilled water, five minutes. 8c. Fifty, 70, 85, 95, 100 per cent alcohol, one minute each. 9c. Absolute alcohol and xylene, five minutes. 10c. Xylene, ten minutes. llc. Mount in balsam.
Celloidin sections on the slide should be exposed to the fumes of the aleohol-ether mixture for half a minute, dried for one minute, and placed in a staining jar of 95 per cent aleohol. All other operations may be carried on as above except that phenol-xylene should be substituted for 100 per cent alcohol.
Counterstaining after staining in bulk. — In order to differentiate the parts of the embryo more sharply, it is often desirable to add a second stain contrasting with the first. The stains that have been employed in the previous exercises are nuclear dyes; that is, when extracting by acid alcohol, the color will persist in the nucleus after it has been washed out of the cytoplasm. The second stains affect the cytoplasm and should contrast in color with the nuclear stain employed. After borax carmine, a 0.5 per cent solution of anilin (Lyons) blue in 95 per cent alcohol is employed; after hematoxylin, a similar solution of cosin should be used.
1. Proceed as in the preceding section as far as 60.
2. Destain in acid alcohol until the color persists only in the nuclei.
3. Restore the blue color to hematoxylin-stained sections in alkaline 80 per cent alcohol.
4, Eighty and 95 per cent alcohol, one minute each.
5. Counterstain lightly, dipping the slide into the solution repeatedly until a light color persists in the sections, one-half to one minute.
6. Rinse in 95 per cent alcohol, dehydrate with 100 per cent alcohol, followed by xylene-absolute, clear in xylene, and mount.
Staining with Delafield and eosin on the slide. — Follow directions given for sections stained in bulk (where stain is too light), as far as step 6c, and follow with directions for counterstaining as given above.
Staining with Heidenhain’s hematoxylin. — This is one of the most important embryological stains. OPPEL’S POLYCHROMATIC STAIN 347
1. Remove the paraffin from the sections and run down the alcohols to distilled water.
2. Four per cent aqueous solution of iron alum, one hour to over night.
3. Rinse in distilled water and place in 0.5 per cent aqueous solution of hematoxylin, same time as in the iron alum.
4. Rinse in distilled water and return to the iron alum until sections are a pale gray. Check from time to time by rinsing in distilled water and examining under microscope to see that the desired structures are still visible.
5. When sufficiently destained, wash in running water for twenty minutes, or in distilled water, with frequent changes, for two hours.
6. Run up the alcohols, clear, and mount.
Fuchsin and picro-indigo-carmine. — This polychromatic stain is especially fine for organogeny.
1. Remove the paraffin and run down the alcohols to distilled water.
2. Stain in basic fuchsin, saturated aqueous solution, twenty minutes.
3. Rinse in distilled water and place in picro-indigo-carmine for five minutes.
Picric acid, saturated aqueous solution................ 50 ce. Indigo-carmine, saturated aqucous solution............ 50 ce.
4. Pass rapidly through 70, 95, and absolute alcohol into xylene-alcohol. The green dye is extracted most rapidly by the 70 per cent alcohol, the red by the absolute. Only experience will teach the right time allowance for each alcohol.
5. Clear in xylene and mount.
Oppel’s polychromatic stain. — This gives beautiful effects with older embryos and larvae.
1. Fix in Bouin.
2. Stain in bulk with undiluted borax-carmine, one to two days. Destain for the same period.
Embed, preferably by the double method. Cut sections, 15-20 u.
Run down the alcohols to water.
Stain in picro-indigo-carmine, 14 minutes. Stain in picro-fuchsin, one minute.
NES OUR 348 PREPARATION OF EMBRYOLOGICAL MATERIAL
Picric acid, saturated aqueous solution................ 50 ce. Acid fuchsin, saturated aqueous solution.............. 50 ce. 8. Wash in distilled water, changed repeatedly, five minutes. 9. Ninety-five per cent alcohol, two minutes. 10. Phenol-xylene, xylene, and mount.
E. TECHNICAL RECORDS
Not the least important part of technique is the keeping of exact records covering every technical operation. For each embryo there should be a card, giving the following data: Kind of embryo and stage of development. Method of fixation, time and date. Bulk staining, time and date. Method of embedding, time and date. Plane and thickness of sections, and date. Slide staining, time and date. Method of mounting, and date. Name of preparator.
