Supplementary information for Altermatt et al. Methods in Ecology and Evolution. DOI: 10.1111/2041-210X.12312

“Big answers from small worlds: a user's guide for protist microcosms as a model system in ecology and evolution”

Altermatt F, Fronhofer EA, Garnier A, Giometto A, Hammes F, Klecka J, Legrand D, Mächler E, Massie TM, Pennekamp F, Plebani M, Pontarp M, Schtickzelle N, Thuillier V & Petchey OL

1.7 Long-term preservation

Below, we first describe the use of Lugol’s solution to preserve dead protists in samples (e.g., for counting/identification, section A) and second describe the procedure to store protists alive, using cryopreservation (section B).

A) Lugol’s solution


Lugol's solution can be used to store samples of protists for several weeks or months. Some cells can be damaged during the procedure, so it is important to pay attention to the concentration of the Lugol's solution you use and not to store the samples for too long. The literature on the effect of the concentration of Lugol's solution on the proportion of cells damaged during the procedure is inconsistent, varying across a few percentage. A specific feature of Lugol's solution is that the protists are stained (they turn to red-brown color; they can be easily seen and counted under a microscope in a bright field) and they are heavy, so they sink to the bottom of the vial. Thereby, one can concentrate the sample by removing part of the liquid above them (or use an inverted microscope to count/observe them). However, Lugol's solution can slightly affect the cell size and shape due to shrinking, which can invalidate comparisons between preserved and unpreserved cells regarding these features.



  • Brown glass vials with screw tops to store Lugol’s solution and samples.

  • Pipettes.


  • Lugol’s solution (also known as Lugol’s iodine) at 5 % iodine potency. This solution can be made of 5 % (weight/volume) iodine and 10 % (weight/volume) potassium iodid (KI) mixed in deionized water, resulting in a total iodine content of 126.5 mg/mL


The aim is to have a 0.5% concentration of Lugol’s solution in the stored sample, higher concentrations lead to the loss of larger percentages of cells. Therefore, to store 1 mL of sample, about 5 microliters of Lugol's solution have to be added to have a final concentration 0.5%.

  1. Take an empty vial and add the right amount of Lugol's solution.

  2. Add the sample with protists that you want to preserve. Adding the Lugol's solution to an empty vial and then adding the sample ensures that it mixes properly.

  3. Close the vial and gently turn it upside down and back to mix the sample (do not shake it too much).

  4. Remember that you cannot store samples in Lugol's solution indefinitely. Storage up for several weeks to a few moths is usually fine.

Important: Lugol’s solution is light sensitive. Store samples in the dark, or (better) in brown glass bottles in the dark.

B) Cryopreservation


There are several reasons why long-term storage of protist cultures using cryopreservation (or cryoconservation, i.e., storage at ultra-cold temperatures, below –130 °C), usually in liquid nitrogen (LN2), is desired (McAterr & Davis 2002; Day & Stacey 2007; Cassidy-Hanley 2012).

Firstly, cryopreserved stocks act as a renewal backup (cell banking) from which standard liquid cultures of strains with a specific interest can be recreated when needed. This is the primary raison d’être of protist culture collections (see section 3.1.1). Recreating cultures from a frozen stock is needed after bacteria/fungal contamination or accidental loss/extinction of the culture in the liquid medium. However, regularly reinitializing protist cultures is also necessary to prevent undesired genetic changes due to evolutionary changes during prolonged vegetative growth. For example, it is advised to restart Tetrahymena cultures every 6 months (Cassidy-Hanley 2012). This is necessary to prevent major genetic changes in the germinal micronucleus, transcriptionally inactive and hence under strong genetic drift. Specifically, this is needed to preserve specific mutations when the wild type has a selective advantage, causing a high risk of the mutation of interest to be lost due to random assortment of macronuclear chromosomes during asexual reproduction (Cassidy-Hanley 2012). It is however important to note that the low survival during thawing makes there is no 100% guarantee of genetic stability even with cryopreservation.

Secondly, cryopreservation of protist cultures can be a key point in some studies, for example in experimental evolution (Kawecki et al. 2012). Indeed, it allows taking a snapshot of a culture/strain under specific conditions and at a certain time. Such cryopreserved cultures can then be subsequently revived by thawing, to be compared on phenotypic or genetic aspects, such as evolved versus non-evolved strains (Kawecki et al. 2012).

