Museum Collection Management Terms and Invertebrate Specimen Processing Procedures: Methods of Fixation and Preservation

VOUCHER COLLECTIONS

A voucher collection should be established early in the taxonomic phase of a project. The greatest number of voucher specimens will be derived from the first cruises or first collecting efforts. However, new specimens will continually be added to the various series as taxonomically important variations are encountered or as new taxa are identified.

The voucher collection for each major taxon should be established by competent, professional taxonomists. Ideally the same taxonomists would be responsible for the identification of all non-voucher specimens. Taxonomic references used in the identification of the voucher series as well as the voucher specimens themselves must be available for use during the identification of the general (non-voucher) collections. All specimens in the general collection identified to any taxonomic level must exhibit taxonomically important characters identical to those characters exhibited by specimens in the corresponding voucher series. The re-identification of a voucher specimen/series by the taxonomic contractors will require that all corresponding specimens in the general collection be re-evaluated and, if necessary, re-identified to insure consistency between the names used for specimens in the voucher collections and those used in the general collections.

DEFINITION OF LOT

A lot is a group of specimens associated at some taxonomic level and are from the same collection locality. As the identification of the specimens is refined, they are still associated as a sample, but are broken up into separate lots based on phylum, class, order, family, genus, or species associations of the sample. All specimens in a lot should be identified to the same Lowest Practical Taxonomic Level (LPTL).

FIXATION AND PRESERVATION OF MARINE INVERTEBRATES

The fixation of biological specimens involves the coagulation of cell contents into insoluble substances with the purpose to prevent autolysis and the degradation of tissue. The optimal fixation of biological specimens is achieved on individual live, preferably relaxed, material. Bulk fixations usually yield mediocre results as various animal taxa must be fixed by variant means, due to the different chemical makeup of each animal group. A good fixation is generally achieved in a brief amount of time (hours to days) and as soon as the animal is collected. After a favorable fixation is attained, the next procedure is the transfer of the specimen(s) into the proper fluid for archival storage. General biological fixation/preservation procedures are discussed below. Specific fixation and processing for molecular analysis, TEM/SEM, histology, and histochemistry require specialized techniques that are not mentioned in this discussion.

Formalin is generally the preferred fluid for fixation and is widely used. Formalin is often sold as 37-40% aqueous Formaldehyde. Formaldehyde is a gas produced by the oxidation of methyl alcohol, whereas 100% Formalin is a saturated solution of this gas in water. To make a solution of 10% Formalin, nine parts of water are added to one part of 40% (aqueous) Formaldehyde. Therefore, a 10% solution of Formalin is the equivalent of a 4% solution of Formaldehyde. Formalin/Formaldehyde solutions and vapors are hazardous. Avoid contact with skin and eyes, and use only in a well ventilated area or in a fume-hood.

Formalin/Formaldehyde readily oxidizes into formic acid, an acidic solution in the pH 2.5 to 5 range. To prevent the dissolution of calcified tissue and other tissue damage, Formalin/Formaldehyde solutions are commonly buffered or neutralized. Since sea water has a pH of 8.2 and in solution with Formalin/Formaldehyde has approximate pH of 7, it is sometimes used as a buffer. However, the buffering capacity of sea water does not last over time. Calcium Carbonate (CaCO₃) or Borax (Sodium Borate) in supersaturated solutions with Formalin/Formaldehyde are commonly used buffers for short term formalin fixation. They should not be used to buffer Formalin/Formaldehyde solutions used for long-term storage since their effects do not last over time and Borax may cause lysis of tissue and clearing of pigments. For long-term Formalin/Formaldehyde storage, a phosphate buffer is preferred. A suitable phosphate buffer (Sörensen’s Buffer) is prepared using four grams of monobasic sodium phosphate monohydrate (NaH₂PO₄ ^.H₂O) and six grams of dibasic sodium phosphate anhydrate (Na₂HPO₄) per liter of a solution of 10% Formalin.

The archival preservation fluid that has been used the longest and is generally preferred is alcohol. The standard is 70-75% ethyl alcohol or ethanol, however, 40-50% isopropyl alcohol or isopropanol is used on some animal taxa. Formalin may be used for some meiofauna and plankton specimens, especially gelatinous forms. "Sorting solution" (1.5 parts propylene phenoxetol, 5.0 parts propylene glycol, 10.0 parts full strength Formaldehyde, & 83.5 parts distilled water) has been used successfully for the long-tem storage of some taxa, but larger specimens may deteriorate over time.

The following table describes recommended fixation and preservation procedures for various invertebrate animal taxa and bulk samples. Specific lengths of time that a specimen is placed in a relaxation, fixation, or wash fluid is a matter of preference and greatly is determined by the size of the specimen. For specific animal taxa, it is recommended that more research on methods of relaxing, fixing and preserving specimens be conducted before specimens are collected and processed. Recipes for various fluids of fixation and preservation (indicated with an asterisk) are shown below.

Recipes