PN OOP Wh Ee
F. OUTLINE OF TECHNICAL PROCEDURE FOR CHICK EMBRYOS
1. Remove embryo from egg in warm normal salt solution.
2. Fix for two hours in Bouin’s fluid.
3. Wash in 70 per cent alcohol (plus lithium carbonate), at least one change, for two days.
4. Pass through 50 per cent alcohol and water, one hour each.
5. Stain in dilute borax-carmine or Delafield’s alum-hematoxylin, four days.
6. Destain in acid 70 per cent alcohol until desired effect is obtained.
7. Wash in neutral 85 per cent alcohol. (The hematoxylinstained specimen is transferred to alkaline 85 per cent alcohol until blue color is restored.) Two days.
8. Dehydrate and clear: 95 per cent, 100 per cent alcohol, absolute alcohol-xylene, xylene, twenty minutes each.
Mount in Canada balsam OR
9. Prepare for embedding by pouring off half the xylene and adding an equal amount of paraffin chips. Keep in warm place up to four days. REFERENCES 349
10. Continue by transferring embryo to melted paraffin and place in paraffin oven for an hour and a half. 11. Embed in fresh paraffin and cool in water. Make blocks. 12. Cut transverse sections 20 u in thickness on microtome. 13. Prepare clean albumenized slide, float sections on this in order, warm until sections are expanded, remove surplus water. Dry for at least two days. 14. Remove paraffin with xylene, and A. Mount in balsam, or B. Run down alcohols to 70 per cent and destain. Run up the alcohols, through absolute alcohol and xylene and xylene, mount in balsam, or C. Run down alcohols to water and restain, dehydrate, clear and mount, or D. To 95 per cent and counterstain for one minute. Dehydrate, clear, and mount.
Baker, J. R. 1933. Cytological Technique.
Ballentyne, F. M. 1928. An Introduction to the Technique of Section Cutting.
Carleton, H. M. 1926. Histological Technique.
Gage, S. H. 1925. The Microscope, 14th Ed.
Guyer, M. F. 1917. Animal Micrology, 2nd Ed.
Lee, A. B. 1929. The Microtomist’s Vade-Mecum, 9th Ed.
McClung, Ch. 1929. Handbook of Microscopical Technique.
Oppel, A. 1914. Embryologisches Practikum und Entwicklungslehre.
Rugh, R. 1934. Induced Ovulation and Artificial Fertilization in the Frog. Biol. Bull. 66, 22-29.
Shumway, W. 1926. Fuchsin and Picro-indigo-carmine, a Polychromatic Stain for Vertebrate Organogeny. Stain Technology I, 1.
Chapter XV Study of Embryological Preparations
During the carly stages of development, embryos are too small to be studied with the unaided eye. Some observations, to be sure, may be made with the dissecting lens, but most embryological work requires the use of the compound microscope. Although the student may be familiar with the use of the microscope from the elementary course in biology, he should nevertheless review this subject before proceeding further. In addition, he should at this time familiarize himself with the simpler methods of measuring objects with the aid of the microscope, as embryological drawings require a strict accuracy as to proportions. A great convenience in embryological work is the camera lucida or some other device by means of which accurate outlines may be traced. Finally, we must consider the methods by which the embryo may be reconstructed in magnified form from serial sections, thus returning, in a sense, to the point where the study of embryological technique was begun.
A. THE USE OF THE MICROSCOPE
Nomenclature of the microscope. — The separate parts of the microscope (Fig. 239) may be grouped into two systems, the mechanical parts, and the optical parts. The principal mechanical parts are the base, from which arises the pillar, attached to which is the arm, which may be inclined at the joint. Attached to the arm, just above the joint, is the stage, upon which the slide is placed for examination, and beneath this, the movable sub-stage equipment, consisting of a condenser-sleeve, and one or two iris-diaphragms, by means of which the amount of light to be used is regulated. At the base of the arm is the mirror, a silvered double mirror, with a plane surface on one side and a concave surface on the other. At the upper end of the arm are two screws, the coarse and fine adjustments, by means of which
the barrel of the microscope may be raised or lowered either 300 THE OBJECTIVES 351
rapidly or very slowly. The barrel is composed of the bodytube, connected to the arm by a rack and pinion, in the upper end of which is enclosed an inner tube, the draw-tube, on which is a graduated scale of millimeters representing the tube length exclusive of the revolving nose-piece at the lower end. The optical parts of the microscope are systems of lenses, the condenser, placed in the condensersleeve, the objectives, attached to the revolving nose-piece, and the oculars, one of which is placed at the upper end of the drawtube.
