Paper - Intra-uterine absorption of ova (1917)
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Meyer AW. Intra-uterine absorption of ova. (1917) Anat. Rec. 12: 293-.
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Intra-Uterine Absorption of Ova
Arthur William Meyer
From the Division of Anatomy of the Stanford Medical School
Seven Figures
While collecting embryological material for other purposes a decade since, my attention was not infrequently arrested by the presence of a degenerating or retarded sheep embryo in an apparently normal uterus the other horn of which contained an apparently normal foetus. Although these embryos were not infrequently quite normal in form they were always decidedly smaller than the normal embryos. In most cases the conditions suggested pathological changes both within the uterus and the embryo. The amniotic fluid was sometimes intensely turbid and even milky and more fre(iuently dark in appearance and very evidently contained degenerating blood. Since twin pregnancies are not so very frequent in sheep, the number of cases seen was necessarily small. In uteri containing normal foetuses in the early stages of development, the embryo, the development of which had been arrested, was often represented by a rather finn fleshy mass of regular form which sometimes showed unmistakable evidences of degeneration even upon inspection with the unaided eye.
While engaged in the determination of the curve of prenatal growth in the guinea pig. Draper and myself found several cases of abnormal or at least regressive ova. All these abnormal guinea pig ova were much smaller than the nonnally developed ones of corresponding age should have been. Indeed, most of them were represented by firm o^'al fleshy masses, some of which possessed protuberances which made them look bicornuate. All were fastened with one end in more or less distinct uterine crypts which were especially evident in one of the smaller ova. The instances met with so far were seen in pigs killed 19, 25 and 37 days after coitus.
In the first case that of guinea pig Xo. 16 only a single ovum was found present twenty-five days after coitus. This ovum was contained in the distal extremity of the right horn and was surrounded by a reddish black fluid. The uterus and adnexa appeared wholly normal to the naked eye. The o\iim was composed of a slightly oval fleshy mass only 5 mm. in diameter although the normal embryo of this age measures 17 mm. It had a smooth regular surface and was still attached to the opened uterus but was easily detached. Xo placenta or foetal membranes were recognizable and the ovum protruded freely into the opened uterine cavity being attached to the uterine mucosa by its base with its longest diameter perpendicular to the latter. The line of attachment on the ovum apparently formed about one-eighth of its total perimeter.
On sectioning, the tissues of this ovmn were found to be decidedly degenerated, the outer layers being composed of nothing but cell detritus. A little beneath the surface, this cell detritus is mixed with degenerating mesenchyme and variously-sized, better-preserved epithelioid cells. Between the latter lie large numbers of erythrocytes. These are scattered about freely and occupy other areas almost exclusively. Polykaryocytes and megakaryocytes in various stages of degeneration and different forms of leucocytes are also present. Some of the giant cells contain very bright golden pigment some of which is found also extra-cellularly. The framework of degenerating embryonic connective tissue, contains scattered cells and groups of cells with extremely large vesicular nuclei and prominent nucleoli.
Deeper beneath the surface remnants of blood vessels and of a reticulum which reminds one of that in young lymph nodes can be seen. In some areas, however, nothing but the degenerating reticulum with a little granular detritus remains. In addition to the giant cells large irregular masses which look like fused giant or other cells are also scattered through the specimen.
Sections made through the middle of the ovum show that the portion nearest the area of contact with the uterine mucosa is best preserved and composed of a syncytium-like mass in which large vesicular nuclei predominate. This portion also contains a large vesicle lined by a low embryonic epithelioid syncytium. The spherical vesicle which in its largest portion comprises more than one half the diameter of the ovum contains nothing but a transparent fluid. Similar much smaller vesicles are also scattered about throughout the rest of this portion of the ovmn some lying isolated at its very perimeter. A similar low epithelioid layer with indistinguishable cell boundaries also covers a portion of the surface of the most degenerated distal portion of the ovum where it also clothes villus-like extensions from the main mass. Some of the sections are almost surrounded by this epithelioid layer.
