Paper - Methods used by C. H. Heuser in preparing and sectioning early embryos
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Heard OO. Methods used by C. H. Heuser in preparing and sectioning early embryos. (1957.) Carnegie Instn. Wash. Publ. 611, Contrib. Embryol., 36, 1-18.
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Methods Used by C. H. Heuser in Preparing and Sectioning Early Embryos
With 3 plates and 12 figures (1957)
Readers of the Contributions to Embryology have long appreciated the remarkable technical ability of Chester H. Heuser as shown not only in the preparations of early and monkey embryos, descriptions of which were published in collaboration with the late G. L. Streeter (:29, 1941), but also in those of human embryos, for example the perfect presomite specimen (Carnegie no. 30) described by Heuser (1932). When, owing to the enterprise and skill of A. T. Hertig and Iohn Rock, the Department of Embryology was entrusted with a unique studies of well preserved human embryos of the first three weeks, it was indeed fortunate that Dr. Heuser was at hand to prepare and section them and, with his unrivaled practical knowledge of very early mammalian embryology, to give advice and counsel on their study. Visiting colleagues who have seen Dr. Heuser working the microtome and have observed the many novel details of his technique have often urged that they be described in print. Dr. Heuser’s modesty and his desire for perfection, however, have deterred him from rendering this service to his colleagues. finally, with his consent, the present account was prepared in consultation with other members of the Department. The author, who was associated with Dr. Heuser for many years, has often assisted in the embedding and sectioning of the embryos. In collaboration with Dr. Heuser, and frequently also on his own initiative, he designed and constructed much of the special apparatus used in this work. Dr. Heuser has reviewed the draft of this paper and has given his approval.
- While this article was being prepared for press Dr. Heuser reached his seventieth birthday. The Staff of the Department of Embryology of the Carnegie Institution of Washington takes pleasure in presenting it in the Contributions to Embryology, which Dr. Heuser's skill has so greatly enriched, as a tribute on this occasion to the lifelong scientific devotion of a beloved colleague.
Collection and Preservation of Embryonic Material
Sources and Types
Human embryonic material received at the Carnegie boratory is of three kinds. The first group comprises iecimens received from physicians outside of Baltimore, irgely in response to periodic appeals circulated by the iboratory. They consist almost entirely of aborted or irgically removed chorions and of Fallopian tubes exised because of tubal pregnancy. The instructions to physicians advise that such specimens be fixed in to ver cent aqueous formol. Although not the ideal fixative, ormol is easily obtained and diluted for use. Specimens are safely kept and shipped in the same fluid without further attention. Furthermore, formol makes the tissues less opaque than do fixatives containing picric acid or nercurial salts, and thus leaves them in more favourable condition for examination and exploration.
The second group comprises specimens obtained by the gynecologists and obstetricians of Baltimore hospitals with whom the laboratory is in close touch. These collaborators are requested to telephone when they have a fresh specimen so that a messenger may be sent to bring the material for immediate attention at the laboratory. Such specimens are fixed, usually in Bouin’s picroacetic formol, by the experienced personnel of the laboratory.
The third group comprises specimens obtained by Member of the Department of Embryology, Carnegie Institution of VVashington,. 1921-1950; Research Associate, 1950-: Professor of Microscopic Anatomy, Medical College of Georgia, 1950-. special collaborators thoroughly familiar with the procedures for fixation and subsequent dehydration, who carry out these steps on the fresh material in their own laboratories. In this category are the embryos obtained by Dr. Arthur T. Hertig and Dr. John Rock at the Free Hospital for Women, Brookline, Massachusetts, and sectioned by Dr. Heuser, which have so greatly contributed to our knowledge of human development during the first two weeks.
It will be convenient to discuss first the handling of material already fixed, and the larger specimens which arrive at the laboratory in the fresh condition. These materials include naked embryos and fetuses; chorions of various sizes and in various conditions—intact or tom open; clots and decidual fragments which may contain embryonic tissues; extirpated uteri containing established pregnancies; and Fallopian tubes extirpated because of ectopic pregnancy.
The preliminary exploration of these diverse materials differs so much from specimen to specimen that only general suggestions can be given. The first step is, of course, to free the gestation sac of obscuring tissues, by cautiously opening the uterus, or by dissecting away clots and debris as the case may require. Chorions less than 5 mm. in diameter must usually be studied and handled under the microscope, as will be described below. Larger gestation sacs may be handled by the usual methods employed with biological specimens of ordinary dimensions. Chorionic sacs found in aborted clots or in intra abdominal clots may simply be washed off with physiological salt solution, freed of adherent organized material as far as is safely possible, studied in a preliminary way, and, if small, fixed by immersion. Chorions 10 mm. or more in diameter should be opened in order to observe the condition of the embryo, and to decide whether to prepare it for sectioning. This statement applies whether the chorionic sac is obtained in an extirpated uterus or as an aborted ovum.
An oviduct (Fallopian tube) containing a gestation sac should always be explored with care, for a good embryo may be present; indeed, it is the tubal pregnancies that offer the best chance of obtaining human embryos in the early somite stages.
A fresh pregnant tube should be dissected at once under physiological salt solution, since fixation adds greatly to the difficulty of exploration by forming adherent, stratified clots about the chorion. If, however, the specimen has been placed in fixing solution by the surgeon or pathologist, the embryologist must do the best he can. With either fresh or fixed material, working under the binocular microscope with finely painted forceps, needles, and scissors, he cautiously removes a portion of the wall of the oviduct so as to make a window into its lumen, and gently pulls away the clots or, when possible, dislodges them with a stream of salt solution. If chorionic villi are seen, he proceeds with great care to uncover the chorion or to locate the embryo if it has been extruded from the chorionic cavity. With a fixed specimen this operation will require patient, piecemeal removal of the clots, small bits being broken away one at a time with the needle.
The subsequent handling of the embryos will be described below, after the recovery of ova and blastocysts has been discussed.
Recovery of Segmenting Ova and Unimplanted Blastocysts
The special methods used by Dr. Heuser are primarily applicable to the material of the second and third classes mentioned above, that is, specimens obtained from local sources in fresh condition and specimens representing very early pregnancies adequately fixed before shipment to the laboratory.
The procedures to be described are naturally not limited to use with human material, but are for the most part applicable to mammalian embryos in general. In fact, they were largely worked out, in so far as they are original with Dr. Heuser and his co-workers, on other species, as will be realized by those who are familiar with the published reports of his studies, partly in collaboration with the late G. L. Streeter, on the early development of the domestic pig and the rhesus monkey. The only technique specifically designed for human material is that for opening the uterus, and even this was developed by experience with the similar though much smaller uterus of the rhesus monkey.
The human ovum, like that of most other mammals, is in the oviduct (Fallopian tube) for about three days after ovulation, and may be recovered by washing out the lumen of the tube. The excised Fallopian tube should be brought from the operating room in physiological salt solution. The investigator holds it directly over a small dish (embryological watch glass, fig. 1) in which a small amount of the salt solution has been placed. The lumen is then irrigated with salt solution by means of a pipette with a large rubber bulb, inserted into the fimbriated end of the tube. Usually the uterine end is sufficiently contracted to cause distention of the ampulla, so that the spaces between the folds are irrigated; if the end of the tube is not contracted, it may be closed by compressing it between thumb and finger until the ampulla is slightly distended. The egg usually emerges with the first fluid that passes into the dish. The free end of the tube may whip about as the fluid is ejected; hence great care must be taken to keep it over the dish at all times in order not to lose even a drop.
Fig. 1. Method used for recovery of tubal ova.
If the egg is not recovered at the first washing, the process is repeated. The tube may be lightly “milked" downward by the fingers, and finally a fine-pointed pipette may be inserted into the narrow lumen of the uterine end and the tube irrigated in the reverse direction.