The condenser. — This is a system of lenses which increases the amount of illumination O thrown upon the object, and is BJECTIVE required only with the higherpower objectives.
The objectives. — These are systems of lenses which produce an enlarged and inverted image of the object under proper conditions. Objectives were formerly marked by arbitrary letters V1¢. 239.— Diagram showing parts of or numbers, with the lowest-pow- ae compound microscope. (From er objectives beginning the series. age.)
To-day they are usually indicated by the equivalent focal length (E. F.), that is, the focal length of a simple lens at 250 mm. or 10 inches, or else by the actual magnification (x) at 160 mm. (Leitz microscopes, 170 mm.). In some of the older microscopes the tube lengths indicated on the draw-tube were calibrated without including the length of the revolving nose-piece, then an accessory part. When setting up these instruments the length of the nose-piece (Leitz, 18 mm.) must be deducted and the drawtube set at the reduced length (Leitz, 152 mm.). The most useful objectives for general embryological purposes are the 25-mm. or 6 X, which will hereafter be spoken of as the lowerpower objective; the 16-mm. or 10 X, which will be called the
4yBuayz-aqny 352 STUDY OF EMBRYOLOGICAL PREPARATIONS
medium-power objective; and the 4-mm. or 40 x, known as the high-power objective. For the study of the germ cells, an oilimmersion objective, of which the front lens must be in contact with the cover glass by means of a drop of cedar oil, is necessary. The most generally used immersion objective is that of 1.9 mm. E. F. or approximately 95 xX.
Oculars. — These are systems of lenses which magnify the real image formed by the objective. Like objectives, these were, in the past, usually numbered or lettered, beginning with that of the lowest power, but now are marked with the E. F. at 250 mm. or the actual magnification at 160 mm. (Leitz oculars, 170 mm.). The most useful oculars are the 50-mm. (5 X) or low-power ocular, and the 25-mm. (10 x) or high-power ocular. When used with the objectives given above, a range of magnification from 30 xX to 450 * may be obtained. A method of obtaining the exact magnification will be described in connection with the directions for reconstruction given below.
The use of the microscope. —
1. Place the microscope squarely in front of you with the pillar toward you and the stage horizontal.
2. Place the low-power ocular in the draw-tube, and adjust this to a length of 160 mm. (170 mm. for Leitz instruments) as indicated on the millimeter scale. Swing the low-power objective into position. Place the mirror bar in the median line and adjust the mirror to secure an even illumination. Use the plane side of the mirror. The concave side is employed only when the condenser is not in use.
3. Place the slide on the stage so that the object to be examined is in the center of the stage aperture, and fasten it down with the spring clips provided. With the coarse adjustment, lower the body-tube until the objective nearly touches the cover glass. Then, with the eye at the ocular, slowly raise the body-tube until the object comes into plain view. With the fine adjustment, raise and lower the body-tube a little at a time until the point at which the smallest details show clearly is discovered. This is the focal point.
4, When using the low-power and medium-power objectives, the condenser should be lowered until the illumination is evenly distributed. With the high-power objective, the condenser should MICROMETRY 353
be raised almost to the level of the stage. The iris diaphragm should be open sufficiently to illuminate about three-quarters of the aperture of the objective. In other words, it is more widely open for the low-power objective than for the high-power objective.
5. If a greater magnification is desired, change to the highpower ocular, which will double the magnification. If this is not sufficient, return to the low-power ocular and swing the mediumpower objective into position, and so on. On most modern instruments, the objectives are par-focal; that is to say, the lengths of the objectives are such that when another objective is swung into place the object will still be visible. If, however, the object is not in focus, it is best to lower the body-tube until the new objective almost touches the cover glass, and focus up until the object comes into view. If the oil-immersion objective is to be used, lower the condenser and place a drop of oil on its upper surface; then raise it until it touches the bottom of the slide. Place another drop immediately over the object on the cover glass and lower the body-tube with great care until the front lens of the objective touches the oil. Focus by means of the fine adjustment only. .
