Wilhelm His Sr. and the development of paraffin embedding

Paraffin histology is one of the most important and commonly-used laboratory techniques in diagnostic histopathology. The discovery of paraffin embedding is often attributed to the pathologist Edwin Klebs. Klebs was following the lead of Stricker, who embedded embryos in a mixture of hot stearin and white beeswax. We show that Klebs experimented with paraffin wax for embedding tumour tissue. But he quickly rejected it as unsuitable because paraffin wax did not infiltrate the tissue. One of Klebs’ correspondents, embryologist Wilhelm His, Sr., learned of Klebs’ experiments and decided to try paraffin embedding. His dehydrated chicken embryos in alcohol, cleared them in lavender oil, and dripped hot paraffin wax onto them. This process allowed His to cut good sections. Here, we have replicated His’s paraffin embedding protocol in order to determine whether His had indeed made the landmark discovery of infiltration embedding with paraffin wax. We followed the protocol that he gives in his 1868 monograph on the early development of the chicken. The protocol described by His failed, in our hands, to yield sections of the quality that he illustrates in his monograph. Typically, the tissue disintegrated when sectioned due to poor infiltration of the wax. Usable sections could only be obtained if His’s protocol was modified by melting the embedded embryos in fresh paraffin wax. One explanation for our findings is that we failed to faithfully replicate His’s protocol. Another is that his protocol was incomplete. We suggest that His is likely to have discovered and perfected infiltration embedding with paraffin wax but did not publish a complete protocol. Supplementary Information The online version of this article (10.1007/s00292-021-00947-4) contains supplementary material, which is available to authorized users.

even with far more complicated techniques.
Some notes on the techniques used by the Würzburg school can be found in the Latin version of the dissertation of Pander. Those notes describe how Pander and his associates opened the eggs under water, a technique that originated with Dollinger, was later recommended by Erdl, and was recently adopted by Moleschott and Dursy. Some maceration in water, and pulling apart with needles, are the only other procedures that appear to have been applied by the older observers. Now, more than ever, the authors of research papers are required to describe the methods that they used. I will try to satisfy this requirement here, and while the methods of eggmanipulation I have used have served me well, there are certainly other methods that may be better suited for some purposes.
Following the Würzburg method recently communicated by von Baer, I cracked the blunt end of the egg, broke the egg open, and then poured the contents into a shallow glass dish 1 .
The chalazae are cut close to the yolk, either before or after decanting the egg contents, so that the innermost, viscous layer of albumen can be stripped from the vitelline membrane as a coherent sleeve. The embryo is caused to lie uppermost by rotating the yolk, the cutting round it in a circle, and lifting clear of the rest of the yolk by means of a large glass coverslip.
This operation fails only if the albumen was not properly removed from the vitelline membrane. If this is done with the necessary care, 2 day embryos can easily be lifted off cleanly and brought under the microscope. In the case of younger embryos, however, a certain amount of yolk comes with the detached specimen. In the case of un-incubated eggs, the entire floor of the germinal cavity, together with some yellow yolk, also comes loose.
The removal of adherent yolk from the isloated embryo, and removal of the vitelline membrane, is carried out using iodine serum [animal serum with iodine as a preservative], which I pipette in a very gentle stream over the specimen. In this and the following operations until dissection, the coverslip is kept [in place] as both a support and a suitable means of transport of the preparation, which under these circumstances suffers little distortion or folding 2 .
I now place the cleaned embryo, still adhering to the coverslip, under the microscope. At low and medium magnifications, it is easy to observe it from the dorsal side, and from the ventral side if one uses the humidity chamber of Kühne, which consists of a slide with an opening drilled through it, and the opening closed at the bottom with a plate. Of course, the [cavity thus formed] must be large enough to accommodate the embryo without the embryo touching the plate when it is turned upside down.
After I have completed my preliminary examination of the fresh embryo, I add a few drops of 0.5% osmium tetroxide until the embryo becomes slightly brown in colour. The osmium tetroxide is then removed by rinsing the embryo with dilute alcohol. Then it is placed, sequentially, in dilute, stronger and finally in absolute alcohol, and after it is sufficiently dehydrated, in essential oil (lavender oil) 3 .
2 Remak, to the best of my knowledge, was the first to use the method of extracting the embryo on a glass plate; he used fresh crayfish blood for rinsing, and I observed him doing this [with excellent results]. Erdl recommends warm salt water, also Moleschott and Dursy in their newest study, find c. 1% salt water to be optimal. The excellent medium of Max Schultze [animal serum with iodine as a preservative] combines the expediency of the former with the convenience of the latter method.
When it has been prepared in this way, the embryo can be sealed between two coverslips in Canada Balsam, and cemented onto a microscope slide with a hole drilled in it. This mounting technique has the advantage that the preparation can be viewed from both sides at high magnification. -If the embryo is to be sectioned, I first bring it under the microscope again and make a drawing of it using a camera lucida at low magnification and wax. We produced, in collaboration, a series of models using the specimens and sections, the aim of which was to achieve the most accurate and naturalistic representation of the embryo.
Those who have never tried plastic modelling can hardly imagine the [level of precision and control] that it provides. Every detail, every apparent irregularity of a section takes on its own special meaning, every flaw [in the section] produces an error in the plastic model, and the detailed working through of such models with compasses and rulers gives a certainty of intuition that can scarcely be achieved in any other way.