Small areas of these sections are practically de\'oid of tissue and contain almost nothing but a faint reticular network enclosing a slightly granular detritus and many polymorphonuclear leucocytes the nuclei of which have a typical horseshoe shape and the protoplasm of which is acidophile. Some of these leucocytes look decidedly degenerate and none seem to be phagocytic. In other often adjacent areas, the place of the polymorphonuclear leucocytes is taken by somewhat large cells with a vesicular nucleus which is circular in outline. The protoj^lasm of many of these cells is acidopliile but here and there groups which look bright golden are seen. Most of these cells are well-preserved but some of them can be seen to be filled with smiilarly staining erythrocytes and what look like fragments and granules of erythrocytes.
Specimen No. 17 taken from a pig pregnant 19 days contained three ova. a normal one in the left horn and two abnoi-mal ova in the right. The normal embryo weighed 35 mgm. and the smaller of the abnoi-mal ova was approximately' as large as the placenta and membranes of the normal embryo which weighed 0.91 mgm. The larger ovum was bicornuate. Both were single masses and no distinct placental portion was recognizable.
Both these ova whicli were no larger than a normal embryo of this age, were regular in form and their surfaces smooth. Upon microscopical examination, however, a few small, villus-like extensions were seen on the distal portion. A few small indentations were also evident but nothing else interrupted the regularity of the rest of the surfaces. The larger of these two ova was very well-preserved and much more vascular than that from pig No. 16. It was covered throughout by a low epithelioid syncytium which evidently was originally composed of a low cubical epithelium for here and there cell outlines are still faintly visible or the free surface of the syncytium is indented quite regularly so as to look crenated (fig. 1). These crenations are evidently the result of projections formed by the individual cells with the indentations located in the region of the former cell boundaries. The slightly irregularly-shaped, evenly-staining nuclei are thickly packed and although the cell boundaries are not clearly recognizable the layer is low and in places contains indistinct lines which look like remnants of cell boundaries and justify one in characterizing the cells as cuboidal. The rest of the ovum is composed of a syncytium containing large vesicular nuclei as shown in figure 2. Cell boundaries are distinct nowhere but the tissue apparently was a large-celled mesenchyme originally. Only a few small areas of almost complete degeneration are present. The tissue is densest and least vascular near the region of attachment to the uterine wall. The most rarefied tissue is found in the two cornua and near the distal portion where the loose mesenchyme contains small bloodvessels. Immediately beneath the investing epithelioid layer the specimen is completely canalized by wide capillaries which form an exceedingly vascular peripheral layer. A bit of tl^^ less ^'ascular l)ortion is shown in figure 3.
This specimen contains no large cavities but numerous smaller epithelioid-lined spaces are found in the cormia and the distal
Fig. 1 Structure of ;i jjortion of the jxTiplicrv of ;i iiiiictccu-daA' ovum. X 475.
Fig. 2 Stru(;tur(' of a jtortion of the pcriplicrv of anollicv ovum of tlu> .same age. X ol")
Fig. 3 Structure of the va.scular i)or(ion of the ovum .shown in fifj;ur(> 2. X475
Fig. 4 Large nueh'i from tlic cciilral iircrotic area of one of these ova. X475
INTRA-UTERINE ABSORPTION OF OVA
297
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298 ARTHUR WILLIAM MEYER
portions. The largest cavity which is hned by a rather low degenerated epithelioid layer is found in the center of the specimen but it is so small that it is not visible with the naked eye in the stained section. Although better preserved these cavities or C3^sts are similar to the very large one contained in the previous specimen. In spite of the condition existing in this specimen the blood is all contained in vessels only a few of which can be distinguished as veins or arteries. Some of the small arteries which are located in the basilar portion of the ovum near the uterine attachment have become completeh' obliterated. The portion of the ovum near the area of attachment is almost non-vascular as in the previous specimen. In some of the outer portions, however, and also near the placental attachment the tissue is of a more fibrous nature and looks far less embryonic. The blood cells are well-preserved and all the leucocytes have round vesicular nuclei in contrast to those found in specimen 16.
The second specimen from No. 17 was somewhat smaller and without cornua but it also was surrounded by an abundantly nucleated syncytium and was canalized beneath its surface by numerous capillaries as shown in figure 1. Portions of the surface were also pitted by crypts which gave the periphery of the sections a fenestrated appearance. All these crypts and vesicles are lined by a similar syncytial layer and all are empt3^ The specimen like the previous one is most vascular near the surface and near the fenestrated portion where the tissue composing it is also much looser. Only a few ^•illus-like ))rocesses are seen in the distal portion.