6. All optical parts of the microscope must be cleaned with lens-paper. After the oil-immersion objective has been used, the front lens, condenser, and slide should be wiped with a bit of lens-paper dipped in xylene and then dried with a fresh piece. Never separate any of the optical parts. The microscope should be lifted by the pillar unless a special grip is provided to the arm. The microscope should be kept in the case when not in use. One of the oculars should be left in the draw-tube at all times to prevent dust getting on the upper lenses of the objectives. Beginners should try to avoid the error of closing the eye that is not in use. Practice will enable the microscopist to work with both eyes open and even to alternate the right and left eye at the ocular.
Micrometry. — The unit of measurement in microscopy is the micron (x). It is the one-thousandth part of a millimeter. Measurement of microscopic objects is performed with the aid of micrometers, of which there are two types, the stage micrometer and the ocular micrometer. The former is a glass slide, in the center of which, under a cover glass, is a line, usually 2 mm. long, 354 STUDY OF EMBRYOLOGICAL PREPARATIONS
divided into 200 equal parts, each of which, therefore, is equivalent to 10 wu. The ocular micrometer is a glass disc, placed in an ocular at the level of the ocular diaphragm, on which is engraved a scale, with arbitrary subdivisions. Some oculars are furnished with a draw-tube so that the upper lens of the system may be focused more sharply upon the scale. The value of the divisions indicated on the scale varies according to the amount of magnification of the real image, and so must be obtained for each objective independently, according to the following method:
1. Arrange the microscope as before, taking particular care to secure the proper tube-length.
2. Focus the eye-lens on the ocular micrometer scale by means of the ocular draw-tube. Focus the objective on the stage micrometer.
3. Make the lines of the stage micrometer parallel with those of the ocular micrometer, and determine the value of the divisions of the ocular micrometer in terms of those of the stage micrometer. Thus, if it requires 10 spaces of the ocular micrometer, and the latter is equal to 0.1 mm., then the value of a single space of the ocular micrometer for that particular objective and at that particular tube-length is 0.01 mm. or 10 ». Determine the value of the ocular micrometer for each objective in the same way.
B. EMBRYOLOGICAL DRAWINGS
Free-hand drawings of microscopic objects can only approximate an accurate representation. However, great pains should be taken to secure at least accurate proportions, neat and cleancut lines, and complete labels. Accurate outlines can be secured by the aid of the camera lucida, various types of projection apparatus, or microphotography.
Equipment. — The student will need a hard lead pencil (4H), a medium pencil (HB), and blue, red, and yellow colored pencils, an eraser, and bond paper to fit the note-book cover used in earlier courses.
Free-hand drawing. —
1. Lay off the space to be occupied by the drawing, by placing four dots at the corners. Rule in two lines, intersecting at the center of this space. These will represent the dorso-ventral ABBE CAMERA LUCIDA 355
and the dextro-sinistral axes, if the drawing is to be of a transverse section.
2. Measure the corresponding axes of the sections by means of the ocular micrometer, multiply by the desired magnification of the drawing, and lay off these magnified measurements on the cross lines already drawn. The following magnifications are recommended: for the twenty-four hour chick, 100 x; for the thirty-three hour chick, 75 x; for the forty-eight hour chick, 50 xX; for the seventy-two hour chick, 30 x; for the 10 mm. pig, 20 x.
3. Draw in a careful outline of the section and of the internal structures, paying particular attention to the proportions, which should be measured with the ocular micrometer and laid off on the axes at the proper magnification.
4, On one side of the dorso-ventral axis, all structures should be colored with the crayons in accordance with the following scheme: ectoderm, blue; mesoderm, red; and endoderm, yellow.
5. Label all structures represented in the section, using broken lines at right angles to the long axis of the paper to connect the label with the structure indicated.
