Paper - Demonstration of the cartilaginous skeleton in mammalian fetuses

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Miller CH. Demonstration of the cartilaginous skeleton in mammalian fetuses. (1921) Anat. Rec. 415-

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This historic 1921 paper by Miller described development of the human skeleton.

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Demonstration of the Cartilaginous Skeleton in Mammalian Fetuses

Charles H. Miller

Department of Embryology, Carnegie Institution of Washington

In 1902 van Wijhe showed that the cartilaginous skeleton could be demonstrated perfectly in small embryos by staining the specimen with anilin dyes, especially methylene blue, then rendering it transparent in xylol and mounting in Canada balsam. His success was based on the fact that cartilage takes the stain intensely and retains it after the color has been extracted by acid alcohol from all the other tissues. Before the publication of this method it had been necessary, in order to study the morphology of the cartilaginous skeleton, to prepare the embryo in sections and model the skeleton by one of the reconstruction methods. The van Wijhe coloring method possesses a great advantage in that the entire skeleton can be prepared quickly and studied directly, with all parts in their natural connection and the contours of the Whole embryo and of the different organs clearly recognizable. On the other hand, the larger specimens, which cannot be mounted in balsam and have to be preserved in xylol, soon lose their transparency and become opalescent.

Two years after the publication of the van Wijhe method, Lundvall introduced some improvements in it that served to make it more adaptable to medium-sized and large objects. He stained the cartilage with toluidin blue in place of methylene blue, and instead of xylol and balsam to render the specimens transparent, he used benzol, from which they were finally transferred to carbon bisulphide. In this fluid even large specimens will remain transparent and can be easily studied.

Both cartilage and bone can be demonstrated by the wellknown potassium hydroxide clearing method of Schultze, which method has been used and experimented with in investigations on the development of the skeletal and vascular systems by Mall, Bardeen, and Hill. Bardeen found that bone and cartilage could be madelto stand out prominently by staining the embryo in alum cochineal and subsequently clearing it in potassium hydroxide. Further experience, however, has shown that such preparations are not as clear as specimens successfully prepared by the van Wijhe and Lundvall technique.

After a considerable amount of experimenting, I have found that very satisfactory results can be obtained by combining the use of the stain advocated by Lundvall with the potassium hydroxide clearing method of Schultze. In this way I have succeeded in making brilliant permanent demonstrations of the cartilaginous skeleton in pig, monkey, and human fetuses varying from 20 to 150 mm. (crown-rump length). In view of the fact that the procedure is simple and inexpensive and that it produces uniformly good results, it is to be strongly commended, and an outline of my experience with it may prove of interest to other workers.


Formalin. Most of the human fetuses with which I have experimented had been fixed in 10 per cent formalin solution. Among them were specimens which had been a long time in the solution and whose original condition was poor, with more or less maceration, as well as freshly fixed and well-preserved specimens. Although in the latter the staining reaction to toluidin blue is a little better, the old material nevertheless gave very satisfactory results. With formalin material other stains (alum cochineal, methylene blue, and hematoxylin) yielded poor results. Resorcin fuchsin (Weigert’s) gave fair results, but much inferior to toluidin blue, mhiclr were uniformly good.

Alcohol. Fresh material fixed for six to ten days in 95 per cent alcohol, so that the embryo was well shriveled, gave excellent results with toluidin blue, as did"alsu specimens that had been preserved for a long time in alcohol.

Bichromate acetic mixture (Mi.'1l1er’s fluid to which 10 per cent glacial acetic acid had been added). Fresh specimens up to 40 mm. long fixed in this fluid, or material that had been fixed in it and preserved in 80 per cent alcohol for a considerable time, gave brilliant results with toluidin blue, fair results with methylene blue, and poor results with hematoxylin and with resorcin fuchsin. STAIN ING

My best results in all of the various fixatives are unquestionably obtained by the use of Gri'1bler’s toluidin blue prepared according to the formula of Lundvall. Smaller embryos are thoroughly stained with it in three days; fetuses 40 mm. long should be left in the stain at room temperature for seven days, which time can be shortened, however, by the thermostat; fetuses 150 mm. long it is necessary to leave in the stain at least ten days. Before staining it is well to take the precaution of neutralizing any residual acid from the formalin by immersing in ammonia water overnight. After staining, the specimen is decolorized in acid alcohol (1 per cent hydrochloric acid in 70 per cent alcohol), which, for small specimens, requires about seven days and for large specimens ten days. The acid alcohol is changed daily until the fluid is only slightly tinged with the extracted stain, and the specimen is then ready for clearing.

