Paper - Preparation of material for histology and embryology with an appendix on the arteries and veins in a thirty millimeter pig embryo (1913)
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McClendon JF. Preparation of material for histology and embryology, with an appendix on the arteries and veins in a thirty millimeter pig embryo. (1913) Anat. Rec. 7(2): 51-62.
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Preparation of Material for Histology and Embryology, with an appendix on the Arteries and Veins in a Thirty Millimeter Pig Embryo
J. F. McClendon
From the Anatomical Department of Cornell University Medical College, New York
The essential of a good course in histology or embryology is good material. Fresh human material should never be allowed to go to waste, but it may be at times very inconvenient to put it up in a variety of fancy fixing fluids.
Perhaps the best general cytoplasmic fixer is formalin of 10 to 20 per cent (4 to 8 per cent formaldehyde). If material so fixed is not soaked too long in alcohol of high concentration, it may be used as fresh tissue in special technique to show fats or mitochondria. In fact the formaldehyde alone makes unsaturated fats and lipoids less soluble in clearing fluids. On the other hand, if the washing in water is omitted, the structure of resting nuclei is well enough preserved for ordinary purposes.
Commercial formalin contains formic acid, which, although developing a more beautiful nuclear structure, may begin to cytolyse the more delicate cells before they are sufficiently fixed by the formaldehyde. This is especially noticeable in erythrocytes — haemolysis, or escape of haemoglobin, occurring in parts of the tissue. Furthermore, acids swell fresh white fibrous tissue. It seems worth while, therefore, to neutralize the formol, and this may easily be done by adding slack lime (CaC0 3 ) or magnesia, and filtering.
Doctor Ferguson first called my attention to the fact that kidney swells in many fixing fluids, whereas it is commonly supposed that the majority of tissues shrink a little. Death of isolated cells as seen under the microscope may be accompanied by swelling (cytolysis) or contraction. In every case, an increase in permeability to some substances occurs, but I found that during the early stages of cytolysis of the sea urchin's egg, it remains very impermeable to salts. Dead animal or plant membranes are more permeable to water than to dissolved substances, but apparently some living cells are impermeable to water. The Fundulus egg, if transferred from sea water to distilled water, does not burst, though it is certainly not capable of resisting the enormous osmotic pressure of its internal salts. Since I found this egg to be impermeable to salts, it must also be impermeable to water (it is permeable to kations, but for every kation that comes out, the electrical equivalent must go in) . If such a cell became, on death, permeable to water, the osmotic pressure of its internal dissolved substances might cause it to swell. If a Paramoecium be killed by an ordinary fixing fluid, even though it be hypertonic, the protoplasm first coagulates, then the whole animal swells a little. This may be what happens to some tissue cells, and I found that it is not always prevented by the addition of 0.9 per cent sodium chloride to the fixing fluid. Therefore I supposed the swelling due to the osmotic pressure of some contained substance of large molecule, and experimented with the addition of cane sugar to neutral formol. By this means the cytolysis of adult convoluted nephric tubule cells is prevented, and the general fixation is good except that some nuclei may be slightly shrunken. This fluid may be used for all adult tissues and embryos, and is easily prepared as follows:
- Formol 100-200 cc.
- Cane sugar : 20-40 grams
- Slack lime (CaC0 3 ) or magnesia about 1 gram
- Water to make 1 liter.
If the shrinkage of a few nuclei is very objectionable use only 20 grams of sugar. This fluid has the advantage that tissues and embryos float in it and therefore do not become distorted.
If the whole kidney of a fetus be fixed in the above mixture or any other fixing fluid, the cells of the convoluted tubules will swell until they fill the lumen. This brings us to a well known point that is often neglected. Tissues should be cut into as thin slices or pieces as is practicable and the cells not injured in the cutting. Fetal tissues are especially delicate. They should be cut with a very sharp thin blade and lifted on the blade into the fixing fluid.
Many workers object to formalin because it "causes" a homogeneous appearance to protoplasm. The ultra microscope has shown that, aside from evident granules, living protoplasm is homogeneous, contrary to Butschli and others. There are persons who now accept formalin for cytoplasmic fixation but say that it "does not fix nuclei well. " Some structures may be seen in living nuclei. I have studied many nuclei with high powers and with the ultra microscope, yet I cannot decide what form of fixation corresponds most closely to the living structure. Both cytoplasm and nucleus of a living erythrocyte of- a frog is homogeneous when examined in serum or uncoagulated plasma with the ultra microscope. Sooner or later bright points or clouds appear on or in the nucleus, but this is usually associated with change of nuclear form and is evidently due to injury.
