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Ethanol concentration for tissue samples (Discussion from NHCOLL; 27 January 2016)

I recently had a graduate student request 100% absolute ethanol to use for preserving tissue samples (fish muscle and/or fins). We routinely use 95% ethanol (and all tissue samples are then stored in a -80 freezer); however, the student is adamant that 100% is the only preservative that is acceptable for tissue preservation. Does anyone have an opinion on 95% versus 100% ethanol for tissue preservation? There is a very steep price difference between the two fluids, and my understanding is that 95% ethanol preserves tissues just as well as 100%. Thanks, Sarah
Sarah K. Huber, Ph.D.
Curatorial Associate, VIMS Nunnally Ichthyology Collection
Office 804.684.7104 | Collection 804.684.7285

When you add tissue to ethanol it is immediately diluted by the water in the tissue so achieving 100% is not possible. In my opinion 95 % should be fine. One method I have used us to pour off the diluted ethanol from the sample then refill with fresh to bring up the concentration.
Paul Sweet | Department of Ornithology | American Museum of Natural History | Central Park West @ 79th St | NY 10023

Sarah,
Although I don't have any data or publications to back it up I think you are correct. There is very little difference between 95% and 100% ethanol and the minimal water content in 95% would have little to no effect on DNA preservation. That being said, both of these will degrade DNA over time and as such we only use this as a transportation fluid when shipping tissues or for field collection where liquid nitrogen is out of the question. All tissues that come into our collection have all fluids drained off before they are placed in our liquid nitrogen dewars to ensure that no further degradation of DNA occurs due to them still being in the fluid. It also ensures that the piece of tissue is accessible and you do not have to hunt through frozen to half frozen fluid to find it in the tube. I have attached publications that talk about extraction of DNA from 95% ethanol preserved specimens.
Hope that helps
Andy Bentley
Ichthyology Collection Manager, University of Kansas, Biodiversity Institute, Dyche Hall, 1345 Jayhawk Boulevard, Lawrence, KS, 66045-7561, USA

Hi Sarah,
Presumably this relates to preservation of DNA? If so, then it was agreed, a few years ago, that using 96% ethanol and stored at 5 deg. C was the best. 99% and absolute were found to be less effective. I am sure there will be other comments relating to this.
With all good wishes, Simon.
Simon Moore MIScT, RSci, FLS, ACR
Conservator of Natural Sciences and Cutlery Historian
www.natural-history-conservation.com

Hi Sarah,
alcohol above 95% is technically dried (to remove the residual water which at this point can hardly be removed by distillation) with low boiling benzines. The resulting ethanol is traded as /High Gradient Grade/ ethanol or similar (99.9 %). We had repeatly bad results using technically dried ethanol when preserving fish tissues (tissue : ethanol ratio was around 1:3 to avoid dilution from released tissue fluids). We do not know the exact reasons, but suppose residual chemicals used during technical drying or subsequent purification to have caused observed degradation of tissues. Since then, we avoid 99.9 % and use 95% only, with good results. Tissues are stored at -25?C, however insects may be more stable under refrigerated conditions ( + 4?C), because released fats may form waxy deposits on the cuticula, which can hardly be removed if "empty" specimens (after extraction) should be prepared for final storage in dry collections.
Hope this helps
Dirk

As Simon points out, it depends what you want the specimen for. On the one hand, in our experience clean DNA (single genes or the whole thing) can be got from specimens in concentrations as low as 70% (though there is some variation between organisms); on the other, 95% will shrink and harden a specimen, making it difficult to do anatomical work without rehydration that itself damages cells. Absolute alcohol (99.9%) has been denatured using chemicals, as distillation only gets purity up to 95%. Any trace denaturant might have unpredictable long-term effects on the specimen.
Paul Callomon
Collection Manager, Malacology, Invertebrate Paleontology and General Invertebrates

DNA storage tubes for -80°C (Discussion from NHCOLL in 2011 and 2016)

In April 2011, the North Carolina Museum of Natural Sciences purchased 1.8 mL NUNC CryoTube vials (cat. no. 368632) for storing tissue collections in our new ultra-cold (-80C) freezers. This vial, which is internally threaded and has a silicone O-ring at the seal, had been used by me (Bryan Stuart) for many years to store amphibian and reptile tissues in EDTA/DMSO salt saturated tissue buffer or in RNAlater (Qiagen), without problems. However, other units at the museum have tissue collections that were preserved in 95-100% ethanol, and we soon discovered after transferring those collections into these vials that ethanol rapidly evaporates from them (on a scale of weeks).

After lodging a complaint with our sales representative, NUNC in Denmark performed an analysis and determined that the vial's silicone O-ring is vapor permeable and therefore not suitable for use with ethanol. In fact, I was also told that none of NUNC and Nalgene's cryogenic vials are intended to store ethanol, but rather were designed for tissue culturing (I broke the news that there are tens or perhaps hundreds of thousands of cryogenic vials being used in natural history collections in the U.S. for non-tissue culture purposes, a large number of which are holding ethanol).

Our sales representative provided us with three alternative vials to test that are externally threaded and lack a silicone O-ring seal (and so are plastic on plastic). These are the Nalgene Cryoware Cryogenic Vials (cat. no. 5000-0020), NUNC CryoTube vials (cat. no. 375418), and Nalgene Cryogenic Vials (cat. no. 5000-1020). In our experiment, we stored 95% ethanol in the original vial with the O-ring and the three alternate makes without an O-ring at four different temperatures (room temperature, 4C, -20C and -80C) for about six weeks. Indeed, the vial with the O-ring dramatically evaporated ethanol, while the other three vials did not. Interestingly, ethanol evaporated fastest at the warmer temperatures. i.e. ethanol evaporated fastest at room temperature and slowest at -80C (I had anticipated the reverse, that freezers were very desiccated and so would evaporate fastest). We also learned that the amount of torque placed on closing the vials with the O-ring seals, i.e. "over-closing" the vials and therefore distorting the O-ring seal, did not have any appreciable effect on evaporation.

In sum, internally threaded cryogenic vials with a silicone O-ring seal such as the NUNC CryoTube vial (cat. no. 368632) should be avoided for storing tissues in ethanol. Instead, we would recommend other models that are externally threaded without an O-ring seal, such as Nalgene Cryoware Cryogenic Vials (cat. no. 5000-0020) or NUNC CryoTube vials (cat. no. 375418).
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Bryan L. Stuart, Ph.D.
Curator of Herpetology & Director of Molecular Genetics Laboratory
North Carolina Museum of Natural Sciences