Taking, preserving, and archiving tissue samples

Excerpt from Winker, K. 2000. Obtaining, preserving, and preparing birds. Journal of Field Ornithology 71:250-297.

Specimens frozen whole on dry ice or LN2 very quickly following death provide tissue samples of high quality for genetic studies. It is important to take tissue samples from these specimens as soon as possible to maintain this usefulness. Whole frozen specimens held at -80 C prior to preparation can be brought slowly up to room temperature by being held in an ordinary (-20 C) freezer for hours or days first, or brought directly to room temperature under close observation. Tissues should be taken and refrozen as soon as the specimen has thawed sufficiently to be worked. Usually, tissues can be taken while the organs are still frozen. With experience in preparation, in small birds the skin can be removed from the carcass and tissues taken before the internal organs have thawed.

Two tubes of tissues should be preserved whenever possible. These should be stored separately, if possible, for both the short and long term to prevent loss of critical material through freezer failure or other catastrophe. Also, tissue tubes should be filled to within 2-3 mm of their tops whenever possible. This maximizes the amount of tissue available for future studies, minimizes wasted freezer space, and prevents tissue desiccation.

Taking tissue samples from each specimen should be a priority for anyone collecting regularly, and can be done by anyone who has the opportunity to work with dead birds. Winker et al. (1996) emphasized the crucial nature of voucher specimens when taking tissue samples. I will not discuss sampling protocols that do not preserve voucher material (e.g., bleeding and releasing live birds). Such cases should be exceptional.

Traditional allozyme studies use several different tissues because of the different proteins available in each. In general, heart, liver, muscle, and kkidney are saved for these studies (Johnson et al. 1984). Although allozyme studies have declined in popularity with the advent of improved DNA technologies, when one is freezing fresh tissue samples it is a good idea to preserve all of these tissues in each tissue tube. Doing so in the order above insures that the types are separable later in the molecular laboratory.

For modern DNA studies, frozen tissues are not absolutely required, and freezing has become less popular because of the extra field logistics required (LN2 or dry ice). This trend is short-sighted, however; frozen tissue samples are still preferable. When freezing tissues is not possible, one can place minced tissues in a vial of buffer (Seutin at el. 1991) or place small pieces into 95% ethanol for dehydration. New archival methods of taking and storing genetic samples will be or are being developed (e.g., using blood-soaked paper that has been chemically treated), but thus far ultracold freezing represents the closest thing to a museum-quality tissue preservation method. Given DNA chemistry, aqueous solutions do not represent long term, archival-quality storage conditions (see Cann et al. 1993, Poinar et al. 1996). Thus, aqueous field storage methods should not be trusted over the long term (years), and should be stored in cryosystems upon return to the museum or laboratory.

Labelling tissue vials presents a special problem in that, for frozen samples, labelling must be completed prior to freezing: one cannot write on a frozen vial. Some curators have gone so far as to give tissue samples final tissue catalogue numbers during preparation (in field or laboratory) by issuing blocks of blank tissue catalogue pages or numbers to collectors and preparators. This requires the preparator to fill out two catalogue entries during preparation, just to issue the specimen a (usually) second unique number. I find this to be needlessly cumbersome and rarely comprehensive - not all specimens entering a collection will be generated using the in-house numbering system.

Cryovial manufacturers make inserts for the vial lids that can be written upon and inserted when samples are finally arranged for cryostorage and catalogued. A specimen does not need multiple unique numbers prior to being catalogued into a collection, and the traditional field catalogue number is sufficiently unique to serve the purpose. This number is also borne by all of the other parts of the animal. Although I haven't seen a duplication in the traditional initials-and-number field catalogue system (see Fig. 1 of Winker 2000), the date, taxon, and locality information accompanying the number make later confusion seem impossible should duplication ever occur. However, given the labelling constraints on tissue vials (even tissues in buffer or alcohol should be frozen for long term archiving), the method outlined in Fig. 3 of Winker (2000) is preferred, since it provides all of the information necessary to assure rapid and accurate sample recovery from the freezer whether the sample has been catalogued into a collection or not. Writing the field number twice is insurance against the loss of information through abrasion, which is not uncommon, especially in LN2 dewars.

Replace caps onto vials very tightly and freeze tissues immediately. Caps that are not replaced very tightly often come off in LN2 and the samples are lost. When you have attained the ability to skin out a bird in about 10 min (separating skin from body), you can take tissues after the body is out. Taking tissue samples and sexing a bird is much easier with the body removed, but tissue freshness is extremely important. I try to get tissues frozen within 30 min or less of thawing to maximize their usefulness in molecular studies. With the carcass skinned, more tissue can be taken in small birds. When skinning small birds, I generally take all large muscles (breast, wings, legs) and all internal organs except intestines and stomach. Lungs and kidneys must be lifted up out of their bony recesses, and with practice can usually be taken out in one or two pieces.

Cann, R. L., R. A. Feldman, L. A. Freed, J. K. Lum, and C. A. Reeb. 1993. Collection and storage of vertebrate samples. Methods in Enzymology 224:38-51.

Johnson, N. K., R. M. Zink, G. F. Barrowclough, and J. A. Marten. 1984. Suggested techniques for modern avian systematics. Wilson Bull. 96:543-560.

Poinar, H. N., Höss, J. L. Bada, and S. Pääbo. 1996. Amino acid racemization and the preservation of ancient DNA. Science 272:864-866.

Seutin, G., B. N. White, and P. T. Boag. 1991. Preservation of avian blood and tissue samples for DNA analyses. Canadian J. Zool. 69:82-90.

Winker, K., M. J. Braun, and G. R. Graves. 1996. Voucher specimens and quality control in avian molecular studies. Ibis 138:345-346.