Left Out in the Lab: What Reagents Survive Ambient Temperatures?

Leaving your things lying around is something parents have lectured about forever, but the rule of tidiness is even more important in a laboratory setting. Biochemical products are a bit more volatile than dirty laundry, so it's important to have all of your chemicals accounted for. This article reviews some of the frequently used substances which may be left unattended and concern technicians – are they still functional, or are they damaged beyond repair? It's necessary to understand which substances are sensitive to neglect, but we hope to assuage some unwarranted panic over robust materials.

What we'll talk about:

1. Antibodies
2. Enzymes
3. DNA
4. PCR-amplified samples
5. BSA
6. ELISA
7. Bacterial cultures
8. Slides
9. Hygroscopic materials
10. Frozen substances

Before proceeding, I want to say that this article is intended to be more informative about products that have been accidentally left out. It is not guaranteed that all reagents will behave the same way and other factors can influence performance. Therefore, do not be careless with products in your lab and always store them according to manufacturer or supplier instruction

1. Antibodies:

• The product: Antibodies have variable shelf-life depending on type, but they’re generally kept with glycerol or sucrose to prevent aggregation and promote functional longevity. While shelf-life can differ, many kept at ideal conditions: temperature, pH, desiccation and lighting specifications recommended by the manufacturer can remain functional for more than a year.
• The situation: You’ve returned to the lab after a weekend and realize you left an entire box of antibodies on the counter, sitting at room temperature. The instructions inform you that storage environment should be 4°C.
• The diagnosis: There's still hope. Don't throw them out before you test them. Suppliers have done experiments on the storage and shipment of antibodies at various temperatures, and leaving them at ambient temperature even for a week did not decrease their effectiveness. Some companies even subject their antibodies to 37°C and higher temperatures for performance quality testing.
• The precautions: Purified antibodies have characteristic stability, but it has never hurt anyone to be cautious and run validation tests to be sure.

2. Enzymes:

• The product: Storing enzymes usually involves -20°C temperatures and the addition of glycerol to prevent protein denaturation. Their use in experiments is sometimes cumbersome, because it’s recommended they be kept frozen in the process.
• The situation: After conducting a reaction, another technician points out your restriction enzyme, no longer in the cooler you used to transport it from the fridge.
• The diagnosis: Enzymes usually won’t be destroyed by a couple of hours outside a freezer, and they’ll survive a power outage too. A test on a group of 23 unmodified restriction enzymes stored and shipped at ambient temperatures revealed they can remain active without being refrigerated for one to three weeks. We won’t recommend leaving them on the bench for nearly a month, though.
• The precautions: Enzyme integrity is damaged by temperature fluctuations, so thawing and refreezing products too many times will cause denaturation. Even leaving enzymes in the door of a freezer can be risky due to unmediated temperatures. Avoid protease contamination when enzymes are in open environments.

3. DNA:

• The product: DNA is incredibly complexand has to be treated with care to prevent contamination and denaturation. This is commonly done by keeping it isolated in temperatures of -20°C or lower.
• The situation: You’ve been using DNA samples since 7:00am. At 5:00pm, you’re struck with the thought that with every minute you’ve had the DNA sample out, the strands have slowly been breaking apart, invalidating the ten hours of work you just put in.
• The diagnosis: DNA stored in a dry room temperature environment will degrade, but the speed of this process is not as rapid as you might think. Long-term storage can be conducted anywhere between -80°C and 4°C, but room temperature is safe for short-term as long as contamination is not a concern with the addition of EDTA and Tris. Stability is maintained longer by buffers.
• The precautions: The freezing/thawing process, if repeated often, can damage DNA just as badly as leaving it in unmediated temperatures. Regardless of storage temperature, samples should be tested for concentration and evaporation.