Identification of 2/13 ovum. The fluid in the dish, adequately illuminated from below through the bottom of thc dish or by a nearly horizontal beam of light through a side, is now carefully examined under a binocular dissecting microscope at a magniﬁcation of about 20 times. The free-lying ovum is usually easy to ﬁnd. As its speciﬁc gravity is slightly greater than that of the salt solution, it sinks, and the microscope should be focused ﬁrst on the bottom of the dish. If there is too much cellular debris in the fluid, it may be pushed aside with a dissecting needle. The ovum when lying in a clear space in the fluid is usually identiﬁable, despite its small size, by its round outline, which is more regular and definite than that of any other organic material present, and also by the bright double contour presented by the zona pellucida. Degenerating segmenting ova may lose their spherical form and become ovoid or even lenticular. Often observers have been misled by the presence in tube washings of pseudomorulas formed from epithelial cells rolled into more or less spherical masses. Such masses may suggest a segmenting ovum so strongly as to require sectioning for identiﬁcation, but with increasing experience it is possible to rule out most of them by inspection at a magniﬁcation of 20.
Fixation and dehydration of these very small objects are described below.
Free Blastocyst in the Uterus
The human ovum is free in the uterus from about day 4 to day 6 after ovulation; it may be recovered from the uterine cavity by the following method taught to Dr. Hertig by Dr. Heuser in 1933 and used by Hertig and others ever since (cf. Hertig, Rock, Adams, and Mulligan, 1954) at the Free Hospital for Women, Brookline, Massachusetts. The present description is based on the joint experience of the two laboratories.
Opening and scare/ring the uterus. The uterus is washed with salt solution to remove excess blood and is placed in a Hat dish. The myometrium is incised with a scalpel through the areas of attachment of both broad ligaments, on each side, from the cervix to the region of the round ligaments, the muscle of the fundus being left uncut. The incision goes down to the base of the endometrium, which at this stage is left intact. Next, the specimen is immersed in a convenient dish, in physiological salt solution, which is promptly changed if it becomes clouded by blood. The uterine cavity is cautiously entered by incising the endometrium with a scalpel and ﬁne straight scissors, the latter being used chiefly t0 Open the interstitial part of the tubes. The incision is made under a binocular dissecting microscope at a magniﬁcation of 3 or 4 and with strong illumination. Hertig has recommended making the first incision into the uterine cavity on the side opposite the recent corpus luteum, so that a free ovum lying near the tubal orifice through which it entered the uterus will sink into the depths of the cavity and not be lost through the first incision. VVhen a small opening has been made, a well rounded pipette with an opening 3 or 4 mm. in diameter is inserted through it, and the fluid in the uterine cavity is pipetted OH and placed in an embryological watch glass for examination under the dissecting microscope. Then the uterine cavity may be very gently rinsed by pipetting a little salt solution in and out, the washings being kept for examination. To avoid injuring an implanted embryo, the pipette should never be inserted beyond the edge of the endometrium.
Handling of Ova and Free Blastocysts
All work with ova and blastocysts is done under magnification of not less than 20, and with adequate illumination. Dr. Heuser uses an illuminator constructed in the laboratory, having a 50-watt 6-volt lamp with an adjustable aspheric condenser forming a well deﬁned beam; it is operated from a toy transformer.
Fig. 2. Method of sealing and storing small specimens in an embryological watch glass with vaseline on ground surfaces.
The specimen under observation is transferred from dish to dish by sucking it into a pipette with a long, slender tip having an orifice at least 1 1/2 times the diameter of the ovum and provided with a small rubber bulb.
The specimen is transferred to a dish (embryological watch glass, ﬁg. 2) of clear salt solution. It is fixed by gently flowing a stream of the fixing solution upon it with a pipette while it is under observation in salt solution. Gradually the salt solution is withdrawn as it is replaced by the fixing solution. The fluid mixture may be stirred by a gentle stream of air bubbles from the pipette, the ovum being watched constantly through the microscope.
With such small objects, fixation will be complete in a few minutes; 5 minutes evidently sufficed for the 2-cell egg described by Hertig, Rock, Adams, and Mulligan (1954). The ﬁxing fluid used routinely with the Carnegie human and monkey ova is Bouin’s picroacetic formol, which gives good preservation of the nuclei and cytoplasm but tends to dissolve or at least to thin the zona pellucida. Investigators wishing to preserve the zona perfectly would do well to experiment with acid-free fixatives.
Should the ovum stick to the dish because of coagulation of albuminous material on its surface, it is gently freed with a suitably ﬁne hair. A hog’s bristle is too large and stiff. For years Dr. Heuser has kept a stock of spines from an anteater skin opportunely provided by a naturalist colleague; these spines have very fine hairlike tips. He has also used a human eyelash, or a hair from a camel's—hair brush. A hair of any sort used in this way must be thoroughly wetted in the alcohol mixture to prevent it from floating unmanageably.
Dr. Hertig once made use of the adhesion by letting it hold the egg in place during preliminary dehydration and transportation from Boston to Baltimore.
Dehydration and temporary storage. These very small specimens are dehydrated by slowly dropping 70 per cent alcohol into the dish and gradually removing the diluted fluid with a pipette, the specimen being kept under observation, until the dish contains alcohol at 70 or 80 per cent concentration. A refinement of this method favored by Dr. Heuser employs two pipettes: the ovum is drawn into the stern of one; with the other, alcohol of higher concentration is added to the dish. The ovum is discharged into the dish and moved about by a pipette-driven stream of the alcohol mixture. This procedure is repeated until the concentration required for temporary storage, photography, and so forth, is reached.
Fixatives other than Bouin's necessitate preliminary washing of the specimen with distilled water, by a similar process of gradual replacement, before dehydration with alcohol.
Ova and blastocysts are best stored in an embryological watch glass with the rim ground Hat; :1 cover of sheet glass made airtight by a layer of vaseline (petroleum jelly) (ﬁg. 2) should be held in place with a spring clip or two small strips of masking tape.
Packing small objects for transport. Ova and blastocysts may be safely transported in glass tubes plugged at both ends with absorbent cotton. Tubes are better than vials because they can be emptied from either end; and cotton is better than cork because it is cleaner and contains no tannin, which might discolor the specimen. The tube should be about 30 mm. long, 6 mm. in diameter. The plugs are prepared by putting wisps of cotton in a dish of alcohol of the same concentration as that in which the specimen is stored. By pressing the cotton against the dish and turning the free ends of the fibers into the center with two pairs of forceps, a smooth, unraveled layer of cotton is formed on the bottom, and this face of the plug is pushed into the tube under alcohol. With a pipette the specimen is introduced well into the alcohol-filled tube, and a second cotton plug is pushed into the other end of the tube. When the plugs are in place there should be no air bubbles in the fluid. The tube is placed in an alcohol-ﬁlled stoppered bottle for further storage or transportation.
Discovery and Preparation of Implanted Embryos
If no ovum or blastocyst is recovered in the above steps, or if the clinical history or the appearance of the uterus suggests the presence of an implanted embryo, after the myometrium has been incised as described above (p. 5), the uterine cavity is opened under salt solution by cutting through the endometrium on each side, beginning at the 0s and working toward the fundus. The anterior half of the uterus is gently pulled away from the posterior half as the lateral cuts progress, in the hope of seeing the implantation site in time to avoid damaging it. This operation of cutting the endometrium and searching the cavity as it is exposed is performed with the aid of a wide-ﬁeld microscope or a binocular loupe. Finally, the two halves of the uterus are completely separate except for the uncut fundus musculature, which should serve as a temporary hinge, the fundal endometrium being left intact as long as possible in case the implantation site is at the fundus. During the whole process the uterus is completely immersed in physiological salt solution, the fluid serving not only to keep the specimen moist but also to support the delicate embryonic tissues.