6. Identify the drawing fully, by means of a serial number, the species, and stage of development, the number given to the series, slide, and section, the type of sections, and the amount of magnification. Example: No. 23, Chick, 48 hours, Series 1102, Slide 2, section 28, transverse section 50 x. If a drawing has already been made of the total embryo or a total mount, indicate on this, by means of a heavy ruled line, the position of the section just drawn, and number this line with the serial number of the section.
Abbé camera lucida. — This is an attachment which reflects the light from the drawing board, by means of a mirror, to a silvered prism, whence the light is reflected to the eye, superimposed on the image of the object which is transmitted through a small hole in the silvered surface of the prism directly above the ocular of the microscope (Fig. 240).
1. Attach the camera to the draw-tube of the microscope in such a way that the mirror projects to the right, and the opening in the prism lies above the center of the ocular.
2. Extend the mirror arm to its greatest length and set the 356 STUDY OF EMBRYOLOGICAL PREPARATIONS
mirror at an angle of 45°. The mirror arm must be parallel to the drawing board.
3. Try various combinations of objectives and oculars until an image of the desired magnification appears on the paper. Magnifications intermediate to those obtainable in this way may be secured by varying the tube-length or by raising or lowering the drawing board. If the stage of the microscope interferes with the drawing, the mirror should be set at an angle of 40° or 35° and the drawing board tilted toward the microscope at an angle of 10° or 20°, respectively, by means of wooden images. If the image is stronger than the reflection of the pencil point, a smoked glass may be placed beneath the prism, or the aperture of the iris diaphragm may be reduced. If the reflection of the pencil is stronger than the image, smoked glass may be placed . at the side of the prism or the amount Fig. 240. — Diagram showing prin- Of light falling on the paper reduced
ciple of the Abbé camera lucida. by means of a screen.
Path of image seen in microscope 4, Draw in the outlines of the sec shown in broken lines, that on, .
drawing paper shown in unbroken tions and the larger internal struc lines. (From Gage.) tures. The details may be added free-hand.
5. Remove the slide and substitute a stage micrometer. Trace in part of the scale by means of which both the magnification of the drawing and the absolute size of the object may be computed readily.
Projection apparatus. — Where many drawings are to be made, as in the case of reconstructions, some form of apparatus by means of which the image of the section may be projected directly upon the paper is very helpful. There are many types of projection apparatus, directions for the use of which may be obtained with the instruments. THE GRAPHIC METHOD (OF HIS) 357
Microphotography. — The photography of minute objects with the aid of the microscope is of great assistance in embryology. However, the methods are so difficult, the apparatus so complex, expensive, and delicate, and the process requires so much technical knowledge and skill, that microphotography has been considered a field too advanced for the beginning student, although a method described by Headland seems to overcome these difficulties to a large extent. In recent years the motion-picture camera has been adapted for use with the microscope, and excellent results have already been obtained.
After an embryo has been sectioned, it is sometimes necessary to reconstruct some part of it from the sections. There are two important methods: graphic reconstruction, in which a geometric projection of a sagittal section, for example, might be made from transverse sections; and plastic reconstruction, in which magnified replicas of each section are made of wax and piled together so as to make an enlarged model of the object to be studied. A complete series of sections of uniform thickness and accurate orientation is required for either type of reconstruction, and an outline drawing of the embryo before sectioning is of great assistance.
The graphic method (of His).— This method can best be described by giving practical directions for a particular problem, e.g., to prepare a geometrical sagittal projection 20 x of the neural tube of a 10 mm. pig embryo from a series of transverse sections 20 pz in thickness.
1. From the lateral view of the embryo drawn before sectioning, make an outline drawing 20 x.
2. Draw a median line corresponding to the cephalo-caudal axis, the length of which, in this case, should be 200 mm.
3. Count the number of sections in the series, in this case, 500.
4. Locate the position of each transverse section which you have drawn on the median line of the outline. Thus if the most anterior section drawn was the fiftieth of the series of 500 sections, it would be located at a point 1/10 of the total length of the axis (200 »), or 20 mm. from the cephalic end.