4. PCR:

• The product: Related to DNA, polymerase chain reaction (PCR) techniques involve mixing DNA with polymerases, primers and other essential compounds to amplify DNA strands. Scientists commonly transfer PCR samples directly to 4°C refrigerators after the thermal cycling process.
• The situation: You start your PCR and walk away. The last cycle finishes two minutes after you leave for your one-hour lunch break. When you come back to the machine, you see that it’s been waiting for extraction at room temperature.
• The diagnosis: Inside of a PCR tube, DNA samples can retain their stability. Successful amplification will allow them to resist ambient environments for weeks, if not longer, without noticeable degradation. PCR itself is used to study ancient DNA samples left exposed to the elements for centuries; if that sample can still generate results, so can the one you left on the counter. It's important to note that many thermal cyclers will keep the tubes cool after the cycling process. Even if the machine were somehow unplugged or turned off, your samples should be OK.
• The precautions: PCR machines must be given the same attention you intend for your samples. If left cooling overnight, the machine's overall lifetime could be impacted. A less reliable device will produce less reliable amplification.

5. BSA:

• The product: Bovine serum albumin (BSA) is a blocking agent commonly used in experiments detecting the presence and relationships of protein. It is rendered from bovine blood serum, reducing residual binding capacity and nonspecific interactions.
• The situation: While conducting an ELISA experiment, you had a good number of tubes out. Your dried BSA was in a particularly small tube, and sometime in the process of things it was misplaced among the others you’d finished using. When you clean the bench at the day’s end, you find the unfinished BSA still among the empty bottles.
• The diagnosis: BSA, especially when dried and used for blocking, will be fine for days without refrigeration. Powders need to be kept dry more than cold. Powder and stock solutions should also be sturdy for a few days if they’re misplaced over the weekend.
• The precautions: Contamination from fluids is more of a problem than heat with this chemical, so it will be more of a concern if the left out container was also open.

6. ELISA:

• The product: An enzyme-linked immunosorbent assay – ELISA – tests for substances in biochemical fluids via an assay of antibodies and color. Antigens are detected in samples by specifically-targeting antibodies, and serum (like BSA) is commonly screened through ELISA kits.
• The situation: Testing for the viral antigen in a sample you’ve created, you put together an ELISA kit and go through the complex-forming process. The lab manager calls you away to perform some “housekeeping” tasks. Two days later, you remember your ELISA test.
• The diagnosis: Whether left on the bench or put in the wrong freezer, ELISA tests are fairly hard to damage in short periods of time. They can sit at ambient temperature without the results degrading, and a kit that has been unintentionally frozen will thaw out with minimal damage.
• The precautions: Extended periods of time can decrease activity in the test, but results should still be reliable. Avoiding contamination as an overall standard procedure is also important when a kit, used or otherwise, is left unattended.

7. Bacterial cultures:

• The product: Bacteria are finicky; too much heat or cold will degrade their value as scientific subjects, because temperature fluctuations damage cells. The necessary storage conditions are highly dependent on strain, but refrigeration is generally suggested. Frequently used samples are ideally stored at 4°C while those kept for longer periods will be frozen or freeze-dried.
• The situation: Your bacterial cultures are just starting to show promise in the last minutes of a Friday lab session. You consider leaving them for the weekend rather than making a fresh grouping Monday. There are risks, right? Mutations, contaminations, etc. Your intern says he once left a sample out for a week and came back to find half a dozen strains in one petri dish (though considering the state of his work bench, you aren’t surprised).
• The diagnosis: Though there are sometimes recommendations to harvest culture DNA or plasmid quite quickly (within 12-24 hours), bacterial strains have been stored for months and years in laboratory settings without adverse effects on their utility. Random mutations will take more than a weekend to transpire. The real concern is contamination or competitive growth in the media if it’s fully exposed to the environment.
• The precautions: Plasmid yields are more promising in fresher samples. Some strains are more sensitive and conducive to recombination than others, so unwanted developments are more likely to occur with a long, unmediated incubation period. It also isn’t out of the question that a large quantity of pelleted strains, left out for a weekend, will cause quite a smell.

8. Slides:

• The product: Microscopic slides are inseparable from techniques in immunology, cellular cultures and organism selection. Microscopy is associated with slide production for viewing single-celled organisms and other small (but microscopically visible) results of experimentation. Slides themselves are fragile, and sometimes so are the samples they contain.
• The situation: You’re a technician assigned to the care of several dozen slides from a friend’s month-long cellular immunology project. You leave the samples on a shelf for the week your colleague is on vacation; needless to say their return initiates a rather awkward conversation involving laboratory protocol and sample storage. Was their research ruined?
• The diagnosis: Slides with properly fixated, embedded and sealed samples can be kept at room temperature as long as the ambient environment is standardly clean and free of agents like persistent mold.
• The precautions: If the container was left open and exposed to light, this could be more of a problem. Artificial and natural light at high concentrations can harm slide integrity. Compare controls to see if anything has deteriorated.

