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Preservation
Morphology: Specimens for morphological study are sacrificed by immersion in 70% ethanol or anesthetized with chloroform (if available). After death, each specimen is fixed by injecting 95% ethanol into the mesosoma, usually in between two of the sternites. Kahle’s fixative, otherwise known as FAA (formalin/acetic acid/alcohol) can also be used: 70% ethanol or isopropanol, glacial acetic acid, and formalin, in a ratio 18:1:1, with 5% glycerol added to keep the specimens from becoming brittle. The chelicerae, pedipalps, pectines, legs and metasoma of each specimen are straightened, and the cheliceral and pedipalpal fingers opened, to facilitate future study and illustration. The specimens are subsequently transferred to 70-80% ethanol or isopropanol for permanent storage. The alcohol is replaced at least once during a fieldtrip or as often as necessary if it continues to discolor.
DNA: Whole scorpions, or part thereof, to be used for DNA isolation, may be fixed by injection and storage in 95-100% ethanol, flash-frozen in liquid nitrogen, or preserved in RNA-later®, depending on the duration of field trips and the restrictions in transportation of such reagents. Live specimens, collected during short field trips or obtained from colleagues, are preferably brought back to the laboratory for tissue fixation at 5°C to –20°C. 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 (Prendini et al. 2002, 2003). Amplification is more difficult from specimens sacrificed by immersion in ethanol at ambient temperature. Well-preserved scorpion tissues are readily identified because the ocelli, articulatory membranes, and internal muscle tissues turn white in colour. Degraded tissues vary from grey to brown or pink (in which case they may be useless for DNA amplification). 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 isolation. When collecting trips endure for more than fourteen days and/or refrigeration facilities are unavailable, tissues have to be fixed at ambient temperature. Simply injecting 95–100% ethanol may be insufficient to guarantee adequate preservation of high molecular weight DNA, especially in large and/or heavily sclerotised scorpions, because the ethanol will be 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 is necessary to make an incision in the pleura 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) is 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 is inadequate if insufficient ethanol is present. Ethanol is replaced within 24–48 hours after the specimen has been prepared, and again thereafter, as often as necessary if it continues to discolor. Larger, more sclerotised specimens, or quantities of specimens, are fixed in larger volumes of ethanol because of the increased dilution effect. They also require more frequent replacement of ethanol, again because of the increased dilution effect. |
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