5. Theoretically, each section is at right angles to the median 358 STUDY OF EMBRYOLOGICAL PREPARATIONS
line, but this angle may be greater or less as a result of variations in technique. Study each drawing of a cross section in connection with the ‘drawing of the total embryo and determine the angle made by that section with the cephalo-caudal axis of the embryo. Draw in, at each point located on the median line, a cross line at the proper angle so determined. These lines represent the dorso-ventral axes of the transverse sections. Their lengths should correspond with those of the dorso-ventral axes of the drawings of the transverse sections previously made at the same magnification, 20 x.
6. Plot in on each section-plane line (dorso-ventral axis) the dorsal and ventral boundaries of the neural tube as determined from measurements of the drawings already made. Interpolate by direct measurement and magnification of these points on intervening sections.
7. Sketch in the contours of the neural tube by connecting up the points which have just been plotted. Compare the drawing with a sagittal section of an embryo in the same stage of development.
Plastic reconstruction. — This method also will be indicated by practical directions for the reconstruction of a particular organ, in this case, a model 50 x of the heart of a 10 mm. pig, from a series of transverse section, 20 u in thickness.
1. Prepare a number of wax plates of the proper thickness. In this case, if every section is to be reconstructed, the thickness of the plates must be 50 X 20 u,orlmm. Nearly as good results can be obtained by reconstructing every second section and making the plates twice as thick. The wax is prepared according to the following formula:
Beeswax... 0... ccc cc cece eee eee e eee eeeeee 6 parts Paraffin, 56° C. mp... ccc eee cceeec eee eeeeeee 4 parts Rosin, white lump. ...... 0. cee eee cece ee eee eee 2 parts
Mix and melt.
Pour 130 grams of this wax into a pan with an inside measurement of 500 X 280 mm., into which boiling water has been poured to a depth of 15 mm. This amount of wax will make a plate 1 mm. in thickness. Bubbles in the wax may be removed by playing the flame of a bunsen burner over the surface as it is cooling. As the surface hardens, cut the edges free from the sides of PLASTIC RECONSTRUCTION 359
the pan. When the wax has set put is still malleable, roll up the plate and remove it to a soapstone slab, where it is unrolled and allowed to cool.
2. With the help of a camera lucida or projection apparatus, prepare outlines 50 x of the heart in all the sections in which it isfound. Number the drawings consecutively and note the serial number of the sections drawn, so that it will be possible to check the drawings later if necessary. Note also whether the right and left sides of the drawing actually correspond with the right and left sides of the embryo or whether this condition is reversed. This is very important, as a mistake at this point would render the reconstruction valueless.
3. Transfer the drawings to the wax plates by means of carbon paper. Place the wax plates on a sheet of glass, and cut out the parts to be preserved with a sharp scalpel, leaving bridges of wax to connect the parts which would otherwise be separated. These bridges are best made in the form of arches bending towards the outside of the section.
4. Pile the sections in order, taking care to avoid the reversal of right and left sides, and to get an accurate fit. It is best to group the sections in piles of ten. A steady pressure of the hand will be sufficient to cause the sections to adhere to each other. The bridges may be cut away and stout pieces of wire substituted. Heat the wire at each end and press into position. After the wire is set, the wax bridges are cut away and the edges of the piece smoothed with a heated scalpel or aluminum modeling tool.
5. When all the sections have been combined in groups of ten, these groups should be united and the completed model smoothed in the same way. Such models may be painted or dissected, and mounted on wooden supports as desired. They are quite permanent if not exposed to high temperatures. Plaster of Paris molds and casts may be made from them in the customary manner.
Belling, J. 1930. The Use of the Microscope. Cage, S. H. 1932. The Microscope, 15th Ed. Guyer, M. F. 1917. Animal Micrology, 2nd Ed:
Headland, C. I. 1924. A Simple and Rapid Photomicrograph for Embryological Sections. Anatomical Record, XVII, 2.
Lee, A. B. 1929. The Microtomist’s Vade-Mecum, 9th Ed.
Mueller, J. F. 1935. A Manual of Drawing for Science Students.
Norman, J. R. 1923. Methods and Technique of Reconstruction. Journal of the Royal Microscopical Society.
Cite this page: Hill, M.A. (2019, November 14) Embryology Book - Introduction to Vertebrate Embryology 1935-5. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Book_-_Introduction_to_Vertebrate_Embryology_1935-5
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