Hot, Frozen, Sublimed and Blown: Biological Sample Storage Methods Summarized – Part One

I’ve recently been doing some lyophilization of biological extracts. While I was preparing for the experiment, I became interested in the number of different methods there are for drying, concentrating and storing samples: freezing, freeze-drying, rotary evaporation, centrifugal evaporation and blow down drying. Here is a brief description of each technique for biological sample storage to bring some clarity and describe how they might be useful.

Freezing

This has the potential to be as simple as popping your sample in the –20ºC or –80ºC freezer; however, there are still a few things to bear in mind to maintain sample quality.

• DNA and buffers: for genomic material, where a little sheering (from ice crystal formation) is not a big deal, or for unstable buffers such as those containing DTT, this is great storage method with no adjustments required.
• Proteins: these are a just bit more tricky as you need to add an anti-freeze. Sucrose or glycerol can work well but the percentage depends (as always!) on the individual protein. I’ve often used 20% sucrose or 10% glycerol. Restriction enzymes can be stored in 50% glycerol to maintain stability. With its lower freezing temperature, this concentration of glycerol prevents the solution from freezing at all!
• Making aliquots: additionally, multiple freeze–thaw cycles can be detrimental, so it’s a good idea to aliquot your samples into appropriate quantities. This goes for DNA (when you want to avoid excessive sheering), most proteins and I also do this for unstable buffers. To take maximum advantage of your prep and minimize waste, aliquoting requires knowing the concentration of your sample and how much you are likely to need in future experiments, so that the aliquots are of an appropriate volume. For tiny volumes, PCR strip tubes, which snap off when the plastic is cold, work well. It’s a bit tedious, but well worth it.
• Microbes (bacteria, yeast and fungi): 10–20% glycerol works well. Higher percentages (i.e. closer to 20%) make for a softer stock, which is easier to sample quickly for streaking out on a fresh plate.
• Higher eukaryotic cells: these require a specialized protocol with time-controlled freezing and an anti-freeze such as DMSO. Details of the protocol depend on the cell line.

Furthermore, some macromolecules maintain functionality better with flash-freezing prior to storage. Flash-freezing is the closest you can get to being a wizard and throwing (should I say gently dropping?) things in a cauldron. To flash-freeze, put on your wizard gear (lab coat, goggles, gloves), gather your sample aliquots and then drop them into a dewar containing a small amount of liquid nitrogen and watch it bubble and smoke (don’t do this in an area with no ventilation!). Once the bubbling has stopped, fish them out (a wire hotpot basket is good for this) and store at –20ºC or –80ºC.

Vacuum Concentration or Centrifugal evaporation

Also known as ‘Speed Vacing’, because of the most popular instrument brand name, this involves centrifuging a sample to keep it in the tube while applying a vacuum that removes evaporating solvent (Figure 1).

You can use it to either lower your sample volume, which will concentrate the macromolecules, or to remove all the solvent to leave a dried sample. The type of vacuum concentrator (sounds posh when you say it like that!) you need will depend on your solvent.

• Benign aqueous solutions are the simplest, needing only the centrifugal force and a vacuum.
• Organic solvents can be categorized into non-aggressive (e.g. ethanol, methanol, acetonitrile) and aggressive (e.g. chloroform, ethyl acetate, hexane), and other solvents include strong acids or bases, or solvents such as DMSO, which have high boiling points. Some of these will require the vacuum concentrator to have a coating on the interior of the instrument to prevent corrosion, vapor traps to collect noxious fumes or, in the absence of a vapor trap, outlets that end in a fume hood.

The interior gets rather warm during the drying process so watch out for heat sensitive samples! Check on the solvent and instrument capabilities before you start – inadvertently poisoning your lab mates would be a sad way to end an illustrious scientific career. Thermo Scientific is a good resource for understanding more about a vacuum concentration instruments.

These two methods might be the first you will use in your laboratory endeavors – they were for me. They are common and usually readily available. In the second section, I’ll tell you about lyophilization, rotary evaporation and blow down drying that offer more or less sophisticated alternatives to concentrating and storing your samples.