Although half — apparently tlio proximal lialf — of this specimen was lost, the structure of the remaining half is practically the same as that of the previous ovum. Its ])reservation is not quite so good, however, for it ccmtains ])artially necrotic and small liquefied areas in its interior. The more necrotic portions of this ovum contained nuclei truly gigantic in size. This will be evident on comparing the magnification of figure 4 with those in 1 and 3. It too is quite vascular and some of the vessels all of which are full of blood, are extremely large. Some portions of this ovum look more like fibrous mesenchyme others
INTRA-UTERINE ABSORPTION OF OVA 299
more like sarcomatous tissue, but cell outlines are nowhere evident.
Specimen 19 in which the period of gestation was twenty-six days contained five abnormal ova, two in the left and three in the right horn. All of these were quite equally-sized, irregular masses but they were only about two-thirds as large as a normal placenta with that duration of pregnancy. They were easily detached and projected freely from the opened uterine cavity as had those in the previous cases. In all except one ovum the placental crypts were very shallow fossae but this specimen was contained in a definite funnel-formed uterine crypt about 3 by 3 mm. in size. Since these crj'pts were not noticed until the ova had been removed from the uterus I am inclined to think, however, that they were formed mainly by the post mortem contractions of the uterine musculature. This assumption is also suggested by the fact that all these ova completely fitted the Imnen of the uterus and formed slight elevations on its surface. No placental portion was recognizable with the naked eye and there was no gross evidence of pathological changes in the uterus.
Although these five specimens of abnormal ova varied somewhat in size this variation was not marked. All were from 4 to 6 mm. long and 2 to 4 mm. thick and in contrast to the preceding specimeuvs the four oxa which were removed from the uterus had a dull fuzzy instead of a smooth shiny surface. They were exceedingly soft and rather irregular in shape. The contracted uterus which looked entirely normal was nodular in consequence of the enlargement opposite the ova. It contained no exudate and upon microscopical examination the ova were found well-preserved. All these specimens were but slightly vascular, the small capillaries being located mainly in the peripheral layers as before. They were all devoid of an outer epithelioid layer and were composed of a syncytium containing large nuclei none of which were nearly as large as those found in the preceding specimens, however.
As in the previous specimen the largest nuclei were found in the interior of t^e ova and the smallest at the surface where they were more elongated and where the s^aicytium took on a more
300 ARTHUR WILLIAM MEYER
fibrous and stratified character because the tissues and the long axes of the nuclei, were arranged parallel to the surface. In one portion of one o\aim the extremely large cells with their large oval nuclei are still preserved and give one a good idea of what the original structure of the ovum, in the earlj^ stages of degeneration really was.
Xo other type of cell was found except that the formation of the giant cell masses is indicated through coalescence of adjacent degenerating cells. The capillaries are engorged with erythrocytes and a few leucocytes with vesicular nuclei, but no vessels larger than capillaries of the ordinary calibre are present anywhere. The structure of these ova at the region of the uterine attachment corresponds to the rest.
One of these specmiens still shows a little of an epithelioid covering in two very small places. In one of these the epithelium is sho^^^i in the form of a tube which may represent the remnant of a crypt or ai mvagination. Although this ovum is completely canalized by a plexus of fine capillaries near the periphery it contains no larger vessels in its interior.
One of these five abnormal ova from No. 19 was left in situ and cut serially in paraffine. It measured 5.5 mm. in diameter after fixation and completely filled the uterine cavity except in a few areas where small spaces were left between the ovum and the uterine wall. A cross section of the uterus and the contained ovum measured 6.5 mm. The musculature of the uterus was thickened nowhere, there were no indications of invasion of it by the tissues composing the fleshy mass nor was there any indication of cellular infiltration. Under low magnification the uterine nmcosa was evident nowhere, however, and as seen in figure 5 there seemed to be no definite line of demarcation between the ovuiii and the surrounding uterine wall. The central portion of tlie fleshy mass was less dense and ccmtained a relatively large incgularly-shaped degenei'ation cavity which contained what looived like a remnant of the embryo some portions of which were in direct contact with the surrounding tissue. The whole ovum was quite unifomi in structure, however, though its vascuhirity varied somewliat.