Standard protocols for the cryopreservation of protists are published (McAterr & Davis 2002; Day & Stacey 2007; Cassidy-Hanley 2012), or are readily available at webpages of culture collections (e.g., Freezing implies a phase of culturing the protists under specific conditions to prepare the cells and ensure the highest cell viability, the use of specific cryoprotectants, and a progressive and controlled cooling down before long-term storage in liquid nitrogen. Cryopreservation in principle works for all protists species, but we focus here mostly on Tetrahymena as a well-developed example. We use it to detail the material, reagents and protocols necessary to implement long-term cryopreservation in LN2 in a laboratory. We go beyond the mere description of freezing/thawing protocols by delivering information about key points for successful establishment of LN2 cryopreservation in the research laboratory, such as consequences of material choice, or the importance of a reliable inventory system.

For a given protist species, changes in the protocol will likely reside in specific points only, such as culture conditions prior to adding the cryoprotectant, or centrifugation force and duration. We advise searching the literature and the internet using species (or genus) names associated to keywords such as “cryopreservation”, “cryoconservation”, “cryogenic”, “freezing”, or “liquid nitrogen” to gather more specific information. It is important to recognize that reviving protists after cryopreservation does not always work, and may be less straightforward than with bacteria. We thus recommend testing survival rates for each specific protist species/strain and cryopreservation method before using it as a routine.

Extra general information on cryopreservation technique, safety, and material (especially recent advances in cryogenic material) can also be obtained from companies selling cryogenic equipment, such as Thermo Scientific (, Thaylor-Wharton ( or Air liquide (

The preferred storage for long-term cryopreservation is in liquid nitrogen (–196 °C), because viability of frozen cells can tremendously decrease in case temperature increases above –130 °C, even for a short period of time. At –196 °C, metabolic reactions are slowed down so extensively that living cells can be maintained for very long time (potentially indefinitely). Handling liquid nitrogen needs careful training of staff and the necessary precautions.

CAUTION: Safety note associated to use and handling of liquid nitrogen (LN2)

It is important that staff is trained in the use of LN2 and associated equipment. Indeed, there are several safety risks associated to the use and handling of LN2 that can be important and should not be minimized, despite they can be largely controlled by enforcing clear procedures and a limited extra equipment:

  • LN2 is extremely cold (–196 °C) and immediately burns skin or eyes in case of contact. Never touch or immerge body parts into LN2, and wear adequate protection equipment (coats, full-face visor and use insulated gloves) at all times whilst handling vessels containing LN2 or manipulating cold items.

  • A very important safety consideration is the potential risk of asphyxiation when escaped nitrogen vaporises and displaces atmospheric oxygen. Oxygen depletion can very rapidly cause loss of consciousness, without any sensation or prior warning because nitrogen is odourless, colourless, and tasteless. Vessels containing LN2 should be kept in well-ventilated areas in order to minimize this risk. In particular, if a pressurized LN2 vessel must be moved between levels, for example for refilling at an external LN2 source, never go in the lift with the vessel to avoid being trapped in a confined space in case of lift malfunction. Large volume LN2 vessels should be accompanied with an oxygen detector triggering an alarm in case oxygen level drops below 19%, or a mechanical ventilation installed in the room holding the LN2 vessel.

  • A third risk is associated to the tremendous amount of force that can be generated if LN2 is rapidly vaporised inside any closed space such as a cryotube. The liquid-to-gas expansion ratio of nitrogen is 1:694 at 20 °C, and this will rapidly lead to explosion of sealed vials. This safety risk must be particularly controlled when cryotubes are stored in the liquid phase of LN2, because LN2 can enter the cryotube. Whereas this risk of explosion is relatively limited in the case of plastic cryotubes with screwtop closure, because accumulating pressure will lead to leaks in the seal that will relieve the pressure, dangers associated to LN2 spraying out of the tube (injury or dissemination of the cryotube content) must be taken into account. To thaw cryotubes kept in the liquid phase, a good practice is to move them in the vapour phase for 24 h, to allow any trapped LN2 to slowly evaporate; an easy way to apply this procedure in a liquid phase cryoconservator (see below) is to keep the top box of a rack above the maximal level of the liquid phase.



We list here the standard equipment needed for successful cryopreservation of protists in LN2:

  • Basic material to work with protist cultures under sterile conditions, e.g., flow hood, autoclave (see section 1.4).

  • Basic material to prepare culture media (see section 1.2) and handle cultures, such as beakers, pipettes, etc.

  • A centrifuge to concentrate cultures, fitted with an appropriate rotor accepting large tubes, such as 50 mL conical tubes.

  • A vacuum pump to aspirate the supernatant after centrifugation.

  • A water bath to heat up medium and cryosamples for fast thawing.

  • A set of tweezers to safely manipulate cryotubes when they float in LN2.

  • A system allowing a controlled –1°C/min cooling rate. The best is a cooling unit that can be programmed for such a precise cooling rate. If such a device is not available, a semi-controlled alternative system, that proved very efficient, combines a –80°C freezer with special cryoboxes for cooling down the samples (e.g., isopropyl alcohol-filled Thermo Scientific Nalgene® Cryo 1 °C “Mr. Frosty”, or alcohol-free Biocision® Coolcell).