When the surface of the entire cavity is revealed, the two halves of the uterus still under fluid are oriented, with their endometrial surfaces uppermost, in the dish in which the dissection was carried out. A beam of light is directed so that it glances across the surface, and the endometrium is carefully examined for a possible small elevation or a local hemorrhage which might mark an implantation site (ﬁg. 13, pl. 1). Once such a site is identified, its salient features are briefly noted and every special feature is sketched. The surface is then flooded with Bouin’s fluid while still under salt solution, and when it has been thoroughly fixed, the half uterus is again examined under the microscope for evidence of implantation that may have been missed in the fresh condition. It is finally transferred to a dish of l3ouin’s fluid without exposure to the air.
It generally will be convenient to transfer the entire half uterus to 35 per cent alcohol and continue the dehydration with alcohol of increasing concentration until a strength suitable for temporary storage (70 to 80 per cent) is reached. At this stage a block containing the implantation site is cut from the half uterus (fig. 29, pl. 3). For this operation a very useful instrument is a suitably fashioned piece of razor blade mounted in a medic holder The implantation site is photographed before the block is cut out, and the block may again be photographed after removal to show the site from various angles.
Dissection of the Larger Chorions
Chorions 10 mm. in diameter or larger are usually dissected in 80 per cent alcohol. The specimen is placed in a medium-sized Stender dish and is completely covered by the fluid. The bottom of the dish is lined with a sheet of cork cut to fit tightly and forced into place; or a removable piece of cork may be weighted by a square sheet of tinned metal placed under it with the corners of the tin bent over the cork. If the chorion is accompanied by and attached to part of the uterine wall, the specimen is fixed to the cork with fine pins (insect mounting pins are useful for the purpose) inserted through the myometrium. More delicate specimens, like naked chorionic sacs, may be held in position for dissection by means of threads, looped over the specimens and tied either to the pins or to small copper eyelets (wires with a loop at one end) stuck into the cork. With very small specimens, the ends of the threads may be held to the cork by mounds of vaseline. One end is fastened down; the specimen is posed; the thread is gently laid over the object, and pressed into the opposite mound of vaseline. Such mounds may also cradle the specimen. A 5-ml. syringe makes a convenient dispenser for the vaseline (fig. 16, pl. 1). Small blocks of glass, standing alone or supported by vaseline, may also serve as temporary supports.
Before the chorionic sac is opened, an effort is made to locate the embryo and the attachment of the umbilical stalk. Transmitted light is directed on the chorion if it is not too opaque, and gentle pressure is applied here and there over the decidua capsularis, if this is still in rim, or over the free surface of the chorion. An area that yields readily to such pressure is presumably safe for incision.
The incision is begun by pricking the tissue with a very fine, very sharp needle, under the dissecting microscope. None but the finest of needles will penetrate the tissue without indenting or damaging it unduly. Only the point of the pricking needle is inserted. When the needle is in position, it is held in one hand, and with the other hand a larger, blunter needle is stroked over the tissue that overlies the inserted point, producing a tiny cut. The fine needle or a favorably shaped piece of razor blade is then advanced and stroked along the incision, and the cutting is continued until the cavity of the chorion is entered. As soon as the orifice is large enough, the cavity is inspected under strong illumination. By means of a small pipette barely inserted within the cavity, loose coagula within the chorion may be gently flushed away with a stream of fluid.
When a short slit has been made it may be held open by small spreaders made from strips of cellophane, so that the hands are freed for further dissection. As soon as the embryo or yolk sac has been located, or when a clear pathway for further safe exploration is discerned, the direction in which the cutting is to proceed is planned, and thereafter the incision may be continued with sharply ground forceps and iridectomy scissors. An incision being made in a small chorion under a relatively high power of the dissecting microscope, and consequently in a restricted visual field, can be directed step by step over the whole distance by choosing under a lower power small landmarks (such as the mouth of an endometrial gland, or a small tag of tissue on the chorion) which lie in the desired direction but within the smaller field. Larger chorions are incised with fine dissecting scissors of the ordinary kind or with a razor blade.
The plan of the opening thus made will naturally be determined by the situation of the embryo in each chorion, the purpose being to open the chorion widely, so as to expose the embryo for photography and to permit excising it intact for sectioning. Usually a circular cut is made, removing a cap which may include half of the chorion. The embryo, if well advanced, is ultimately detached by severing the umbilical cord; the area of attachment containing the body stalk of an early embryo is separated from the rest of the chorion by a small cir cular cut in the region of the body stalk, on which a few overlying villi are left undisturbed.
Dissociation of Embryos with Mechanical Stirring
Delicate objects and tissues, such as are handled by the embryologist, require very gradual change of the concentration of alcohol in order to avoid damage. The conventional method of transferring the specimen from one dish of alcohol to another of higher concentration consumes overmuch time and attention if the changes are sufficiently gradual. For this reason the Carnegie laboratory has used with success two methods utilizing mechanical stirring and continuous change of alcohol concentration. Specimens small enough to be enclosed in a vial are subjected to mechanical stirring as follows.
The specimen, in weak alcohol, is enclosed in a “trap," for example a glass cylinder closed at both ends with linen cloth or with aluminium foil pierced with fine needle holes (figs. 3, 4). The trap is immersed in a closed vial or wide-mouthed bottle (fig. 5) containing alcohol of higher concentration. The bottle is placed on a rack or similar device designed for holding several such vessels (fig. 15, pl. 1), and the rack is slowly rotated by a motor so that the fluids in the jar and in the inner trap are agitated and gradually intermixed through the mesh or needle holes. The advantage of the closed system is that once the specimen is placed in the trap it need not be touched again until dehydration has reached the desired point.
Fig. 3. Specimen trap with perforated foil and air bell.
The rate of mixing, which is determined by the size of the holes and the rate of movement, has been arrived at empirically. When mixing is complete, there is no convection current when the two fluids are united at the end of the run. A trap of 10—cc. capacity in a 25—cc. suitable size is chosen; for small embryos in the limb— bud stage, for example, a piece 12 mm. in diameter and 25 mm. long will serve. The ends are flared outward by heating and reaming. Caps are made by crimping disks of aluminium foil down over the smaller end of a cork that fits the tubing easily. While the foil is still on the cork, numerous fine holes are pricked in it with a needle. The cap has the shape of a truncated cone with a skirt at its base. It is pushed into the open tube like a cork, and the skirt is crimped over the flared end. In this way the fine projections about the needle holes are aimed outward, leaving a smooth perforated surface next to the specimen. Before use, caps are thoroughly cleaned with distilled water and alcohol. The volume of the tube is measured for subsequent use in calculating the strength of celloidin during the infiltration process.
For holding a small, delicate embryo, such as one of the early somite stage with attached yolk sac, the vial may be molded by heating and indenting its walls to container exchanges within 24 hours. It is convenient, therefore, to replace the alcohol in the container, once a day, with alcohol 5 or to per cent stronger. W'hen absolute alcohol and ether-alcohol are reached, several changes of each, rather than one change per day, are required.
Fig. 4. Elements of a specimen trap: :1, flared glass tube; 12, aluminum foil cap viewed in section; c, end view of cap; (1, cap in position on flared end of glass tube.
Fig. 5. Wide-mouth specimen bottle with glass closure.
Experience has shown that rotation of the rack bearing the containers at a rate of 2 to 4 times per minute will not traumatize or abrade small embryos. Figure 17 (pl. 1) demonstrates the perfect dehydration, without collapse or distortion, that can be achieved by this method.
To make the trap (fig. 4), a piece of glass tubing of make rounded supports; or narrow rectangular strips of cellophane, thick enough to be springy, and slightly longer than the inner diameter of the vial, may be sprung into place at the ends of the specimen, to prevent excessive movement. Another method of immobilizing a specimen is shown in figure 6. These contrivances must be fashioned according to the size and shape of each embryo. Thus, when the vial is gently stirred by rotation of the rack, the specimen can move a little, but its weight will not fall upon such delicate structures as the yolk sac or the roof of the fourth ventricle. fluids entering through the perforated foil at the ends of the vial freely exchange with the fluid in the vial through the spaces around the embryo and the supporting bosses and strips.