INTRA-UTERINE ABSORPTION OF OVA
301
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Fig. 5 Uterino wall with a i^ortioii of X\w i)('rii)luM-y of an ovum of twent>five days. X 1340
Under higlier magnification it was seen that glands of the uterine mucosa were present in a zone of loose mature connective tissue which probably took its origm from the submucosa and the inner portions of which were mingled with the tissues at the periphery of the ovum. The latter were also fibrous in character but much looser and more vascular and looked quite like normal emhiyonic tissues. They stained more with orange G,
302 ARTHUR WILLIAM MEYER
however, while the maternal submucosa which contained some uterine glands stained deeply with fuchsin.
The outer portions of this ovmn also, were composed of a loose fibrous connective tissue containing numerous capillaries filled with blood the erythrocytes of which were well-preserved. The thickness of this outer thin layer varied somewhat and from it to the center of the mass there was a gradual transition to large epithelioid cells which were plainly necrotic in places around the cavity which contained a remnant of the embryo. The latter was represented by very irregularly-shaped, folded hollow tubes composed of one and two layers of epithelioid cells which also showed signs of degeneration and apparently represent the ectoderm and entoderm. In one portion, shown in figure 6 a-65 and a-101 an indication of the mesoderm also seems to be present.
In some portions this embryonic tube was two-layered being composed of an outer layer of cubical and an inner of polygonal cells with large vesicular nuclei as shown in figure 6 a-1. In other portions the order of these layers seems to be the reverse. There were no evidences of phagocytosis and giant cells were not seen.
The next specimen was obtained from a guinea pig killed thirty-seven days after coitus. There were four fetuses, two in each horn. The distal one in the left horn was very evidently considerably smaller than the other three. The three large unopened apparently normal specimens weighed 12.2, 13.1 and 13.1 grams and the respective embryos measured 4.3, 4.5 and 4.4 mm. The fourth specimen which was abnormal weighed only 0.1 grams. A remnant of the foetus seemed to be contained in what looked like the greatly folded and collapsed membi'anes. The placental disc measured 1.5 cms. in diameter as compared to the normal ones which measured 2 cms. From these measurements and also from the weight of this intact spechnen it is evident that the embryo in this ovum must have degenerated almost completely. This inference is also borne out by the microscopic examination. But the most interesting thing was the fact that abortion had not occurred.
INTRA-UTERINE ABSORPTION OF OVA
303
Moreover, the fact that this specimen was contained not only in what appeared to be a perfectly normal uterus but was implanted within less than 1 cm. of a perfectly normally developed embryo is equally interesting and significant. The latter was as heavy and practically as large as the larger of the two embryos in
(Z/O/
Fig. 6 Sections from an embryo contained in tlie ovum a portion of Avhich is shown in figure 5. The numbers under the ilhistrations represent the number of the section which was drawn. The sections Avere cut 10 m thick. X 47.5
ova side by side with normal ones in apparently perfectly normal uteri. Although the abnormal ova found by Huber were very much younger than the specimen here recorded, the significance of the facts maj' be the same.
An incision made through this fixed specimen showed a necrotic hemorrhagic area in the center of the u-shaped mass of the
304 ARTHUR WILLIAM MEYER
fixed ph'centa. Upon microscopic examination the most striking thing was the remarkable phagocytic activity in the center and the entire absence of comparable phenomena in the periphery of the placenta. There is an entire absence of phagocytosis and of hemorrhagic areas here although the nuclei in the mesenchyme are extremely large and degenerate.
It is as if absorption of this embrj-o and placenta were taking place from the interior of the specimen. The portion of the placenta directly beneath the embryo is decidedly hemorrhagic
Fig. 7 A portion from the degenerating area of the placenta directly beneath the remnant of the ovum showing the remarkable number of macrophages, y 515
and very necrotic. Many of the large mononuclear phagocj^tic cells found in the central area and shown in figure 7, are so degenerate that they are mere shadows. Others are full of vacuoles and still others of erythrocytes and cell detritus. Many of them look so necrotic that one must doubt their ability to have remained actiA'ely phagocytic much longer even if they possess cell inclusions. The miclei of the larger cells are usually small and not very evident but in the small cells which possess fewer or no cell inclusions they are relatively larger and are also stained better Most of these macrophages have an acidophile
INTRA-UTERINE ABSORPTION OF OVA 305
protoplasm. Although much blood is contained in this necrotic area only very few poljTnorphonuclear leucocytes are seen and these do not show evidence of phagocytic activity. In some areas these large phagocytes lie in a wide-meshed reticulum. For a discussion of the origin, relation and properties of these cells the reader is referred to Evans '15.
