Clearing the Specimen

The general procedure we adopted was to clear the tissues to semi-transparency by placing the specimen in the styrine monomer. (The catalyzed resin completes the clearing process). The specimens may again need to be placed under vacuum, and should be allowed not only to sink to the bottom of the container but to remain there for some hours longer. Overnight is satisfactory for small specimens of dimensions 7 × 5 × 3 cm. This ensures a thorough penetration of the monomer into the tissues.

We use, however, a slightly different technique for specimens that have been macerated in potassium hydroxide. With this material we have a further series of clearing baths. The first bath is a 1:1 mixture of monomer and polymer. The second a 1:3 mixture and the final bath a 1:6 monomer/polymer mixture. Again the specimens should remain in each grade of the series for two or three days at least after they have sunk to the bottom of the container. The mammalian embryo (noted above on page1) remained in the final stage of the clearing series for 14 days after it had sunk to the bottom. This technique not only assists materially in giving and retaining tonicity in the specimen, but it eliminates air bubbles that may remain even after vacuum treatment, beneath the skin and in the oral and nasal cavities.

Nonetheless, we have not had 100% success in eliminating air bubbles in the hardened block with this type of material (Fig. 12). This is particularly the case with young embryos 2 to 5 cm in length that were not eviscerated prior to maceration and in which the newly formed bone is very porous. Our main problem has been the prevention of very tiny air bubbles forming in the marrow cavities and lungs. This may happen even from one to three days after the block has set sufficiently hard to be removed from the mould, but not hard enough for polishing. The after-clearing method we have found most successful is given on p. 6 under the heading "Insertion of the specimen in the catalyzed resin".