FIG. 6. Trap with plastic insert for immobilizing specimen to prevent injury during dehydration and infiltration.
The cleaned trap, closed at one end, is placed in a dish containing the specimen in alcohol of known concentration. The specimen is coaxed into the open end, which is then closed. If the transfer is properly made under fluid, there will be no air within the trap; if desired, however, a small bubble may be introduced by a capillary pipette through one of the perforations in the foil or through a mesh of the linen cap.
In this method, no longer much used, the specimen lies at the bottom of a jar, in an alcohol solution stirred by a motor—driven glass plunger moving it1 a perforated glass cylinder. Alcohol of a higher percentage drips into the jar at the rate of about one drop per second, the rate being controlled by a plug of cotton packed against indentations in the drip tube.
An intermittent siphon drains excess fluid from the jar. The entire assembly is enclosed in a dust—proof cabinet, except the motor, which is mounted outside. The stirring action is varied by a simple eccentric mechanism on the motor shaft.
This method is subject to loss by evaporation, especially with strong alcohols and alcohol-ether.
Ova and embryos are photographed with a camera and stand (fig. 14, pl. 1) developed at the Carnegie laboratory to meet its specific requirements. An early form of the apparatus first used by Dr. Heuser has been described and illustrated by Heuser and Streeter (1929); its final form, constructed by O. 0. Heard, was reported by Hcuser and Heard (I942). The camera is mounted vertically on a hinged support; below it a binocular microscope is so mounted on a pivot that the microscope and camera may be moved alternately in and out of position on the optical axis of the camera. The essential feature of the apparatus is the ability to position the specimen in the pivotal plane. The swinging microscope is set to focus on this plane, and the level of the camera front (lens board) is adjustable. Lens adapters are interchangeable and designed for parfocal adjustment through the range of lenses used, so that, no matter which lens is selected, the camera when in position will be nearly in focus in the same plane.
The specimen, mounted on a stand which can be raised and lowered by a screw, is brought into the focal plane by adjustment of the stand. Thus the specimen may be posed and the lighting arranged with the aid of the microscope (fig. 24, pl. 3). The microscope is then swung away and the camera is swung into position. Now the image of the specimen falls centrally on the photographic plate and is approximately in focus.
To permit stereoscopic photography, the camera is pivoted at its focal point. By this means it can be tilted laterally, without going out of focus, to positions right and left respectively of the vertical, at the proper angle to yield correct stereoscopic fields.
Magnifications from less than I to 50 are obtained by means of photographic objectives (for example, Microtessars). The camera is provided with stops and temPlate rods to facilitate the use of various lenses to obtain precise magnifications. For magnifications above 50 the compound microscope is used; stereoscopic photography b tiltin the camera is then hardly feasible.
For photographing embryos by reflected light, a Stender dish is set in half a Petri dish, which serves as a moat to catch spilled alcohol. If a light background with a shadow of the specimen is desired, a piece of opal glass is placed in the bottom of the dish, and a disk of clear glass is mounted at a short distance above it. It may be convenient to put the opal glass in a small Petri dish inside the Stender dish and let the rim of the Petri dish support the clear glass. The extent of the shadow cast on the opal background depends on the distance between the clear and the opal glass.
The specimen is carefully freed of small specks of dirt and organic debris with a camel’s-hair brush and a cleaning pipette as described in the next section. It is put in the Stender dish, which is placed on the focusable stand of the camera mentioned above (fig. 14, pl. 1), and is posed under the binocular microscope of the photographic apparatus. To immobilize and support the embryo, small mounds of Vaseline are extruded from a small syringe (fig. 16, pl. 1) onto the glass bottom plate. The Vaseline may be built up to the right height and molded with needles until the embryo is in the desired pose. The dish, completely full of alcohol, is then covered by sliding a flat glass plate over it, excluding all air.
Two focusable spotlights are desirable, one to provide the general illumination and the other at a somewhat greater distance to balance the shadows and bring out particular (letails. Diffusing plates of ground glass may be interposed (fig. 24, pl. 3). The use of two lights has replaced a former practice of mounting a curved reflector of white paper in the dish near the specimen,
to balance the light of a single lamp. A glancing beam is ellective for showing fine details, as on the surface of a blastocyst, or for emphasizing surface modeling of an embryo.
Transmitted light has been used in photographing cleared specimens (to show the skeleton or injected blood vessels, and so forth) with the aid of a special condenser held horizontally in a modified microscope stage.
Photographing Free Ova
A cell (Fig. 7) is prepared from an ordinary slide (1 inch by 3 inches), a square cover slip, and four small glass slips made by cutting a cover slip into narrow strips. A square area somewhat smaller than the whole cover slip is marked off on the middle of the slide by extruding a line of vaseline from a syringe. The small strips of thin glass are pressed down onto the vaseline and covered with a second line of vaseline. The slide is placed in a shallow finger bowl and covered with alcohol of exactly the same concentration as that in which the ovum is stored. The concentration can be tested by adjusting the dilution so that no diffusion currents are produced when a drop of one alcohol mixture is placed in the other. If the two mixtures were not alike, the egg might be lost by spinning away in the diffusion current.
Fig. 7. Special mount for free ova built on a I by 3 inch slide, composed of pieces of glass cover slip and vaseline.
Transferring the Specimen
If the ovum is stored in a dish rimmed with vaseline and covered with a glass plate, the lid must be partly slid off (the operator watching the egg through the microscope) so that a little of the fluid can be pipetted OH to prevent alcohol and the egg from flowing over the rim of the dish.
Any spicules of dirt near the egg are cautiously removed under the microscope. For transferring ova a glass pipette with a long narrow tip is used. It is chemically cleaned and rinsed with distilled water and with alcohol of the same concentration as that in the dishes. The tip is partly filled with alcohol by suction; it is then put into the dish containing the egg, a drop or two of alcohol is extruded, and under the microscope the tip is brought up to the egg. The egg is sucked into the pipette by lightly releasing the bulb. It is kept in view through the wall of the pipette at all times. If the pipette is chemically clean, the egg will not stick in it; if it does stick, gentle pressure on the bulb may release it. If not, the pipette is scored with a diamond pencil and broken off at a safe distance from the egg, which may then be freed by a hair inserted into the tip.
Before the ovum is transferred to the specially prepared cell, the cell is examined under the microscope to make sure that the area within the square is free from dirt. A cover glass is placed on the end of the slide ready for use; it should be slightly smaller than the width of the slide, to lessen the danger of accidental dislodgment in subsequent handling. The ovum is ejected from the pipette into the cell. The cover glass, picked up with forceps, is held at one side by a needle and gently lowered onto the top of the glass—rimmed square, and is pressed down to ensure that the vaseline seal is complete (fig. 8).
Fig. 8. Method of transferring egg to special mount totally submerged in a glass finger bowl.
The slide may now be lifted out of the bowl. Excess alcohol is blotted off, and the slide is ready to be placed under the photographic microscope. The egg may be kept safely in the cell, for days if necessary, until microscopic study and photography have been completed, by simply reimmersing the cell in the bowl of alcohol when it is not in actual use. The bowl should be covered and sealed with vaseline against evaporation.
A useful device for removing small particles of foreign matter from the vicinity of an ovum or from the surface of the specimen itself is a thin—walled glass tube (3-mm. bore), drawn out at one end to form a fine capillary tip which is bent at 45°. The other end, serving as handle, is fitted tightly with a cork and is provided with a rubber tub; with this pipette, particles of debris can be flushed away or pjcked up by suction. When not in use the delicate pipette is protected by putting it tip down into a 50-cc. bottle so that it is held by the cork.