From figure 6 it is quite e\'ident that although these ova may have developed normally up to a certain point they must subsequently have developed abnormallj'. The disproportion between the size of the embryo and the placenta alone shows this. Furthermore, since the very early rat o^'a described by Huber already showed degeneration phenomena it follows from this as well as from the relatively large size of the placenta that the life of the embrj'o must ha\'e been prolonged for a considerable period of time.
The cause of death of these ova must, to be sure, remain a matter of conjecture although the gross and microscopic character of the uteri would seem to indicate that the cause probably was intra- rather than extra-embryonic if it was not due to defect in the corpora lutea. At any rate death of the o\'um did not seem to be due to a defecti\'e placental de\-clopment although it must be borne in mind that it is possible even if not probable, that the uterine site upon which implantation occurred may nevertheless have been pathological. Such an assumption is made very unlikely, by both the apparently normal development of the placentae and b}- the surprisingly extensive development of the latter. The latter fact also seems to indicate that the early placenta even possesses considerable independence of the embryo.
Although the question as to whether or not abortion would finally have occurred in these cases must remain a matter for conjecture, I am ready to believe that such a termination would not have occurred. To be sure, such an assumption presupposes the gradual absorption of these ova, embryos and placentae and also raises the question as to what percentage of pregnancies terminate spontaneously in this way even after considerable development has occurred. If such a regression and
306 ARTHUR WILLIAM MEYER
absorption occur in man also the surprising percentages of spontaneous terminations of pregnancies given by Mall '08 and '10 would be increased still further.
It will be recalled that Frankel '03 was able to cause death and intra-uterine absorption of ova in rabbits up to the twentieth day of pregnancy through destruction of the corpora lutea. Frankel found that abortion did not occur in these rabbits and described the gross changes as follows. The eggchambers which became less tense because of a decrease in the production of amniotic fluid also became folded longitudinally, wrinkled and changed in color from a very red to a pale yellow. Frankel considered the reduction in the quantity of amniotic fluid as the first sign of regression and noted that the spherical chamber became more elongated, cylindrical, firmer and nodular. The embryo also became drier, smaller and more unrecognizable, finally being dissolved and represented only b}" an amorphous grayish white 'Schmiere.'
The placenta was preserved the longest and could be recognized as such for several days later. But it also became dry and pale red, only a few fragments finally remaining in the longitudinally folded and slightly swollen mesometrium. In this stage the uterus was only slightlj^ firmer and showed but a minimal enlargement. After fourteen days even these evidences of a past pregnancy had disappeared only an anemic ring remaining and after three weeks not the least indication was left of the interrupted pregnancy.
Although the intra-uterine absor])tion of all guinea pig ova belonging to a single pregnancy could be due to defective de\'elopment of the corpora lutea or entire lack of development of the latter it is much more difficult to see how regression and absorption of a single ovum lying between apparently normal ova could occur. It is conceivable, of course, that the growth of the coi'pora lutea might be sufficient for the development of four and not for six or more ova but one would expect all to suffer a corresponding retardation ratlier than have one or two destroyed and the rest preserved.
INTRA-UTERINE ABSORPTION OF OVA 307
Since the ovaries of the guinea pigs concerned in these incidental observations were unfortunately not preserved I am unable to report on their condition. Nevertheless, even this small series of cases indicates that intra-uterine absorption of ova is not a rare phenomenon in guinea pigs. This conclusion is also in entire accord with Frankel's observation regarding rabbits. According to Koebner, Frankel concluded that the physiological regression of one chamber is very common, and emphasized that in many cases not all foetuses reach normal development. Individual fetuses die and are resorbed together with the placenta, while the rest go on to maturity. From these observations of Frankel which so far as I can learn were drawn from experimental work, Koebner observes that the rabbit is apparently less disposed to abortion in the first twenty days of pregnancy than to a "dry degeneration and resorption" of the fetus. Koebner '10 found that the bones also are absorbed under experimental conditions in the rabbit, and Williams '16 while discussing the subject of missed abortion in women states that "In very exceptional instances the entire product of conception may be absorbed without a sign of external discharge. Polano and L. Frankel have reported cases in which this occurred after the pregnancy had advanced as far as the fourth month and Koebner has demonstrated its possibility by animal experiments."
