5. Amphibians & Reptiles

5.1 Voucher Requirements

The identification of most species of amphibians and reptiles can be adequately confirmed from photographs (provided that diagnostic features are clear), so collecting is not required during inventory projects for identification purposes. Concern for declining populations of several species of amphibians necessitate that communication between involved agencies occurs before any collecting is done. Generally, collections should not be made unless there is a need for tissue, i.e. DNA work, disease analysis.

Diversity Inventory

A photograph (showing diagnostic features) of each amphibian or reptile species encountered should be taken to provide documentation of identification.

Directed Inventory

If collecting is required to obtain tissue for a project, then the number to be collected must be determined on a project to project basis. Consult the research technicians at the lab you will be working with to determine how much tissue will be required.

5.2 Data Needs

• Required Data Fields
• Field collection number (Collector's number); Collector's name; Collection date; Detailed location (gazetteered location name as well as Latitude and Longitude or UTM; Elevation (m); Genus; Species; Identifier; Date of identification; Sex; Age; Snout to vent length (cm or mm); Collection method (capture method).
• Enter sample data including location and physical site information and specimen data including taxonomy and number of collections separately (both data sets can later be linked by the collector's number).
• Use a .dbf file format to record data digitally.
• Labels
• Write data in dark pencil onto appropriately sized label paper.
• Minimum label data required: Field collection number; Collection date; Genus; Species; Location description; Latitude / Longitude or UTM.

5.3 Preparation and Care of Specimens - Amphibians

5.3.1 Photographs

• Photographs of amphibian species encountered should be taken with a macro or close-up lens, and they should show features used for identification.
• Photographs should be submitted with the pertinent raw data.
• It may be necessary to take more than one picture of one specimen from different angles.

5.3.2 Whole Specimens

Killing

• Amphibians are most efficiently killed by immersing them into a solution of chloretone, made by dissolving a few grains of hydrous chlorobutanol crystals (available at a pharmacy or through Fisher Scientific) in a litre of water.

Fixing

• Use a container with a tight fitting lid. (A "Tupperware" type plastic container approximately 33 x 21 x 6 cm works.)
• Line the bottom with a white paper towel or cheesecloth soaked in 10% buffered formalin and position the animal so measurements can easily be taken and examined for key features. Formalin penetrates the body cavity of small amphibians quickly but large frogs and salamanders will require injections into the gut, body cavity and large muscle masses.
• When the floor of the tray is covered with specimens, blanket them with a second paper towel wet with formalin and carefully fill the tray with formalin to about one third its depth. Be sure that labels with field data are assigned to the proper specimen. Most specimens will have hardened enough to maintain their shape after a few hours.
• Attach field tags to the specimens. The tags should be tied with a square knot above the knee on the right rear leg of frogs and large salamanders and around the neck of small salamanders. Larvae should be placed in small vials with buffered formalin.
• Transfer specimens to a jar where they are immersed in 10% buffered formalin.
• Larvae should be placed in small vials with 10% buffered formalin. A field label should be put into the vial and another field label should be attached by a string and tied to the vial under the lid.
• Amphibian eggs require special care, as they are easily damaged. Single, short strings or small clumps of aquatic eggs can be placed directly into small bottles or vials of 10% buffered formalin.

5.4 Preparation and Care of Specimens - Reptiles

5.4.1 Photographs

• Photographs of reptile species encountered should be taken with a macro or close-up lens, and they should show features used for identification.
• Photographs should be submitted with the pertinent raw data.
• It may be necessary to take more than one picture of one specimen from different angles.

5.4.2 Whole Specimens

Anaesthetizing

• Reptiles may be killed by injecting them with the anaesthetic Nembutal, diluted 1:5 to 1:10 depending on the size of the specimen. Specimens die quickly and are ready for fixing. Muscle contractions and kinking may occur if too much is injected.
• Ether is another method of killing reptiles. Place a cotton swab soaked in ether in a covered container and insert the animal.

Fixing

• Reptile skin inhibits preservatives from entering the body quickly enough to prevent rotting, so injections must be used.
• Small lizards should either be injected with 10% buffered formalin into the body cavity or have a cut made on the left ventral (underside).
• Larger lizards should be injected with 10% buffered formalin in each leg segment and at the base of the tail just underneath the skin.
• Snakes should be injected with 10% buffered formalin at three or four points between the snout and vent. The tail is injected separately. The hemipenes should be everted and tied off.
• Turtles should have the head and neck extended from the shell and a piece of wood or plastic put into the mouth to keep the jaws open. With the snout up 10% buffered formalin should then be injected into the neck, limbs, tail and deep into the body cavity and lungs to keep the specimen from floating in the preservative.
• Place specimens into a hardening tray which can be a plastic container with a tight fitting lid approximately 33 x 21 x 6 cm. Line the bottom of the tray with white paper towels or cheesecloth that have been soaked in 10% buffered formalin solution. The specimens should be positioned in a way which allows key features to be seen readily, the maximum number of measurements to be obtained and permits placement in a bottle.
• Cover specimens with a layer of paper towels soaked in formalin. Carefully fill the tray with formalin to about one third its depth. Keep track of the animals in the tray so that the correct label with field data is assigned to the proper individual. Most reptiles require soaking for several hours or overnight in the hardening tray.
• When the specimens are hardened, remove them from the tray and tie on their field tags. Snakes should have their tags sewn on.
• Reptile eggs may be put directly into a vial or bottle of 10% buffered formalin. Large eggs should be injected with a 10% buffered formalin.

5.5 Museum Accessioning

The collection manager at the museum needs to know approximately when the voucher specimens are to arrive. Close contact should be maintained to ensure efficient handling of the specimens. Any interim reports or field notes must accompany the voucher collection upon arrival.

Royal BC Museum Contact: Kelly Sendall, Invertebrate Zoology Collection Manager

Phone (250) 387-2932 Fax (250) 387-5360

Email ksendall@rbml01.rbcm.gov.bc.ca

5.6 Materials and Costs

Table 4. Materials associated with preparing whole amphibian and reptile specimens.

Item	Specifications	Supplier
Jars	125ml, dim. 51 x 102mm Cat. No. 21749	Anechemia, Richmond, BC
	250ml, dim. 62 x 127mm Cat. No. 21750	Anechemia, Richmond, BC
	500ml, dim. 76 x 145mm Cat. No. 21751	Anechemia, Richmond, BC
Lids	48mm for 125ml jar (1300/box)	Lukian Plastic Closures, Oakville, ON
	58mm for 250ml jar (1800/box)	Lukian Plastic Closures, Oakville, ON
Liners	polyethylene, 45mm for 125ml jar	Premo Plastics, Victoria, BC
	polyethylene, 54mm for 250ml jar	Premo Plastics, Victoria, BC
Fixative	37% formaldehyde	Northwest Labs, Victoria, BC
Preservative	95% ethanol (not denatured)	Stanchem, Vancouver, BC
Label Paper	78 lb. Permafibre	Coast Paper, Vancouver, BC

5.7 References

Heyer, W.R., M.A. Donnelly, R.W. McDiarmid, L.C. Hayek and M.S. Foster, eds. 1994. Measuring and monitoring biological diversity, standard methods for amphibians. Smithsonian Institution, Washington, DC. 364 pp.

Simmons, J.E. 1987. Herpetological collecting and collections management. Society for the Study of Amphibians and Reptiles. Herpetological Circular #16. Univ. Texas. 70 pp.