Museum collection Protocol

Specimen Preparation for National Museum of Natural History

Protocol for Shenandoah and Big Bend National Parks Amphibian Monitoring Field Crews

At a minimum, we will collect a) one adult voucher specimen of each species at Shenandoah and Big Bend National Parks, b) two to five larvae of each species from various stages of development at each Park, and c) one section of an egg mass of each species at each Park. These will serve as voucher specimens for the study and will be useful for identification.

Note: Always wear gloves and work under a hood or in a well-ventilated area while working with formalin.

Amphibians

To euthanize tadpoles or adults, place in solution of either Chloretone (1 teaspoon/1 Liter water) or MS-222 (0.05% solution). It may help to add a few drops of 95% ethyl alcohol as solvent to either of these solutions. For large adults such as bullfrogs, rub a little Anbesol (toothache benzocaine ointment) onto forehead.

After amphibian is euthanized, place adult amphibian in 10% buffered formalin such that the entire specimen is covered and that there is at least twice as much solution as animal. Use a designated shallow tupperware container for this. Line the tupperware with paper towels (white only) and soak the towel with 10% formalin. Position amphibian according to McDiarmid (1994). Tuck elbows and legs and spread toes such that webbing is evident. You never have to inject amphibians with formalin. Once the amphibian is in position, place a formalin-soaked paper towel over the specimen.

Leave the specimen in position in the formalin for 1-2 days in the container. Then, place the amphibian in a jar of formalin for one week. After a week in the formalin, transfer the adult specimen to 70% ethanol.

Fill out data sheet for each individual adult. For tadpoles, fill out one data sheet per group of tadpoles collected at a site.

Place Tag with Specimen:
To prepare tag, wrap string around a field notebook and cut 8 inch strands. Place string through tag and tie a square knot approximately 0.25 - 0.5 inches from the tag.

	For Adult Frogs and Large Adult Salamanders Place tag using a square knot above the knee on the right rear forelimb. These specimens are are transferred to 70% ethanol for long-term storage.
	For Small Adult Salamanders Place tag around neck. These specimens are transferred to 70% ethanol for long-term storage.
	For Salamander Larvae and Anuran Eggs and Tadpoles NEVER tie a tag to a salamander larva or anuran eggs and tadpoles. Simply place the tag in the bottle with the specimens. These specimens stay in 10% buffered formalin for long-term storage.

To Prepare 70% Ethanol

Measure 70 ml of 95% Ethanol in Graduated Cylinder
Add Distilled Water to 95 ml level

To Prepare 10% Buffered Formalin:

9 parts Water (e.g., 900 ml water)

1 part Formaldehyde (e.g., 100 ml formaldehyde)

1/2 teaspoon per Liter of Magnesium Carbonate (buffer)

Reptiles

Follow same protocols as for amphibians, but for reptiles you have to inject formalin into the body. Use 10 - 20 cc syringe with 23 needle gauge and inject formalin along the length of the body (into gut).

Reference

McDiarmid, R.W. 1994. Preparing Amphibians as Scientific Specimens. Pp. 289-297 in

Measuring and Monitoring Biological Diversity: Standard Methods for Amphibians. Heyer,

W.R., Donnelly, M.A., McDiarmid, R.W., Hayek, L.C., and Foster, M.S. (Editors).

Smithsonian Institution Press, Washington D.C.

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