LITERATURE CITED
Evans, H. M. 1915 The macrophages of mammals. American Journal of Physiologj', vol. 37.
Frankel, L. 1903 Die Function des Corpus luteum. Arch, fur Gyn., Bd. 68.
HuBER, G. Carl 1915 The development of the albino rat Mus nor\-egicu3 albinus. II. Abnormal ova; end of the first to the end of the ninth day. Wistar Institute of Anatomy and Biology, Philadelphia.
Koebner 1910 Knochenresorption bei intra-uterinen Eischwund. Arch, fiir Gyn., Bd. 91.
Mall, F. P. 1908 A study of the causes underlying the origin of human monsters. Wistar Institute of Anatomy and Biology, Philadelphia. 1910 The pathology of the human ovum. Keibel and Mall, Manual of Human Embryology. Lippincott Company, Philadelphia.
Williams, J. Whitridge 1916 Obstetrics. New York and London.
THE ANATOMICAX, RECORD, VOL. 12, NO. 2
METHODS OF MOUNTING SECTIONS IN GELATIN
J. B. JOHNSTON ANT) EDNA G. DYAR
University of Minnesota
Gelatin has been used to some extent by various European workers for mounting sections, but has not come into general use. Our experiments show that gelatin possesses three properties which render it undesirable for this purpose until overcome b}' special treatment. Gelatin is non-hydroscopic, brittle and inelastic, and instable. In the extremely dry winter climate of Minnesota the gelatin dries and shrinks imtil it bends or breaks the glass slide on which it is spread, or cracks and peels off the slide. In very moist sunmier weather the gelatin may become whitish-opaque from moisture. ^Vhen these qualities are corrected by the addition of glycerin to render the film hydroscopic, sugar to make it elastic and phable and some hardening agent to render it stable, a satisfactory and permanent mounting medium is secured. To render the gelatin insoluble in water, formalin, chrome alum, chromic acid, tannic acid or potassium bichromate may be used with varying results. The most satisfactory product is obtained by means of potassium bichromate or chrome alum. After the addition of one of these salts the gelatin is rendered insoluble upon exposure to light. There is also produced a very shght gra^-ish-green tint by the chrome alum and a deeper greenish tint by the bichromate, but this does no harm in thin films. Perhaps the best results are obtained by protecting the gelatin by means of an insulating varnish. The varnish seals the gelatin against the action of atmospheric vapor and also prevents the evaporation of water from the hardened gelatin film, the presence of water being necessary to maintain elasticity and pliability.
The advantages of a gelatin mounting medium are: saving of expense of dehj'dration for mounting in balsam and of the cost of cover glasses, and availability in some cases where an aqueous mediima is necessary. It can be used on sections stained "with haematoxj'hn, carmine and some but not all of the anilin dyes (e.g., not with acid stains soluble in water). Its use in films without glass may prove to have some value.
SOLUTIONS TO BE EMPLOYED
A. Best quality photogelatin 5 grams. Distilled water 100 cc. Add glycerin 5 cc. Let stand two hours. Raise to 50°C. Gelatin dissolves. Filter through canton flannel. 309
310 J. B. JOHNSTON AND EDNA G. DYAR
B. Hydi'ate 2 grams, gelatin in 45 cc. distilled water. Add 10 cc. glycerin and 15 cc. corn syrup. Raise to 50°C.
When gelatin is dissolved add 18 grams gelatin hydrated and
dissolved in 55 cc. distilled water. Filter through canton flannel.
C. Prepare as in B, using 2 gi-ams gelatin and 45 cc. water and
23 grams gelatin and 55 cc. water.
D. To render A, B, or C insoluble add to the solution prepared
as above 1| cc. of a 10 per cent solution of potassium bichromate or chrome alum to every 100 cc. of the gelatin solution. This solution must be used at once, since it will not melt after being allowed to harden. In making the above solutions a temperatm-e of 45° to 48°C. is necessary to dissolve the gelatin. After the gelatin is dissolved the solution may be lowered to 30°C. without causing the gelatin to set. In mounting sections it is necessary to keep the gelatin sufficiently warm to secure penetration, but advantage should be taken of the fact that the gelatin remains fluid at lower temperatures which are less likely to harm the tissues. In the following directions it is intended that the gelatin itself shall be kept at about the temperatures indicated, whether by means of an oven or a constant temperature plate.