9. Hygroscopic materials:

• The product: Hygroscopic compounds are defined as such for their water-attracting characteristics. They are able to absorb and hold water molecules from the air. It's important to keep them in desiccated states or in the presence of desiccants.
• The situation: Your dimethyl sulfoxide (DMSO) has been out in the lab for a good couple of weeks before you notice it sitting on the table. It’s conveniently placed about two feet from the laboratory sink.
• The diagnosis: Hygroscopic compounds, if encountering high amounts of moisture in an improperly sealed container, are going to be damaged. Even in a properly desiccated environment, leaving these substances for too long without changing desiccants will expose them to excessive moisture. Being attentive to indicators is important, and if they signal compromised material, your product probably isn’t usable anymore.
• The precautions: Desiccated materials can very sensitive. They require special protection from air, not because of temperature, but because of moisture, and this makes it harder to distinguish good from bad until they're quality-tested.

10. Frozen substances:

• The product: Biochemical samples are almost universally kept cool. Frozen substances are further defended from heat-provoked transformation; they’re thawed before use and returned to consistently low temperatures. This category ranges from bacterial strains to pure compounds.
• The situation: You have a stock of bacterial plasmids frozen for communal use in the lab. In the course of a day, you’ve removed the stock from the freezer, placed it on the bench, realized you needed to reorganize the entire freezer and come back to the stock seven hours later to find it in liquid form. Are the other techs going to ostracize you?
• The diagnosis: There is no one answer here. There are circumstances when a chemical or sample is frozen to prevent degeneration, and allowing it to thaw out and maintain an ambient temperature will destroy it. Alternatively, recoveries of frozen E. coli strains after hurricane-produced power outages have been up to 94% successful even after over a month. Your bacterial plasmids should also be fine. Cases must be judged individually based on the substance’s function and traits.
• The precautions: As previously stated, some chemicals will only function for as long as they are protected from temperature fluctuations. Check the labels to see how they're meant to be stored, and always be aware of your freezer settings.

A concluding thought: vigilance is essential for chemical preservation and accurate science, but it’s equally as important to consider the durability of accidentally neglected materials. Don’t rush in your panic to discard all evidence of a wandering scientific mind – if you have safe methods, good controls and experience with the substance, you should be able to identify if something is still operable versus when it belongs in the trash.

Resources

Best long term storage method for DNA? (2013, July). Retrieved June 28, 2017, from https://www.researchgate.net.

Clark, J., March, J. B., & Mdegela, R. H. (2000). Extended stability of restriction enzymes at ambient temperatures. Biotechniques, 29(3), 536-542. Retrieved June 19, 2017, from https://www.ncbi.nlm.nih.gov/pubmed.

Cody, W. L., et al. (2008). Skim Milk Enhances the Preservation of Thawed -80°C Bacterial Stocks. Journal of Microbiological Methods, 75(1), 135-138. doi:10.1016/j.mimet.2008.05.006.

Johnson, M. (2012). Antibody Shelf Life: How to Store Antibodies. Materials and Methods, 2(120). doi://dx.doi.org/10.13070/mm.en.2.120.

MiniPCR. Debunking the 4 degree myth: PCR can be left at room temperature overnight. (2016, December 2). Retrieved June 28, 2017, from http://www.minipcr.com/classroom-tips.

Moran, E. (2013, May). Using Enzymes at the Bench — Keep it in the cooler? On ice? or at RT? Retrieved June 19, 2017, from http://bitesizebio.com.

Wu, J., Kim, L., T. K., Huang, C., & Anakella, B. (2009, May). Stability of Genomic DNA at Various Storage Conditions [Scholarly project]. In SeraCare Life Sciences. Retrieved June 19, 2017, from http://www.colorado.edu.