The following procedure, a modification of Davis and Gore (1936) can be used for staining and clearing small vertebrates such as worm snakes, frogs, and fetuses. It is possible by this method to render the muscle transparent and the skeleton a cleat brilliant red. The procedure is not difficult but, due to their fragility, specimens must be handled carefully. To prevent complete deterioration of the specimen, the digestion and staining procedures should be accomplished as rapidly as the specimen will allow. The worm snake and frog should be digested and stained in 36-38 hr. The fetuses of pigs, cattle, and rabbits should be completed in 48 hr.

I. Fixation

1. Due to the use of KOH and strong sunlight in this process, glass containers are preferred. Place specimen in the container and "kill" by adding 70% ethyl alcohol. This will leave the specimen in a relaxed position.

2. Those specimens with hard skin and pigmentation such as the worm snake or frog are skinned. Fish must have thier scales removed from the body. The scales are dermal in origin and will stain. Fishes should be eviserated if they are very small. Fetuses are also eviscerated. Specimens which are not previously preserved should be placed in formalin alcohol acetic acid (FAA) solution for at least one week.
 

3. Pour off the FAA solution and wash gently in H₂O for one hour by decantation.

Note - Specimens which have already been preserved in formalin or alcohol should be soaked in water for one hour.

II. Digestion

Add 2% potassium hydroxide to specimen (2 g KOH, 100 ml H₂O) (Humason 1962). The KOH should equal twice the volume of the specimen. Because of the delicate muscle composition of the frog and worm snake, a weak solution of KOH must be used. With fetuses of pigs, rabbit, rat, sheep, and cattle, a 4% KOH solution (4 g KOH, 100 ml H₂O) works effectively.

The time required for digestion of the specimen will vary with the size of the specimen. A pig fetus 12.5 cm in length should be kept in potassium hydroxide approximately 18-24 hr. A worm snake, 20 cm in length was kept in KOH for 6 hr.
 
 

III. Staining

Saturate by adding Alizarin Red S slowly while shaking.

2. Add by drops 1 ml Alizarin Red S stain for each 100 ml of KOH to the specimen. Keep the specimen in the stain until the bones are colored red. Check the ribs of the worm snake or the mandible of the fetus for stain penetration. The staining process will not take longer than 4-6 hr., depending on the density of the muscle of the specimen.

3. Decant stain and add fresh KOH. This will remove excess stain from the muscle and permit you to check the small bones which are embedded deeper in the tissues.

4. Initial de-staining can be complete in 12 hr for small specimens; 24 hr for large specimens such as cattle fetus 14 cm in length. Keep the specimens in KOH on a white surface (a white paper under the glass container) in strong sunlight to accelerate the de-staining of the muscle. Pour off the KOH solution and start clearing procedure.

IV. Clearing

The specimens must be cleared of fluid and replaced by glycerin. This is accomplished in three stages using these solutions:

1. Pour off KOH and add solution No. 1 to specimen. Retain specimen in this clearing solution until it becomes partially translucent; snakes and small fetuses, 3 days; pigs or cattle fetuses, 10 days.

2. Decant clearing solution No. 2 and add clearing solution. Keep in solution No. 2 from 1-3 weeks, using your judgment concerning the correct length of time. Remember that the object is to slowly replace the fluid of the specimen with glycerin.

3. Decant clearing solution No. 2 and add clearing solution No. 3 to the specimen. The specimen should remain in this solution the same length of time that was used in the second step. It should reach its maximum transparency during this period.

4. Store specimen in pure glycerin. To prevent mold growth add a crystal of thymol. The addition of glycerin to the specimen has toughened it and it can now be handled. If desired, the specimen can be embedded in plastic.

This is a simple effective method of bone evaluation in small embryos and fetuses. The specimen is digested in KOH stained with alizarin and cleared by adding glycerin to the muscle of the specimen. When completed there is a brilliant red skeleton structure in the transparent muscle. The specimen can then be stored in glycerin or embedded in plastic.

Davis, D. Dwight and Ulys Roy Gore. 1936. Field Mus. of Natur. Hist. Technique Series, No. 4 Oct. 1936, p. 1.