Preserving Techniques
Specimens for morphological study are typically preserved by immersion in 70% ethanol. Larger specimens are fixed by injecting them with 95% ethanol. Kahle’s fixative, otherwise known as FAA (a mixture of formalin/acetic acid/alcohol in a ratio 18:1:1, with 5% glycerol added to keep the specimens from becoming brittle), can also be used to fix specimens, although it renders them unsuitable for DNA extraction. Whichever method of fixation is used, the chelicerae should be opened to facilitate future study and illustration. Once fixed, the specimens should be transferred to 70-80% ethanol or isopropanol for permanent storage (ethanol is preferred, as it is better at preserving color). The alcohol should be replaced fairly soon after the initial preservation, and again as necessary if it continues to discolor. It may be possible to temporarily transfer specimens into propylene glycol (found in some antifreezes) for shipment or transportation on airplanes, although with airline restrictions subject to frequent change, the acceptability of this practice should be verified first. Be sure NOT to use ethylene glycol as a preservative if DNA extraction is intended.
Solifuges that are to be used for DNA extraction may be fixed by injecting them and storing them in 95-100% ethanol, flash-freezing them in liquid nitrogen, or storing them in RNAlater®. The choice of method for preservation may depend on whether one is in the field or in the lab, and upon restrictions in the transportation of reagents. Live specimens collected during short field trips or obtained from colleagues are preferably brought back to the laboratory alive for tissue fixation at 5°C to –20°C. Specimens fixed by freezing alive in 95–100% ethanol must be retained at low temperature for at least 7–10 days to ensure adequate fixation, after which the ethanol (now diluted) must be replaced before the samples are dispatched or processed for DNA extraction. This method has been found to significantly increase the yield of high molecular weight DNA, presumably because low temperatures retard degradation of the tissues during the period when fixation occurs. Cryo-shippers (dry-shippers), which employ a double vacuum wall to absorb liquid nitrogen, freeze specimens in vapor and will retain a temperature of -150°C for up to three weeks. These can be used in the field when available. Because there is no residual liquid, they may be transported on airplanes. Alcohol-preserved specimens that are stored in 95% or 100% ethanol typically must be shipped through a courier service, as they are not permitted on airplanes.
When collecting trips extend for more than three weeks and/or refrigeration facilities are unavailable, tissues will have to be fixed at ambient temperature. Unfortunately, amplification is more difficult from specimens sacrificed by immersion in ethanol at ambient temperature, especially during hot weather, so any available means of keeping the specimens cool (such as ice chests or 12-volt refrigerators designed for use in vehicles) should be employed. Simply injecting 95–100% ethanol into the specimen may be insufficient to guarantee adequate preservation of high molecular weight DNA, particularly in large solifuges, as the ethanol is unable to diffuse through the specimen before the soft tissues have degraded through autolysis (a process that occurs within a very short period of time, usually a matter of hours). In such cases, it may be necessary to make an incision in the opisthosoma or remove a pedipalp and/or legs from one side of the specimen, to allow the ethanol to diffuse more rapidly into the internal tissues. A sterile scalpel and dissecting tray (e.g. a petri dish) should used for each dissection to avoid contaminating one specimen with tissues from the preceding specimen(s). It is also important to use a high ratio of ethanol to tissue volume for each specimen or specimen-lot because fixation will be inadequate if insufficient ethanol is present. Ethanol should be replaced 24 to 48 hours after the specimen has been prepared, and as often as necessary thereafter if it continues to discolor. Well-preserved specimens are readily recognized because articulatory membranes and internal muscle tissues turn white in color.
Complete and accurate collection data should be associated with each preserved specimen or lot. This should include, at minimum, an accurate record of the geographic location where the specimen was found (including GPS data if possible), along with the date of collection and the name of the collector.