Talk:Genetic Resources

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

Labels Inside Vials, including Rite-in-Rain Paper (Discussion from NHCOLL; 6 January 2017)

Rite-in-Rain or "all weather" field paper is used by the many agency biologists depositing specimens and genetic samples with the MSB Division of Fishes and other repositories. Recently, the USFWS Southwestern Native Aquatic Resources and Recovery Center has requested that I find out if there are any issues with using this paper for genetic labels in the field or otherwise. They tried to get information from the Rite-in-Rain company re: what was in the paper and coating (formalin?) to determine if the paper would pose a problem for genetic samples. The company was not forthcoming re: Rite-in-Rain product specifics.

We have had many genetic samples received at both the Center and the MSB with these paper tags made by tearing pieces of paper out of the RIR field books, writing the sample number on RIR tag with pencil, and stuffing the tag into a cryotube. Not optimal in terms of contamination issues, etc. but getting word out to field crews, etc. can be difficult. For many projects, I preprint labels on polyspun paper (DataMax) for researchers, but there are many opportunistic collections that receive these temporary labels.

Any thoughts or insights? Does anyone have an idea of how this paper is manufactured/treated?

Thank you.
Alexandra M Snyder, Collections Manager-Fishes
Museum of Southwestern Biology MSC03-2020
University of New Mexico Albuquerque NM 87131
PH./FAX 505.277.6005<tel:(505)%20277-6005> amsnyder@unm.edu<mailto:amsnyder@unm.edu>

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We have a policy here at KU that nothing gets put inside the tube except the tissue and preservative (if necessary). There are just too many possible avenues of contamination from the ?paper? to the ?ink? to the residue left on the paper by human contact. All labels are external and usually adhered before going in to the field using a Brady labeling system and cryo safe labeling material.

Andy
Andy Bentley
Ichthyology Collection Manager
University of Kansas
Biodiversity Institute
Dyche Hall
1345 Jayhawk Boulevard
Lawrence, KS, 66045-7561
USA

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Hi Alexandra,

I am not sure about the composition of RIR, but if uses formaldehyde e.g. in the pulp to glue it together, the paper might be reactive if it gets into contact with formalin, e.g. start to swell, get sticky / stick together. I once tried a high quality archival paper and put a small bundle of labels into formalin. As soon as they entered, they started to swell and stuck firmly together; it was impossible to detach individual labels from each other again.

But there are also other chemicals that could do harm (e.g. shifting the pH). Normally, if a paper is some sort of water resistant, either the surface and bottom and/or the pulp is hydrophobised. Normal paper already uses up to 30 chemicals, waterproofed paper surely more. Even if you use RIR only temporarily in the field until samples are returned, you may already have a chemical cocktail inside because 96% EthOH is a fairly good organic solvent, too.

For me, externally pre-numbered tubes seem the safer option; or tubes that can be marked externally with pencils. If those internally RIR tagged samples fail during extraction or give bad sequencing results, you add just one more unknown to the black box of weird lab mysteries making samples fail.

I guess the only other option would be to specifically test for effects of internal RIR tags by subdividing a tissue to two tubes, one externally labelled, the other with internal RIR, returned under identical conditions to the lab, and compare e.g. DNA yields after extraction, PCR results, etc. if you want to be sure. For sure still not the answer you are looking for, but hope it offers some sort of help.

All the best Dirk

Dirk Neumann Tel: +49-89-8107-111 Fax: +49-89-8107-300 email: Dirk.Neumann(a)zsm.mwn.de postal address: Bavarian Natural History Collections The Bavarian State Collection of Zoology Dirk Neumann, Section Ichthyology / DNA-Lab Muenchhausenstr. 21 81247 Munich (Germany)

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.. apparently RIR is using

pure barium sulfate (insoluble in water and pH neutral) as paper brightener (cf. wikipedia section); barium is a heavy metal and it might be worth testing how much barium gets solved if immersed in 96% EthOH https://urldefense.proofpoint.com/v2/url?u=https-3A__en.wikipedia.org_wiki_Barium-5Fsulfate&d=CwID-g&c=-dg2m7zWuuDZ0MUcV7Sdqw&r=CLFZJ3fvGSmDp7xK1dNZfh6uGV_h-8NVlo3fXNoRNzI&m=5JM_j-aYlqQ-PWhGjALnFjjsWtXgyTb3v9_aeeOwNMI&s=AjJo-zB3Snzj22ifaJoDVfeQygAgZjjX1bcIwuy118s&e=

Lucidene?605 seems to be not pH neutral (> 8.0), and might cause pH shifts, especially in 96% EthOH which barely has any buffering capacities, especially since RIR paper omits any calcium carbonate in the paper https://urldefense.proofpoint.com/v2/url?u=http-3A__www.hydrite.com_Files_resources_Paints-2DCoatings-2DInks-2D-2DAdhesives_GraphicArtsIndustryBrochure-2Dv5.pdf&d=CwID-g&c=-dg2m7zWuuDZ0MUcV7Sdqw&r=CLFZJ3fvGSmDp7xK1dNZfh6uGV_h-8NVlo3fXNoRNzI&m=5JM_j-aYlqQ-PWhGjALnFjjsWtXgyTb3v9_aeeOwNMI&s=TE424emySUQKGDknSD6qQqYyZ3uyMO1dlCLz_00NxlM&e=

and one acrylic monomere (which they don't exactly want to disclose; they are citing a broader range of such acrylates); these are small acidic and highly reactive molecules, as the denaturant methyl ethyl ketone (MEK) based on a propylene. It is the simplest unsaturated carboxylic acid, and miscible in water, alcohols ethers and chloroform. https://urldefense.proofpoint.com/v2/url?u=https-3A__en.wikipedia.org_wiki_Acrylic-5Facid&d=CwID-g&c=-dg2m7zWuuDZ0MUcV7Sdqw&r=CLFZJ3fvGSmDp7xK1dNZfh6uGV_h-8NVlo3fXNoRNzI&m=5JM_j-aYlqQ-PWhGjALnFjjsWtXgyTb3v9_aeeOwNMI&s=y7goomOq6zi4mkPu0unz-DoVq69dtQZm8Kelvt8B4vM&e=

Waterproof sheet od RIR are "/derived from monomers selected from the group consisting of styrene, butyl acrylate, 2-ethylhexyl acrylate, acrylic acid, and a mixture thereof/"

Acrylic acid reacts with alcohols and forms the corresponding esters; respective esters and slats thereof are known as acrylates, which are highly reactive; it also readily combine with themselves to form polyarcylic acid or other suited monomers (containing ammonium) to acrylonitrile or acrylamid.

It is not unlikely that it dissolves into the ethanol of the tissue tubes and at least may cause pH shifts, but may also otherwise spoil the DNA of included organisms.

If the overall pH remains neutral because of the Lucidene remains to be tested, but from the ingredients that are disclosed, I would not use it inside tubes, even though this might be handy in the field or researchers love it the easy way.

Thanks for sharing the patent link, Dean - good to know that you can look up patents online

All the best Dirk

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Hi All,

I agree with the opinion that one should avoid putting anything in tubes with samples if at all possible. That said, I collected a lot of fish tissues in the early 90?s and put a RITR label in every tube with 90% EtOH, 10% TE. I was able to extract DNA from every sample and the DNA was in fine shape. A friend here in Tacoma (where J.L. Darling is located) is the former co-president of RITR. I have a call in to him and will let the list know if I learn anything new or different from what has already been posted. Best, Peter

Peter Wimberger Director, Slater Museum of Natural History Albertson Professor, Biology and Environmental Studies University of Puget Sound Tacoma, WA 98416-1088