Paying the Ovun
Positioning very small, round specimens such as segmenting ova and blastocysts presents difficulties, because the specimen is necessarily mounted in a closed cell for photography under the compound microscope. Trial and-error methods must be used. Experience shows that an egg tends to rest in one position in the cell but can be moved by various means. The slide may be removed from the stage and cautiously jolted or tilted, the result being checked with a hand magnifier. A bubble in the cell may be efficacious in moving the ovum about. A bubble may be deliberately left when the specimen is first mounted in the cell; or, by submerging the slide in the finger bowl and slightly displacing the cover slip, a small air bell may be introduced with the cleaning pipette. When the slide is tilted the bubble will move the egg.
Very small objects, like unsegmented and segmenting eggs, are usually best photographed with transmitted light. Sometimes a modified dark-field illumination reveals details of single cells in morulae with as many as 8 cells. Later stages yield better results with reflected light, either vertical or glancing, as determined by trial. A spotlight with focusable beam is essential. Negatives are made with differing poses and illumination in order to record the totality of details that cannot be shown in any one view.
The ordinary paraffin method has been employed at the Carnegie laboratory for small embryos and will no doubt continue to be used in special cases, although double embedding in celloidin and paraffin is now preferred for the more valuable specimens.
The procedures in paraffin embedding are the conventional ones, except that the apparatus and instruments are adapted to the individual handling of small, delicate, and very valuable specimens requiring accurate orientation for serial sectioning.
Specimens of relatively large size (5 mm. or more) after being photographed are carried through the later stages of dehydration (80 per cent to absolute alcohol) in the mechanical apparatus described above. They are then transferred by hand. Binocular microscopic observation is essential while very small embryos are being transferred from dish to dish; otherwise, there is real danger of losing them. Such transfers may be made by pipette, improvised spoons, or cups so that specimens may at all times be totally submerged and observed with convenient magnifications, as in figure 9. Orientation in the paraffin block is accomplished by the usual method: when the paraffin is on the point of congealing, the specimen is placed in the desired position, in the dish or paper box to be used for embedding, by means of an electrically heated copper needle.
Very Small Object:
segmenting ova can be more easily kept under observation during transfer through the alcohols and embedding if they are deeply stained with eosin after the in tato photographs are made. Drops of alcoholic eosin may be added to the dehydration alcohol at any stage, preferably as soon after photography as possible.
During the infiltration with paraffin and the actual embedding, heated instruments are required, and convenient apparatus (fig. 9) must be at hand to keep the paraffin liquid and at the proper temperature. A brass or copper plate, electrically heated and thermostatically controlled at a constant temperature, is supplemented by overhead heat by radiation from electric bulbs. Over one end of the warm plate a binocular microscope is placed so that the dish and specimen can be observed and all transfers from one fluid to another made directly under the lenses. The eggs are transferred in a pipette wound with resistance wire and heated by electric current closely regulated to keep the contained paraffin at the melting point.
The eggs are embedded beside a small piece of stained tissue which serves as a marker. The piece is cut from some soft tissue large enough to be plainly visible to the unaided eye. It is first embedded in the usual way in a small mass of paraliin which is allowed to harden. When the egg is ready for embedding, the paraffin is melted with an electrically heated copper needle, bent at an angle of 45° a few millimeters behind the working point, until the marker is almost free, and the egg is deposited close to one end of it. If the electric needle is carefully used, there is little danger of superheating the paraffin and thereby injuring the specimen, since it rests upon the congealed paraffin in a layer barely above the melting point. The orientation cannot be accomplished with the copper point of the same electric needle that melts the paraffin because the heat might injure the specimen if the needle should touch it, and moreover the hot needle causes eddies in the melted paraffin which might dislodge the egg from the desired spot. A very useful embedding tool is one provided with two working points, one hot and one warm. It is made by soldering at the bend of the copper point a short piece of fine German silver wire, bent in the same plane but in the opposite direction, so that a gap of I or 2 mm. separates the two points. The specimen is oriented with the silver point, which does not conduct the heat as well as copper and is not hot enough to melt the paraffin. Should the paraffin begin to congeal around the egg too soon, the instrument is rotated and the copper point quickly liquefies
CELLOID1N—PARAFfi~.\' Relatively Large Specimen:
Years of experience in the sectioning of mammalian embryos have resulted in preference for double embedding in celloidin and paraffin. This method protects fragile tissues against the risks of loss of sections and of fragmentation that occur with paraffin alone. A lowmelting—point paraffin can be used. Furthermore, the it again. Sometimes a loop of resistance wire heated by passing the electric current through it is mounted on the arm of a lens holder equipped with a rack and pinion, and so placed as not to touch but to rest just above the embedding mass of paraffin. By regulating the distance between the loop and the specimen, the depth to which the paraffin melts can be controlled; and by manipulation with a needle passed through the loop, the egg can be accurately placed in relation to the marker. As long as the paraffin is melted, the egg can be seen sufficiently well under the microscope to permit placing it in the correct position. The marker can be seen even after the paraffin congeals. The block is secured in the microtome so that the marker lies between the knife and the object. Sections are cut until the first part of the marker is reached, and these are discarded. The remaining part of the block is then cut; the sections containing the specimen itself are located by inspecting the ribbon with a microscope, and these alone are mounted.
Fig. 9. Warm plate with electrically heated instruments and controls arranged for microscopic examination while specimen is being embedded in paraffin.
celloidin-paraffin method, as will appear below, is essential in Dr. Heuser’s procedure for orientation of embryos.
The double embedding facilitates the wet-knife method of sectioning, which reduces compression in cutting.
Celloidin- paraffin embedding in its practical aspects was introduced to Dr. Heuser in 1931-1932 by a guest investigator, Dr. Peter Mihalik, who had familiarized himself with the method in Europe. Experience has suggested various modifications.
After the specimen has been photographed, it is dehydrated and carried into the ether—alcohol mixture (equal parts ethyl ether and absolute ethyl alcohol) by means of the mechanical agitator described above. At this stage, support of the more delicate embryos by molded vials and by cellophane retaining strips is especially desirable.
Celloidin is added in solid form to the ether—alcohol and allowed to dissolve, so that the concentration is raised very slowly and infiltration is gradual.
The celloidin currently used at the Carnegie laboratory is prepared by the Mallinckrodt Chemical VVorks, St. Louis 7, Missouri. The chips are washed in absolute alcohol, inspected, dried on filter paper, and stored in a clean glass-covered dish.
The volume of the vessel in which the embryo is to be infiltrated (see next paragraph) having been ascertained, enough of the clean celloidin is weighed out to make an 8 per cent solution when dissolved in the contents of the vial. The proper amount of celloidin is divided into four approximately equal parts for successive addition.
The vessel used by Dr. Hcuser is a small, widemouthed specimen bottle. The opened end is ground flat and sealed with a glass plate held in position with :1 metal spring clip. A circular sheet of cellophane is shaped over the small end and the sides of a cork with a hot spatula, to form a basket, which is perforated with numerous holes in its end and wedged into the neck of the bottle. This arrangement is suitable for all except the very smallest specimens, which are more conveniently handled with a miniature rocking device described on page 14. One-fourth of the weighed celloidin is placed in the vessel, to be gradually dissolved in the ether-alcohol as the fluid is gently stirred by the machine. When the first quarter part of the celloidin is dissolved and the embryo is, therefore, in a 2 per cent solution of celloidin, another quarter is added, and so forth until the desired concentration of 8 per cent is reached.
Boxing. A rectangular box, open at the top, is made of cellophane sheeting by folding a piece of the requisite size around a wooden block. The corners of the box are pressed flat with a warm knife blade, any fullness at the sides of the box being pressed toward the corners. The sellophane used at the Carnegie laboratory is du Pont MST-3W; any other transparent plastic sheeting should be tested to make sure that it is not soluble in alcoholether or in chloroform. A dish with a neatly fitting cover is chosen, a little larger than the cellophane box, and a small amount of alcohol-ether is placed in it. The cellophane box is put into the dish and is partly filled with 8 per cent celloidin. The embryo may then be transferred to the box from the vial in which it was infiltrated, with a little dipper made of foil. Dr. Heuser prefers, however, to pour the contents of the vial gently into the box, letting the embryo be carried with it. Here again the embryo must be submerged at all stages of the process in order to avoid unequal pressure and consequent deformation.