Next to toluidin blue, methylene blue gave the most §atisfactory results. The disadvantage of this stain, however, is that there is some danger of carrying the decolorization too far. The color is extracted more rapidly by the acid alcohol. Small embryos should not be left in the decolorizing fluid more than two days. A further disadvantage is that the color of the specimen subsequently fades much more rapidly than after toluidin blue. Formalin specimens, stained in resorcin fuchsin, gave fair results. This stain is more resistant to-“acid alcohol than either methylene blue or toluidin blue, is more difficult to thoroughly extract the color from the other tissues; hence the specimens are not so brilliant. Both hematoxylin and alum cochineal were followed by poor results, owing to the difficulty in securing uniform decolorization and to the fact that, in clearing the specimen in potassium hydroxide, the remaining stain was completely removed.


Small embryos can be easily rendered transparent by passing them through the graded alcohols, then benzol, and finally into oil of wintergreen, after the Spalteholz method, or into carbon bisulphide, according to Lundvall. With larger fetuses (over 110 mm. long) we have experienced some difficulty in making them completely transparent. On account of this, resort was had to potassium hydroxide, which usually cleared even the largest specimens and without any detriment to the stain. In one instance (a human fetus 258 mm. long), although the greater part of the specimen cleared satisfactorily, there remained some irregular milky patches which would not clear. Resort was had to ‘absolute alcohol and ether, on the basis that the failure was due to the subcutaneous fat. This treatment, however, did not remove the difficulty, which is still unexplained.

Specimens may be transferred directly from the acid alcohol to 2 per cent potassium hydroxide, but this tends to soften the tissue. It was therefore found better to thoroughly harden the specimen in 95 per cent alcohol before clearing. Embryos 20 mm. long will clear in 2 per cent potassium hydroxide in two days at an average room temperature, after which they should be transferred to 20 per cent glycerin. For fetuses 40 mm. long four days is sufficient. The rapidity with which the specimens‘ become clear is influenced greatly by the temperature; in summer the process is much more rapid than in winter. With large fetuses it is well to start with 2 per cent potassium hydroxide and after two changes to increase the strength to 3 per cent. They should stay in the latter solution until they are transparent, which may require seven days or longer. On transferring to glycerin, if it is found that the specimen is not completely transparent, it can be safely returned to 3 per cent potassium hydroxide for further clearing. After the specimen is cleared it is passed through graded glycerin, beginning with 20 per cent and increasing 20 per cent every two days, until it is finally stored in pure glycerin, to which has been added a crystal of thymol to prevent mold. The clearing of larger specimens is facilitated by removing the brain through the anterior fontanelle and the viscera through a median incision in the abdomen. In the permanent storing of such material it is well to take the precaution of protecting it from the light. I have some specimens that have been kept in subdued light for two years and which show scarcely any indication of fading.


The complete method that we are now using in this laboratory for preparing specimens for the demonstration of the cartilaginous skeleton may be summarized as follows:

1. Formalin material. is first washed overnight in water to which a few drops of ammonia have been added.

2. Transferred to 70 per cent alcohol, where it is left for from seven to fourteen days, changing the alcohol daily for the first five days. 4

3. Stained in Lundvall’s solution of toluidin blue (Gri'1bler’s toluidin blue, 1 grain; alcohol 70 per cent, 400 cc.; HCl, 4 cc.).

4. Decolorized in 70 _per cent alcohol, 100 cc.; HCl, 1 cc., for from seven to ten days, until the alcohol is but slightly tinged with the stain.

5.’ Transferred to 80 per cent alcohol, then to 95 per cent alcohol, three days in each.

6. Transferred to 2 per cent KOH in distilled water, where it is left until cleared, usually two or three days.

7. Transferred to glycerin, 20 per cent, 40 per cent, 60 per cent, and 80 per cent, respectively, in distilled water, two days or more in each; the larger the specimen, the longer it should remain in each grade of glycerin.

8. Stored in pure glycerin to which have been added a few crystals of thymol to prevent mold.

Literature Cited

LUNDVALL, H. 1904 Ueber Demonstration embryonaler Knorpelskelette. Anat. Anz., Bd. 25, S. 219~223.

Scnurrrzn, O. 1897 Ueber Herstellung und Conservierung durchsichtiger Embryonen zum Studium der Skeletbildung. Anat. Anz., Bd. 13 (Verhandl. der Anat. Gesellsch.), S. 3-5.

VAN WIJHE, J. 1902 A new method of demonstrating cartilaginous microskeletons. Proc., Koninklijke Akademie van Wetenschappen te Amsterdam, May 31.

Cite this page: Hill, M.A. (2024, April 12) Embryology Paper - Demonstration of the cartilaginous skeleton in mammalian fetuses. Retrieved from

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