[NHCOLL-L:5722] Re: CryoTubes

Thu Nov 3 05:58:20 EDT 2011

Dear Gabriela, Bryan

we had similar problems with TPP-brand Cryo vials, which resulted in up 
to 100 % evaporation losses before arriving in the field ... (maily 
related to underpressure conditions in smaller aircraft enrout to the 
final sampling locations). We did then compare several Cyrotubes 
(O-Rings vs. no O-Ring, internally vs. externally threaded, the whole 
variety) by measuring evaporation losses of ethanol tubes (2 ml standard 
volume in each vial, stored for three weeks in 56°C warm water bath). 
Results were devastating, except (surprisingly) for common Eppendorf 
safe lock tubes (!) and NUNC storage vials (cat. no 264300).
http://www.nuncbrand.com/us/page.aspx?ID=267.

Because of higher evaporation pressure at room temperature, it is not 
surprising that your results indicate higher losses in vials kept at 
room temperature compared to vials stored frozen. You should also 
consider that ethanol below 80 % volume evaporates as pure ethanol, 
leaving more residual water in your vial. In combination with the water 
coming from the included tissues/samples, the drop of the absolute 
ethanol concentration  might be even more critical then the actual 
evaporation loss itself (= general loss of fluid level).

Some CryoTubes seem to require deep freezing (-80°C) for hermetical 
closure depending on technical tolerances required for the lids when 
kept frozen at -80°C (corresponding shrinkage of tubes and vials); 
therefore, _Cryo_Tubes should not be your first choice for field 
sampling or storage at room temperature.

We found that Nunc Storage vials work perfectly in  the field (up to + 
40° C, underpressure conditions) and in our deep freeze chambers (-25°C; 
which is sufficient for tissue storage). For storage vials (cat. no 
264300), NUNC guarantees temperatures down to -40 °C. One advantage of 
these vials is that they do not have O-Rings (which may over time 
crumble apart as not all O-rings are made from silicone but sometimes 
from a mixture of different plastics which include softeners). Vials and 
caps are made from HDPE and LDPE respectively, and therefore should be 
inert for leaching polymers or softeners.

After replacing some 6000 cheap apex tubes (majority with miserable 
O-Ring caps) we decided to invest some more bugs in high quality tubes 
while saving working ours for monitoring & replacement. Sadly, we first 
replaced with high quality Cyrotubes with the already indicated results ....

For DNA-storage (-80 °C), we use Thermofisher 2D barcoded V-bottom tubes 
(cat. no 3792MTX).
see 
http://thermoscientific.com/ecomm/servlet/productsdetail_11152_L10857_80494_12811553_-1  
but with different vials in matrix racks; cat. no 3792MTX is not 
displayed for any reseason on the Thermofisher hompage, but racks are 
available under this cat. no.

Some further reading in 
http://www.abctaxa.be/volumes/volume-8-manual-atbi/volumes/volume-8-manual-atbi/chapter-22/chapter_22.pdf 
(free access)

Dirk

Am 31.10.2011 22:47, schrieb Hogue, Gabriela:
>
> I am posting this for Dr. Bryan Stuart. Please send any 
> questions/comments to him at bryan.stuart at ncdern.gov 
> <mailto:bryan.stuart at ncdern.gov>
>
> 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 internally threaded without an O-ring seal, such 
> as Nalgene Cryoware Cryogenic Vials (cat. no. 5000-0020) or NUNC 
> CryoTube vials (cat. no. 375418).
>
> ---
>
> Bryan L. Stuart, Ph.D.
>
> Curator of Herpetology & Director of Molecular Genetics Laboratory 
> North Carolina Museum of Natural Sciences
>
> 1626 Mail Service Center, Raleigh NC 27699-1626 USA (postal address)
>
> 4301 Reedy Creek Road, Raleigh NC 27607 USA (physical address for 
> FedEx, UPS, etc.) t 919.733.7450 ext. 751  f 919.715.2294 Lab Webpage: 
> http://www.bryanlstuart.com Museum Webpage: http://www.naturalsciences.org
>
> E-mail correspondence to and from this address may be subject to the 
> North Carolina Public Records Law and may be disclosed to third parties.
>

-- 
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