Formaldehyde not only does not coagulate protoplasm but renders it more difficult to coagulate. It also makes lipoids less soluble in clearing fluids. However, I find an after-treatment with Miiller's fluid or some other -oxidising fluid necessary for the preservation of lipoids, the amount of oxidation necessary depending on whether mitochondria, myelin or fats are studied.
Ordinary staining depends on the fact that all protoplasm treated with acid, stains with acid dyes, whereas certain parts take also basic dyes. Many staining solutions contain free acid, but tissues stain more quickly if they are previously treated with acid. For this reason we put everything into the formol mixture and after a few hours transfer part of it to Bouin's fluid. This tissue is finally stained on the slide in haematin and eosin. The alum haematin lake is usually so strong that it stains in three minutes, but the eosin is so much diluted that twelve hours are required to stain and in this time smooth muscle stains less intensely than white fibrous tissue The acid in Bouin's fluid causes the tissue to stain more brilliantly but if the fresh tissue is put into Bouin's fluid the blood in some of the vessels will be laked. Part of the material is transferred from the formol mixture to Miiller's fluid and subsequently stained with iron hematoxylin to show the lipoids (mitochondria, etc.).
Ordinarily, the student is shown two dimensions of a piece of tissue or embryo, and left to imagine the third. Though whole mounts of chick embryos are handed out, cleared pig embryos, and blocks or thick sections of certain tissues are even more useful. For a solid mount, the object should be placed in a dish of balsam or damar dissolved in benzol and protected from dust until it evaporates down to sirupy consistency, then mounted in the usual way. By this means the necessity of rings or other supports to the cover glass is avoided, and drying out or great shrinkage prevented.
All of the solid mounts turn yellow with age, but a number of highly refractive fluids may be obtained that are colorless. These are listed, with their refractive indices, in Landolt-Bornstein; Behren's Tabellen; and Lee's Vade Mecum. The higher the refractive index the better, for if in any case a lower index is desired, this may be obtained by the addition of paraffin oil or xylol (or water in case of aqueous media). It may be noted here that, whereas the process of clearing in a mixture of oil of wintergreen (Gaulteria) and benzyl benzoate has been patented in Germany and is widely known under the name of the patentee, wintergreen was first used by Stieda in 1866, and the synthetic oil (methyl salicylate) recommended by Gueguen in 1898, and is noted in various books on technique.
Methyl salicylate is permanently colorless, and comparatively inexpensive, and ideal for a fluid mount. If rings are cemented on slides with shellac or liquid glue and allowed to dry, they are not loosened by the oil. Paper rings soaked in shellac or glue will do, but rings may be cut from lead pipe with an ordinary saw or a bone saw if the proper size of glass rings are not at hand. The shellac must be dry before adding the oil, which must be free from alcohol. I prefer glue.
If the tissue is hardened in alcohol, thick sections may be cut free-hand. Thick sections are often better unstained, especially if injected, and much detail may be made out by partly closing the diaphragm of the microscope. If stained with very dilute haematin containing much acid, connective tissue is colorless and cytoplasm nearly so, whereas nuclei may be readily distinguished. In this way blood vessels and glands in areolar tissue are caused to stand out sharply.
Whole mounts and thick slices are especially useful in embryology, and are a necessity unless one is contented with teaching the third dimension with models. The larger the embryo, the more attention must be paid to the clearing medium in order to distinguish internal structures. Methyl salicylate is admirable for pig embryos of all sizes and even for small fetuses. I found ethyl salicylate to be as good if not better, but it is more expensive. Canada balsam has about the same refractive index ( n D = 1.535) as methyl salicylate ( n D = 1.536), but darkens with age.
Embryos may be placed directly from absolute alcohol, benzol, xylol, toluol or chloroform into methyl salicylate, but in order to obtain the proper refractive index, the preliminary fluid must all be removed. This may be evaporated, or washed out with more wintergreen. Benzol is to be recommended because it is cheapest and evaporates out most easily. The evaporation may be hastened by an air pump, which also removes any air bubbles that may get into the specimen. These bubbles expand and are absorbed after the pump is disconnected, or by successive pumpings. An ordinary air pump will cause the benzol and air to boil out. A water-suction air pump (aspirator) will suffice but a float valve and safety bottle should be interposed between the pump and the specimen to prevent the back flow of water. An exhaustible desiccator is convenient for holding large embryos while they are being pumped out. If the cover is well ground, the oil will seal it sufficiently, and vaseline should not be used.