For paraffin sections
Bring into distilled water on the slide. Place slide on constant temperature plate at 35°C. and cover with sufficient solution A to make a complete covering film when dry. After a few minutes on the warm plate set in a horizontal position to harden.
For celloidin sections up to a thickness of 100 microns
Carry the sections on paper ('onion skin' best). Clear in glycerin and water over night, followed by glycerin several hours.
Immerse sections in solution A in flat dish at 35°C., 20 minutes. Clean and flame^ a slide and place on warm plate. Spread on slide a small amount of solution A and immediately place section on it, removing the paper.
Carefully press out all air bubbles.
Add more solution A and then drain to secm-e a thin but complete covering for the section.
Keep on level warm plate until gelatin is evenly spread.
Place in horizontal position to dry at room temperature.
'Flaming the slides is necessary to remove the thin film of organic material which is taken up from the air by slides e.xposed for any length of time. The usual cleaning solutions do not wholly remove this.
MOUNTING SECTIONS IN GELATIN 311
To secure a sufficiently thick and even covering, when the five per cent gelatin is used it is best to make the first coat thin and apply a second and third coat to the slide when cold, draining each time.
For thicker celloidin sections (1 to 2 mm.)
After clearing in glycerin immerse the sections for at least three hours in solution A at as low a temperature as practicable (30°C.) and mount in solution B by the method just given.
For mounting sections in films without glass
Fasten together two very thin celluloid films by means of snaps on one edge, place the section or sections between them and immerse in water or glycerin at 35°C. Drain, separate the films and sections as they are immersed in solution B in a flat dish on warm plate. Abundance of gelatin solution must be used, to secure complete irmnersion. After twenty minutes carefully press out the air bubbles with the fingers or a rubber wedge. Attach snap hangers to all fom- corners of the film and hang up to dry at room temperature. The hangers at the lower end serve as weights to prevent warping.
For thick sections (1 to 2 mm.) in films, immerse first in solution A at least three hours and then mount as just directed, using solution C. As this thick solution does not drain off readily, the excess gelatin should be removed from the surface by stripping the film between thumb and finger.
Solutions A, B, and C may have thymol added and be kept cold and remelted when wanted.
Finally, in each of the above cases either an insoluble preparation (solution D) should be used or the finished preparation should be covered with a varnish, as soon as it is dry enough not to be sticky. One may use the Zapon varnish found in the market. The same may be made by dissolving 5 grams celluloid in 20 cc. amyl acetate and 80 cc. acetone. A good varnish which is more pleasant to use is made by Abney as follows:
Alcohol 20 ounces
Ether 40 ounces
Pyroxj'lin or celloidin 400 grains
In applying the varnish care should be taken to have the gelatin completely covered. Films should be dipped in the varnish, and on slides the varnish should be spread with brush or spray beyond the edges of the gelatin.
December 8, 1916
THE VALUE OF ABSOLUTE ALCOHOL FOR REMOVING ADHERENT PARAFFIN SECTIONS FROM PAPER OR PASTEBOARD TRAYS
LOUIS H. KORXDERi From the Anatomical Laboratory of the Northwestern University Medical School
Laboratory workers in embryology-, histologj^, and nem-ology, and especially those whose work calls for the mounting of serial paraffin sections, have all at some time been annoyed by the adhesion of their sections to the paper or pasteboard tray on which they had been placed prior to mounting. This occurs, of course, most frequently during the warmer months of the year or where sections are not mounted for a long time; thus leading often to a complete loss of a valuable series.
Because of this it may be of value to call attention to the use of absolute alcohol as a means for overcoming this difficulty. The amount of alcohol used is very shght, being just sufficient to moisten the sections completely and in addition overrun on the sides so as partially to impregnate the paper or pasteboard to which thej adhere. After this, several minutes time should be allowed for evaporation of the alcohol. This requires only a brief period, but can be hastened if a current of cool air is allowed to strike the tray. Where the sections adhere with especial firmness this last is particularly desirable.