The dish is covered to allow air bubbles to rise to the surface and be dispelled by the alcohol-ether vapor from the layer in the bottom of the dish. The alcohol-ether is then pipetted off and chloroform is substituted, or the box is lifted to another covered dish containing a little chloroform. W'hen the celloidin begins to congeal under the influence of the chloroform, the box may be tilted and held at various angles, so that the embryo is coaxed into the desired position and finally held there when the celloidin becomes firm enough to prevent it from sinking. Use of a needle or other tool at this stage would introduce ineradicable bubbles and thus ruin the block. There should be clear celloidin on all sides of the embryo to a thickness of 6 mm. or more, to allow for trimming of the block. Considerable time and patience must be devoted to this tedious maneuvering of the specimen into correct position.
Clearing t/2e block. The celloidin block is left in the dish in chloroform vapor until it is hardened throughout, usually overnight. It is then cleared in several changes of cedarwood oil or oil of pine. It may be stored indefinitely in either of these clearing oils.
Orientation. The specimen will, of course, have been oriented in the block as accurately as possible. Orientation is a relatively simple matter if the long axis of the embryo is observable and if the head form, limb buds, and other externally visible features are available as aids. A block of uterine wall containing an implantation site can be oriented with reference to the edges and surface of the block. A small intact chorion, on the other hand, presents no such information. The general position of the early embryo or germ disk within the chorion may be ascertained by observations during the first preparation and subsequent observations of the specimens, for instance by transilluminating the chorion and noting the shadow of the germ disk, body stalk, or yolk sac. By relating this information to small details of the surface, some degree of approximate orientation will have been attained. If no inner structure has been seen, it may be useful to orient the block with reference to the longest axis of the chorion. The germ disk usually but not always lies at the deepest point of the chorionic cavity.
When the block is to be mounted it is studied with the microscope under clearing oil. Trying various powers of the microscope, illuminating the block from various angles, and studying it in many positions, the embryologist takes note of all available clues that may enable him to choose the most advantageous plane of section. In this part of the task previous experience is extremely helpful. Dr. Heuser’s conspicuous success in cutting the Hertig-Rock embryos and other early specimens has depended upon his accumulated knowledge of the form of young embryos and their situation within the chorion. Even be, however, is not able to determine the exact plane for sectioning an embryo of the prcsomite or early somite stage in the intact chorion. Assurance is possible only if the chorionic cavity is opened.
Reference mar/(3 (pins). When after careful study the plane of section has been decided upon, it is marked by inserting two pins through the celloidin block at right angles to each other, in planes parallel to the proposed plane of section, far enough apart so that they do not strike each other, and at a safe distance below the embedded embryo (fig. 25, pl. 3).
The pins are the thin, stiff kind employed by insect collectors. The points are shortened by grinding so that they can be pushed through the celloidin without risk of accidental diversion caused by bending of a long, finely tapered point; but the shortened points must be made sharp to avoid cracking or distorting the celloidin. One of the two pins is bent at a right angle near the head to distinguish it from the other.
The block is held with forceps in a dish of clearing oil, and, while the embryo is closely observed under the dissecting microscope, the two pins are pushed through the block, one at a time. If the specimen is not very symmetrical, it may be difficult to place the pins suitably.
Fig. 10. Wide-field binocular microscope goniometer for measuring the angles of orienting pins in relation to a predetermined plane of section.
Dr. Heuser frequently pushes short pins part way into the block as temporary marks before inserting the two long pins.
Correction of the plane. The placement of the pins is checked by restudy of the block under the dissecting microscope, with reference also to whatever notes and sketches the embryologist has made while choosing the plane of section. After much experience Dr. Heuser has found that he can usually place the pins within 2° to 3° of the desired plane.
Errors in placement must now be measured (as will be described in the next paragraph) and recorded in order that they may be finally corrected when the block is mounted on the carrier of the microtome. If there is an obvious error (6° or more), however, it is best to withdraw the incorrect pin and reinsert it, because compensating for an error of this size by tilting the block may displace the second pin from its position in the correct plane.
Small errors of placement are measured with a special goniometer (fig. 10) consisting of a brass plate mounted on one eye tube of the dissecting microscope just below the eyepiece and projecting fanwise laterally. An are at the far end is graduated in degrees, and a pointer is fixed to the eyepiece. As the eyepiece is rotated, the amount of rotation is read on the are. In the eyepiece is a glass disk engraved with a single fiduciary line or a grid of parallel lines. The block being at rest in clearing oil under the microscope, the fiduciary line is placed in the desired plane, then rotated until it is aligned with the pin, and the angular correction is recorded. By another observation at right angles to the first, the error of placement of the second pin is also recorded. The record consists of a careful diagram showing the block, the embryo, the two pins, and the respective corrections. Before the development of the home-made special goniometer, similar corrections were made by projecting the images of the embryo and the pins with a camera lucida onto ruled paper, on which the correction angles could be measured with an ordinary protractor.
Fig. 11. Device for infiltration by mechanical stirring, with a simple adjustable eccentric and wire connector to facilitate adjustments.
With angles of less than 6° of arc, the subsequent correction of the error of one pin will not materially displace the other pin if it is in the correct plane.
Very Small Specimen:
Very small objects (ova and blastocysts) are infiltrated in anembryological watch glass. To the ether-alcohol mixture in which they lie, celloidin is gradually added, either as drops of a thick solution or as small, dry chips. To facilitate solution and uniform mixture the dish is placed on a small motor-driven rocking apparatus resembling a miniature seesaw (fig. II). The rate and magnitude of rocking are controlled by a cam revolving at a selected speed. The watch glass is provided with a :over tightly clamped to its upper ground surface (fig. 2) 1nd sealed with dilute celloidin. When the desired concentration (8 per cent) is reached, the specimen remains in it for 48 hours, after which it is boxed and the celloidin is hardened.
Boxing. A cellophane box of suitably small size, constructed as described above, is placed in a covered dish zontaining a little ether-alcohol. The box is filled with :elloidin of exactly the same concentration as that finally attained in the dish in which it has been infiltrated; a difference of concentration at this stage might damage 1 delicate specimen by collapsing or tearing it. To this )0); the ovum or blastocyst is transferred by a pipette of adequate bore, care being taken to avoid getting bubbles nto the celloidin. The dish is covered for a few minutes :0 allow trapped air to escape. It is not safe to put a drop )f ether-alcohol on the surface of the celloidin to dispel bubbles because of the danger of altering the concentra:ion and thus causing collapse of delicate structures.
The alcohol—ether is removed and chloroform is substituted, or the box is transferred to another dish conaining chloroform, until the surface has congealed. The zedious but essential task now begins, to get the specimen Jositioned centrally in the celloidin block, or at least well way from any surface. The method is the same as de:cribed above for larger specimens: turning the box over Ind over, letting the ovum sink in one direction and mother until the position is fixed.
If the specimen does not settle rapidly enough, an air bubble may be introduced by pricking the bottom of he box with a needle. The bubble may then be used to push the ovum into a favorable position; once the specimen is in the center of the mass, the bubble will float up ind away from it. The location of the specimen (if it is arge enough to see clearly) and its preliminary position ing in a suitable plane may be controlled by observation under the dissecting microscope.
When the celloidin is hardened throughout, the box is left in chloroform, usually overnight, and is then cleared in oil of cedarwood or oil of pine.
