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I know that a -80°C freezer is the recommended means of long-term cell-line storage, and that cells will typically not last long in a -20°C freezer. But how long will a typical bacterial strain (e.g., E. coli DH5-alpha) actually stay good in a -80?

Searching around via Google, I'm finding a wide range of answers, anywhere from only 3 months to indefinitely, so I'd like to try to get a better grounded way of estimating longevity.

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    $\begingroup$ For what is worth, our 6 year old agrobacterial strains in -70 freezer seem to grow just fine. However, I am not sure I would notice if some percentage of cells died, since we don't take precise amount of the mixture when taking a bit for growing. $\endgroup$
    – BagiM
    Mar 8 at 14:32
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    $\begingroup$ Exactly: I'm finding lots of anecdotal data points, like this, which makes me very uneasy about predictability of lab operations. $\endgroup$
    – jakebeal
    Mar 8 at 14:37
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    $\begingroup$ How predictable do you need your storage to be? There are many things that can happen in several years time. Freezer can fail without people noticing until morning. Someone can forget bacteria outside after using them. If my strains die I can make new ones with the same plasmid. For important stuff, there are backups. $\endgroup$
    – BagiM
    Mar 8 at 15:02
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    $\begingroup$ To be frank, there probably isn't one answer. It will likely depend on the organism and possibly the specific strain you're preserving, how they were cultured, the concentration and type of cryoprotectants used, and the starting concentration of viable cells, among other possible factors. In any case, the decline in viability is likely to progress slowly enough at -80C that you'll notice before total loss of viability, as long as you're using a sound practices. $\endgroup$
    – MikeyC
    Mar 8 at 15:45
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    $\begingroup$ There are viral strains preserved from 50+ years ago. RNA is generally more sensitive to temperature. $\endgroup$
    – Fizz
    Mar 9 at 0:55
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The reason you haven't found a definitive answer is that it depends on several factors. The first factor that comes into play if we consider only ultra-low freezers (- 80 °C) is the organism you're freezing. Thermofisher gives an approximate time of bacterial cultures viability between 1 to 10 years :

$$\begin{array}{|l|c|c|} \text{Condition} & \text{Temp (°C)} & \text{Time (approx.)} \\ \hline \text{Super-cooled freezer} & \text{-80} & \text{1 - 10 years} \\ \end{array}$$

For ATCC, the shelf life is even shorter :

-80°C is sufficient for most bacteria and fungi for short-term storage (5 years or less)

However, in Table 1 from She and Petti (2015), we can see that the shelf life - for an ultralow-temp freezing storage (-70 to -196) - varies greatly both between the organism groups and inside each group : up to 1 year for the Streptococci group and up to 3 decades for the gram-negative bacteria for example.

$$\begin{array}{|l|l|c|} \text{Organism group} & \text{Cryopreservative} & \text{Storage duration (yr)} \\ \hline \text{Streptococci} & \text{Skim milk} & \text{0.2 - 1} \\ \text{Mycobacteria} & \text{Skim milk} & \text{3 - 5} \\ \text{Spore-forming bacteria} & \text{Skim milk, glycerol} & \text{2 - 30} \\ \text{Other Gram-positive bacteria} & \text{Skim milk, sucrose, glycerol} & \text{1 - 30} \\ \text{Gram-negative bacteria} & \text{Sucrose, lactose, glycerol} & \text{2 - 30} \\ \text{Filamentous fungi} & \text{Glycerol, DMSO} & \text{2 - 30} \\ \text{Yeasts} & \text{Glycerol, DMSO, skim milk} & \text{2 - 30}\\ \text{Protozoa} & \text{Blood, nutrient medium with DMSO or glycerol} & \text{} \\ \text{Viruses} & \text{SPGA} & \text{1 - 30} \end{array}$$

How the stocks are made and used are also important factors :

  • Among other things, ATCC recommends a gradual freeze and a quick thaw :

In addition to using a cryoprotectant, the rate of cellular dehydration during the freezing process can be managed by using a -1°C/minute cooling rate

Thawing should be rapid at 37°C

  • ThermoFisher recommends a high cell density as cell death will occur no matter what :

Also, the greater the cell density, the better the recovery is after thawing the cells. For most bacteria, a density of 107 cells/mL will result in adequate recovery if all conditions are properly maintained.

Cell death during storage is inevitable but should be minimized as much as possible

  • The number of freeze/thaw cycles is critical and thawing even partial should be avoided :

Try not to freeze/thaw your glycerol stock too many times. Placing the glycerol stock on dry ice while streaking onto LB agar will prevent it from thawing completely and will improve the shelf life. from addgene.org

Coming back to the initial question, based on the information above and some of the feedback in this discussion stocks of the typical E. coli DH5-&\alpha& stored at - 80 °C would probably last up to 2 or 3 decades maybe more but depending how you make them and use them it can be much less. To go further, depending on your workflow you may want to consider having working and stock vials to limit the number or freeze thaw cycles or using freeze-dried stocks for longer term storage.

Edit : One thing to keep in mind too is that frozen stocks shelf life is not all about cell viability upon thawing but also about "cell integrity". As mentioned by She and Petti (2015), van Griethuysen et al. have observed the loss of the mecA genes in 2 years old Staphylococcus aureus isolates frozen stocks.

The mecA gene was lost in 36 (14.4%) of 250 methicillin-resistant Staphylococcus aureus isolates after 2 years of storage at −80°C with the Microbank system (Pro-lab Diagnostics, Austin, Tex.). Further analysis of 35 of these isolates confirmed loss of the mecA gene in 32 isolates. This finding has important implications for the management of strain collections. I personally experience a similar issue with a routinely used strain that lost its adhesive phenotype so we had to use the backup stock.


References :

  • She, Rosemary C., and Cathy A. Petti. "Procedures for the Storage of Microorganisms." Manual of clinical microbiology (2015): 161-168.
  • Van Griethuysen, Arjanne, et al. "Loss of the mecA gene during storage of methicillin-resistant Staphylococcus aureus strains." Journal of clinical microbiology 43.3 (2005): 1361-1365.
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Richard Lenski's lab at Michigan State university has been doing long-term evolutionary research on E. coli and maintaining a "frozen fossil record" of their experimental populations every 500 generations since 1988. They still periodically release research papers comparing the physiology of evolved and ancestral populations. For example, this recent paper looks at populations collected at 2000, 10000, and 50000 generations. According to this timeline, they used populations frozen over 3 decades ago.

This doesn't guarantee that your specific organism will last that long, but it does show that freezer stocks can remain viable for longer than some research labs will even exist. Most of their protocols are on the website. I've had lunch with Dr. Lenski, and he's a pretty approachable guy. I'm sure if you emailed him with specific questions that aren't covered on the website, he'd try to find an answer for you.

References:

Changes in Cell Size and Shape During 50,000 Generations of Experimental Evolution with Escherichia coli. Nkrumah A. Grant, Ali Abdel Magid, Joshua Franklin, Yann Dufour, Richard E. Lenski, Journal of Bacteriology Mar 2021, JB.00469-20; DOI: 10.1128/JB.00469-20

Lenski Lab homepage: http://myxo.css.msu.edu

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