Most of the internal organs may be distinguished in unstained embryos by cutting down the light. The individual cells of mesenchyme, cartilage and blood may be seen; the cellular structure of the neural tube is indicated by radial striations and the larger nerves appear as bundles of fibers. Some organs in smaller embryos are made more distinct by staining with very dilute alum haematin containing a large amount of acid.
Even in quite small embryos, many of the blood vessels may be traced by the blood cells, and the large empty veins followed as cavities. However, with the smaller vessels this becomes more laborious than serial sections. On the other hand, the injection of small embryos for class use means quite an outlay of time. Therefore, it seemed necessary to find some way to fix the haemoglobin, and keep the vessels full, in order to distinguish the vessels by the color of the blood. I found that the same method that prevents the cytolysis of nephric tubule cells prevents haemolysis.
Living embryos are obtained, the amnion opened, the placenta squeezed to force the blood into the embryo, and the umbilicus tied or clamped. Artery clamps are too strong and pinch off the cord. (I made clamps out of wire (lower part of fig. 1) in order to avoid tying so many cords at the slaughter house. The clamp may be removed in half an hour and used again.) The embryo is dropped into the neutral-formol-sugar mixture described above, and left until thoroughly fixed. In case of a fetus, part of the skin should be torn off after the superficial blood vessels are fixed, to insure penetration of the formaldehyde. A hole may be made in the skull by slicing off a small piece tangentially or by a sagittal cut near (to the right of) the median plane. Large fetuses, unless skinned completely, will have to be scraped to remove the pigment layer.
Transfer the specimen after washing, or directly, from the fixing fluid to alcohol of about 70 per cent. After they have hardened in 80 or 95 per cent alcohol it is well to split the large specimens by a sagittal cut a little to the right of the median plane with a very thin bladed knife. The dehydration with higher alcohols should be slow enough to prevent shriveling.
By this method the blood retains its color, and although it does not take the place of injection, it is a great help to the student. I have inserted three figures to show what can be seen in such specimens.
Figure 1 represents a pig embryo about 8 mm. long. The nerve tube, fore gut, mesonephros, liver, heart, eye and ear are clearly seen. The arterial system and part of the cardinals and subcardinals can be distinguished. The notochord is distinct, and the 5th, 7th, 8th, 10th, and 11th cranial nerve roots can be made out. Figures 2 and 3 are described in the appendix.
I have prepared hundreds of pig embryos and fetuses in this way, and also injected many with india ink and cleared them in wintergreen oil. A completely injected fetus can only be studied in comparatively thin (freehand) sections. Various degrees of partial injection are very useful to show the larger vessels, but these may be seen in the uninjected fetuses. The left side of an uninjected fetus which has beeD cleaved a little to the right of the median plane, will show the general circulation, except in the liver. The larger vessels in the liver may be seen by removing the lateral portions and passing a strong light through the remainder (an arc light is excellent), or the liver may be removed and cut into slices. In injected specimens the liver is hopeless.
I washed with alcohol the blood out of the vessels of a fetus 4 inches long and cleared it in wintergeen oil, then injected it with mercury. This method has the advantage that the extent of the injection may be watched and controlled.
The injection may be limited by using a coarse granular pigment that will not go into the capillaries. A gelatine mass is not absolutely necessary to hold the pigment. A light colored opaque pigment has the advantage that it may be seen by transmitted or reflected light.
The arteries may be injected and the haemoglobin fixed in the veins, giving handsome specimens. If it is desired to show only the injection, no formalin should be used. Much of the haemoglobin may be dissolved out by putting the fresh specimen into weak alcohol or alcohol and acetic acid. All of the haemoglobin may be removed with dilute acetic acid provided an injection is used that is not affected by this acid.
Appendix on the Arteries and Veins in a 30 mm Pig Embryo
The method of fixing the haemoglobin and clearing in wintergreen oil to show the course of the vessels has been especially successful in case of pig embryos of about 30 mm. length Figures 2 and 3 show the larger vessels of the median plane and left side of one of them. The courses of most of the vessels approach the type of the adult pig and show distinctions in topography from those in man. The common carotid artery and (right) innominate artery arise from a common trunk, the brachiocephalic artery. The posterior inferior cerebellar artery arises from the basilar instead of from the vertebral.
Notwithstanding the great development of the vena cava, the left posterior cardinal is of considerable size. The right cardinal (not figured) is smaller. The thoraco-epigastric vein is divided into two parts, one of which drains anteriorly into the internal mammary.
The vessels of the limbs could not be completely followed, but enough was seen to demonstrate that they differ very much from those in the adult.
Besides the vessels, the mouth cavity, brain, eye, endolymphatic labyrinth, lungs, mesonephros, kidney, testis and penis are outlined in the figures.