Orientation. Very small specimens are oriented like larger embryos, the block being trimmed if necessary to a size that will permit observation under sufficiently high powers of the microscope for accurate orientation. Dr. Heuser was able thus to cut sections of the 2—cell Hertig—Rock ovum, Carnegie no. 8698, at right angles to the cleavage plane and through the nuclei of both blastomeres.
If the specimen is so small that it will be invisible after the block is infiltrated with paraffin, a third pin is inserted above it, the distance between specimen and pin being measured with an eyepiece micrometer and recorded. When the mierotome knife reaches the track of the pin, the embryologist will know how far he has to go before the object is reached.
Infiltration with Parafiin
When the preliminary orientation procedure has been completed, the celloidin block is roughly trimmed at top and sides with a razor blade. It is then passed through three changes of toluene and one of paraffin-toluene mixture, which is fluid at room temperature. This part of the procedure can be carried out on the mechanical agitator. The actual infiltration in three successive changes of paraffin is done in dishes in the paraffin oven. The block is placed in a small, perforated metal tray suspended from a lever by which it is slowly lifted up and down in the paraffin bath. The lever is operated by a camshaft revolved by an electric motor outside the paraffin oven.
When infiltration is complete, the block is removed from the paraffin bath and put into cold running water to harden the paraffin.
Mounting the Block
The block is now to be mounted on the carrier post If the mierotome so that the knife will traverse it in the )lane indicated by the two pins. A special post holder :designed and constructed by O. 0. Heard), which re)l21C6S the usual adjustable holder, permits the specimen 0 be placed in the predetermined plane. This special holder is an accurately machined metal block with a vertical hole into which the post, a steel cylinder 12.; mm. n diameter (shown in fig. I2), fits and is clamped by a etscrew, thus being held on the mierotome in a fixed vertical position. By means of the apparatus next to be described, the specimen is adjusted to the correct position or cutting, when the celloidin—paraffin block is attached 0 the post.
The positioning apparatus is shown in figures 26, 27, 28 (pl. 3). It consists of a base (fig. 27), in which the post is held vertically by a setscrew, and of a bracket mounted on an arm clamped to the post after the post is positioned. The bracket carries at the top an arm which terminates in a plate borne on a universal joint by which the plate may be set in any desired plane. In the latest model, Dr. Heuser has fitted a built-in protractor by which the plate may be accurately set at the proper angle.
The celloidin— paraffin block is temporarily attached at its top (the end which is to be struck first by the knife) to the movable plate by embedding short pieces of copper wire in the top of the block and then in a bed of paraffin on the plate.
The position of the plate is now adjusted so that each of the orienting pins is brought into a plane at right angles to the vertical axis of the post: first, the pin is positioned in line with the horizontal line of the protractor; then the small angular correction, called for by the measurements previously made when the pins were inserted, is introduced with the aid of the protractor, the plate being moved so that it deviates from the base line of the protractor by the required angle.
Fig. 12. Special post holder, designed for a no. 860 Spencer Precision microtome, and used in conjunction with goniometer and positioning device shown in figures 21, 22, and 23 (pl. 2).
When the block has been placed accurately, the base is removed. The post, on top of which a layer of highmelting—point paraffin has been fused, is freed by releasing the setscrew on the main arm, and is pushed up until it touches the bottom of the block. To this it is fused by melting the paraffin with a warm needle or small blade.
Before being sectioned, the block is accurately trimmed in a mechanism (devised and constructed by O. O. Heard) consisting essentially of a small sledge microtome modified by fastening a Hat metal plate to a sliding knife block. Through a square notch in the front edge of the plate the celloidin- paraffin block is presented for trimming, the post to which the block is fused having been fixed in the object carrier of the microtome. A short microtome knife in a suitable holder is pushed manually along the flat plate to plane off the block as the block is racked upward by the ratchet wheel and screw of the microtome.
The object holder of the microtome is modified so that it bears two sockets for the post. One of them (fig. 18, pl. 2) presents the top of the block for trimming; the other presents the block horizontally, so that its vertical sides may be trimmed (fig. 19, pl. 2). This socket assembly is mounted on a disk which can be rotated through 360°; the disk can be held at 90° intervals by a spring stop, so that accurate trimming of each of the four sides of the block in succession is possible. When the excess celloidin-paraffin has been removed, only enough being left to support the object, one corner of the block is slightly chamfered by a cut at a 45° angle to the sides. The cut serves to mark one corner of each section as a guide in arranging the sections on the slide in case any of them is accidentally turned around or turned over. The diagrams previously made after placement of the pins are consulted in choosing which vertical angle of the block is to be so marked, and the choice is recorded on the diagrams.
The post is taken to the sectioning microtome and Fixed in the socket of the rigid carrier block previously mentioned. As a result of the various maneuvers just described, the block is now properly positioned for sectioning in the desired plane.
The doubly embedded blocks are sectioned on a sliding microtome, by the “water-on-the-knife” method introduced by the late Professor Carl G. Huber, of the University of Michigan. The only previously published description of this valuable method appears to be a brief account by Heuser in McClung’s Handbook of Microscopical Technique (1929, 1937).
Care of the Knife
The cutting edge of the knife must be well sharpened. The condition of the surfaces as well as of the facets of the knife is also very important, for in the Huber-Heuser method the upper surface must be wet to the very edge, while the lower surface is dry so that water will not How onto the block. In the practice of the Carnegie laboratory, as will be described in another paper, there is a special reason for keeping the block dry, namely the photographic recording of its upper surface for orientation purposes.
The considerable experience acquired by the author of this article, in the sharpening and surface treatment of microtome knives to meet the above requirements, and also to aid in reducing distortion in section cutting, has been recorded in a recent publication (Heard, 1953).
For cutting serial sections of embryos, the knife is sharpened and surface-treated as outlined in the article just cited. The condition of the edge is checked under the microscope at a magnification of I()O, and a record of any small nicks that may remain is made on a strip of paper fastened along the knife. The record is filed with the knife until needed.
The knife is mounted so as to make a slicing cut with the edge at an angle of about 45° with the direction of movement, and at a rake angle of about 60° with the plane of section. \Vith the paper record in position, the knife is set so that nicks will not traverse the block. It is helpful though not absolutely necessary to spread a drop of Mayer’s albumen fixative over the portion of the upper surface of the knife that is to be used; it serves as a spreading agent for the fluid used to wet the knife.
The wetting fluid is not actually water, but a 0.1 per cent solution of sodium chromate, which acts as a rust inhibitor also. Three or more small dishes of this solution are at hand to receive the sections as they are cut, and a larger one serves as a reservoir for replenishing the fluid on the knife. Two artist’s brushes are provided. One of them, small and finely pointed (fig. 20, pl. 2), is used in handling the sections. A clean sable brush, dipped in water and given a quick shake, acquires a fine point. Incidentally, pointing the brush in the mouth is to be avoided, because it transfers squamous epithelial cells to the sections.
With the larger brush the knife is thoroughly wetted on the upper surface of the portion that will pass through the block. As the knife enters the block and cuts a corner of it, the section will begin to roll up. Dr. Heuser’s practice is to rest his left hand (see fig. 20, pl. 2) on an auxiliary post at the level of the knife so that, as he draws the knife toward him, his brush (the smaller one) touches the corner of the block. This contact is maintained until the knife moves forward and barely enters the block. The brush is then raised above the forming section but does not touch it as it slides up the slope of the wet knife. The brush prevents the rolling up of all but a very small part of the section, which can easily be unrolled when the sections are placed on the slide; if, as may happen with thick sections, the tissue—free corner will not unroll, it can be cut off.
If a section rolls up tightly as it is cut because of lack of synchronization of movements, it can be successfully unrolled at once or, if supported in a small dish of water, after all the sections have been cut. When the section is ready to be recovered, it is transferred to a wet but not flooded slide under the dissecting microscope. A free corner of the section is held down with a needle, and the rest is gently unrolled with a pointed sable brush. It is important not to press downward upon the rolled part with the brush, but only against the side of the roll where it bends upward from the flattened portion held C.l0\VI1 by the needle. If the sections are thick, 10 microns )r more, very cautious warming by the heat of a lamp focused upon a rolled—up section will help unroll it.