It is of advantage when deahng ^^^th large sections to take a sectionhfter or fine-bladed scalpel and run it along under the edge of the sections before the alcohol "has completely evaporated. After this procedure sections which adhered firmly loosen almost always with ease.
The use of 95 per cent alcohol is not recommended since with it satisfactory results are less often obtained. It does not evaporate with the rapidity and thoroughness of the absolute alcohol. The latter vaporizes very readily and to this its action of loosening paraffin sections from paper or pasteboard trays may possibly be ascribed.
> Contribution No. 47, November 1, 1016.
312
PRESERVATION OF ANATOMIC DISSECTIONS WITH
PERMANENT COLOR OF MUSCLES,
VESSELS AND ORGANS
A SUPPLEMENTARY NOTE, DESCRIBING ANOTHER METHOD, THE CURING METHOD
EDMOND SOUCHON
Professor Emeritus oj Anatomy, Tulane University of Louisiana
Since the printing of the original manuscript in the Anatomical Record, Vol. 10, No. 1, November, 1915, I have found out a new and simpler method, the cm'ing method. It consists in the f ollow-ing : After the completion of the dissection, if the muscles present a dark brown color, the preparation is immersed in G33C1 for three days. G33C1 means a solution composed of 33 per cent of glycerine, 1 per cent of carbolic acid and 66 per cent of water. It registers 10° Baumd.
At the end of the three days it is taken out and exposed to the air in a room until the muscles become black. This requires about ten days. If some muscles do not blacken as fast as the others, they should be painted daily with pure glycerine until they become as black as the others.
As soon as all the muscles are black the preparation is immersed in Ch.F75, i.e., a solution of chloride with 75 per cent of formal, as previously described.
Then it is placed permanently in Ch.F5.
The results are quite satisfactory. The advantage of this method is that it does away with the use of calcium chloride, described previoush^, which is not always uniform.
Pale muscles do not do so well by this method. They should be painted in preference.
I have also brought out the following points which are of some assistance in the work.
In using tallow for distending the arteries, it is best to use beef tallow from around the kidneys. It is obtained from the butchers at the market. Tallow from corn fed beef is the best.
When the dissection is completed, if the muscles present a dark brown color they are suitable for the curing method. The}' are comparatively scarce. If the muscles show a lighter color than dark brown they are suitable for the paint method. They are much more common than the dark brown.
313
314 EDMOXD SOUCHON
In the final preservation of curing preparations Ch.F5 will do as well as Ch.F.20; thus effecting a marked saving in the cost of the permanent solution.
I found that the following mixture of paint gives better results: Tuscan red, half teaspoonful; turpentine, 2 teaspoonfuls ; lamp black, 2 grains. The lamp black tones down the Tuscan red w^hen too bright in the solution. This quantitij' is more than enough to paint two or three times all the muscles of the upper extremity. Try the mixed paint on 1 or 2 inches of a muscle to see how the color will show and modify the paint accordingl}'.
When using lamp black, any dark red paint will do. Tuscan red is not so essential.
Two or three thin coats of paint are better than one thick coat. Thick coats make preparations look like daubs.
When the preparation is placed in the solution, if the color is not satisfactory, too hght or too bright, do not hesitate to take it out, expose to the air for a few hom'S to ch-y and then repaint, with a suitably prepared paint. Do this two or three times if necessary. Final success depends on it.
Painted preparations do better decidedly on A10F.5 than in AlO alone. They do not require filtering as often and filter better. The F.5 prevents the bacterial cloucUness. It does not seem to affect the color of the paint any more than AlO or A20.
Artists' oil paints (Winsor and Newi:on) are better for the vessels than any other. Deep Vermilion for the arteries and Permanent Blue for the veins.
WTien the paint remains in the cup over night, even with a lid, it becomes too thick and turpentine must be added to it before using it.
A flat brush of fine bristles about | inch wide is quite handy to paint large surfaces.
Keeping solutions clear all the time is the foundation of the preservation of the preparations.
When solutions have been filtered t\\'ice in succession, if they do not come out clear, they should be changed for fresh solutions.
Solutions verj' cloudy or very discolored filter badly. They clog the filters. It is best to make new fresh solutions.
When solutions get old (two or three years) they should be changed unless thev remain clear.
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