To lift the section from the knife, where it floats on :he wetting fluid, the brush, loaded with fluid, is touched :0 an edge of the section with a gentle twisting movenent, so that the section is lightly wound onto it. By reversing the twist, the section is deposited on the slide or on the surface of the fluid in one of the small dishes previously mentioned (fig. 21, pl. 2). The next section is placed in the next dish, and so on to the third or fourth dish. By this time the assistant will have taken over the first section, leaving its dish free for the next section in order. Recently O. O. Heard has improved this procedure by the introduction of a revolving tray like :1 “Lazy Susan” to keep the dishes moving in proper sequence.
Mounting the Sections
The assistant picks up each section in turn with a brush and transfers it to a special mounting dish fitted with a metal rack as shown in figure 23 (pl. 2) and figure 30 (pl. 3). This rack is made with two end pieces of brass held together by 3-mm. rods. Two wire clamps hold the slide on a slope partly submerged in the water. Four Nichrome wires under the slide delimit the area within which the sections must be mounted. The slides are 2 by 3 inches (50 by 75 mm.), and the cover slips 43 by 50 mm.; the utilizable area is such as to leave a margin of about 2 mm. between the edge of the cover slip and the nearest points of the sections. If for very small objects a 3 by 1 inch slide (75 by 25 mm.) is used, two slides are placed on the rack and the fluid is brought up partly to cover the upper slide, on which the sections are mounted. The water level in the dish is maintained by a large rubber bulb connected by tubing to a glass siphon (fig. 23, pl. 2). The bulb serves as a reservoir, and the level is held by a clamp on the tubing. Neither bulb nor tube must be allowed to contain air bubbles, which might disturb the sections when the level is changed. Another means of controlling the water level is a reservoir bottle suspended by a rack and pinion device.
The assistant first brings the water level up on the sloping slide to the line along which the upper row of sections is to be mounted. The sections are successively slid with a brush out of the water and onto the slide. \Vhen the first row is full, the level is lowered and the second row is mounted, and so forth until the slide is filled. During this process the sections in the upper rows must be kept moist. Dr. Heuser uses a narrow strip of filter paper, dipped in thin celloidin solution (to prevent loose fibers from getting onto the section), and placed on the slide just above the line for the first row of sections. The strip is helpful in aligning the first row, especially of small specimens, and it holds the sections in place when water is added with a brush from time to time along the upper edge of the slide.
A filled slide is replaced by an empty one, and the work goes forward. A team of two persons accustomed to working together can operate with a satisfactory rhythm, but it is helpful to have a second assistant who takes over the freshly mounted slides. He flattens or removes the small corner rolls; checks and, if necessary, adjusts the alignment of the sections; removes air bubbles from under the sections by brushing them out; and sees to it that there is a narrow space between adjacent sections to allow for the small amount of spreading that occurs.
If a second assistant is not available during the sectioning process, the final arrangement of the sections may be postponed. The slides bearing the sections in correct order are stored under Petri dishes until they can be arranged and dried. Recently we have devised moist chambers from cut-off 3—liter reagent bottles, 13 em. in inside diameter. Racks, designed to hold twenty-five 2 by 3 inch slides, are made of Bakelite and sheet brass to stand upright in Petri dishes. The filled racks a11d Petri dishes, the latter containing distilled water, are covered by the cut-off bottles. Sections are still movable after three days’ storage.
The mounted slides are dried at 44° C. 011 :1 thermostatically controlled warm plate protected by a cover from direct air currents and from dust. They are usually left on this plate overnight at 36° C., after which they may be stained. Figure 31 (pl. 3) illustrates one of a sagittal series of sections through a presomite embryo, Carnegie no. 8725, handled by the above technique.
For routine purposes a modification of the hematoxy1ineosin staining method is used. It will be outlined here to illustrate the general nature of the staining procedure.
- Remove paraffin with three changes of xylene.
- Remove celloidin with absolute alcohol-ether (the usual procedure; celloidin may be allowed to remain if necessary to protect very delicate objects).
- Dip slides in 1 per cent celloidin, and dry in air briefly until a film has formed; this step prevents washing off the sections.
- Immerse in alcohol, 70 per cent, or chloroform, to harden the celloidin coating.
- Change to 95 per cent absolute alcohol, 5 per cent chloroform; the chloroform is necessary to prevent solution of the celloidin coating by the strong alcohol.
- Rehydrate in alcohol from 80 per cent to 30 per cent by steps of to per cent, then distilled water.
- Stain with Ehrlich’s hematoxylin.
- Rinse in distilled water.
- Decolorize and sharpen stain in acid alcohol.
- Rinse in distilled water; turn the sections blue in tap water, or in a weak alkaline solution if local tap water is not sufficiently alkaline.
- Rinse in distilled water.
- Dehydrate to 70 per cent alcohol.
- Counterstain in 1 part eosin, 4 parts phloxine, 500 parts 70 per cent alcohol.
- Rinse quickly in 95 per cent alcohol (containing 5 per cent chloroform if the celloidin is to be retained).
- Dehydrate (and differentiate the eosin-phloxine stain) in absolute alcohol.
- Immerse in absolute alcohol—ether if celloidin coating is to be removed; otherwise, pass directly to xylene, 2 or 3 changes.
- Mount with Clarite, and dry overnight at 36° C.
The foregoing account, simplified as it is, represents the major effort of a professional career devoted to embryology. The quality of the morphological reports in the Contributions to Embryology is largely founded on the painstaking procedures originated or perfected by Dr. Heuser. It is hoped that this account will make his methods for handling, preserving, and preparing early and delicate specimens available to fellow biologists who have not had the opportunity to profit by watching him at work and observing his skill and precision.
The author acknowledges with thanks the editorial assistance of Dr. George W. Corner in preparing this article for publication.
Because the author was not familiar with all the sources, written and verbal, from which Dr. Heuser obtained information on which various parts of his technique may have been based, the latter should not be held responsible for omissions in the following list.
HEARD, O. O. 1931. A photographic method of orienting serial sections for reconstruction. Anat. Rec., vol. 49, pp. 59-70.
1953. The influence of surface forces in microtomy. Anat. Rec., vol. 117, pp. 725—739.
H1-111115, A. T., I. ROCK, E. C. A1).-\.\1s, and \V. I. Mu1.1.1oAN. 1954. On the preimplantation stages of the human ovum: a description of four normal and four abnormal specimens ranging from the second to the fifth day of development. Carnegie Inst. VVash. Pub. 603, Contrib. to Embryol., vol. 35, pp. 199-220.
HEUSER, C. H. 1932. A presomite human embryo with :1 definite chorda canal. Carnegie Inst. Wash. Pub. 433, Contrib. to Embryol., vol. 23, pp. 251-267.
and O. O. Hmnn. 1942. A vertical stereocamera for biological photography. Iour. Biol. Photog. Assoc., vol. 11, pp. 5-12.
and G. L. STREETER. 1929. Early stages in the develop ment of pig embryos, from the period of initial cleavage to the time of the appearance of limb-buds. Carnegie Inst. Wash. Pub. 394, Contrib. to Embryol., vol. 20, pp. 1-30.
1941. Development of the macaque embryo. Carnegie Inst. Wash. Pub. 525, Contrib. to Embryol., vol. 29, PP- ‘5"S5
HUBER, G. C. 1901. Studies on the ncuroglia. Amer. Iour. Anat., vol. 1, pp. 45-61.
McC1.uNc., C. E. (ed.) 1929. Handbook of microscopical technique, for workers in both animal and plant tissues. 495 pp. New York.
1937. Handbook of microscopical technique, for workers in both animal and plant tissues. 2d ed. 698 pp. Oxford.
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