  • A LN2 cryoconservator, which is essentially a deeply insulated jar where LN2 is stored, creating a liquid phase down and a vapour phase up; often the limit between the two phases can be adjusted by the user to favour one or the other phase. An extensive range of sls in available, with smaller ones having capacities of 80 to 90 cryotubes placed on aluminium canes, to huge vessels with a capacity > 20,000 cryotubes placed in cryoboxes. Cryotubes can be either stored in the vapour or the liquid phase of LN2, each with advantages and disadvantages. This choice has important consequences for the selection of an appropriate cryoconservator and must not be neglected. For safety reasons, it is often recommended, especially by companies selling cryogenic equipment, to use vapour phase storage. Indeed, this limits the risks associated to LN2 entering the tubes when submerged, which may lead to cryotube explosion during thawing (see safety note above) and/or cross-contamination between samples if contaminants float in the LN2; this latter risk is extremely important when working with biologically hazardous organisms. However, storage in the vapour phase is accompanied by a trade-off limiting either cryoconservator capacity (big liquid phase & small vapour phase) or its autonomy (small liquid phase & big vapour phase), because autonomy straightly depends on the quantity of LN2 in the liquid phase. Furthermore, temperature is less stable and forms a vertical gradient in the vapour phase (from –180 °C to –140 °C), which might be critical for some protist species. Recently, a specific type (dry phase) of cryoconservator has been developed, where LN2 circulates into a closed circuit, with thermal transfer elements ensuring cryotubes are maintained at appropriate low temperature; this technology ensures cryotubes are not in direct contact with LN2, either liquid or vapour. Despite attractive in its principle, this design may have two major disadvantages for some laboratories: dry phase cryoconservators are largely more costly than liquid/vapour phase ones, and their autonomy in the absence of external LN2 refilling is usually very short (a few days only). Whatever its type, a fortiori for dry and vapour phase or when external supply of LN2 can be erratic, a LN2 cryoconservator should be constantly monitored and alarmed for temperature and LN2 level, because any failure in maintaining the minimum level of LN2 in the cryoconservator will lead to irremediable loss of the frozen samples. Note that electronic ultra-low (–135 °C) freezers exist, but their mechanical complexity requires an external LN2 backup in case of failure, and their temperature is high compared to LN2; so they are currently rarely used for protist cryopreservation. Regular advances in technology might lead to changes in the perspectives expressed here in a near future, so we advise laboratory planning to acquire a cryopreservation system to enquire about the most recent available equipment and their features before choosing for a specific solution.

  • An external source of LN2 for regular refilling of the cryoconservator. Depending on the local availability, the LN2 refilling could be performed manually, by pouring LN2 into the cryoconservator (but see safety note above), or manually/automatically from a pressurized source of LN2 attached to the cryoconservator. Many modern cyoconservators can indeed be fitted with automatic LN2 level monitoring systems that trigger refilling from the external source when needed (often user adjustable). Except in the rare cases where a pressurized LN2 circuit is available, this external LN2 source is a pressurized tank, which must itself be refilled either from a larger tank or directly from a truck. Local constrains about the regular delivery of LN2 must be taken into account with prime importance when choosing the cryopreservation system to ensure sufficient autonomy even in adverse conditions. A LN2 cryoconservator can often survive absence of electricity power for a prolonged time (even up to a month), but in case of shortage of LN2, there is no way to maintain the integrity of cryosamples.

  • Cryoboxes and sterile plastic cryotubes. Cryotubes in the 1.2 to 2 mL volume range (e.g., Thermo Scientific Nalgene® #5000-0020 or Nunc® #340711) have been proven adequate for protist culture freezing; tubes with external thread limit the risk of contamination from handling compared to internally-threaded cryotubes. A large variety of cryotubes and cryoboxes exist; specific features of some brands and models are worth mentioning. A small cryotube size allows using cryoboxes holding 100 (10*10) or even 169 (13*13) tubes boosting the overall capacity of a cryoconservator compared to the classical 81 (9*9) cryoboxes with limited extra cost. Also, cryotubes and cryoboxes with integrated barcode can be useful for easier referencing (see inventory control system below). Be sure to use cryotubes and cryoboxes suitable for LN2 storage, as some can only be used in freezers at temperatures above –100 °C.

A reliable inventory control system, designed to organize the contents for ease of location and retrieval, is vital for efficient cryopreservation in the laboratory (as well as being important in other techniques). The key point is that small cryosamples cannot be kept out of LN2 for more than 30 s to 1 min, making hunting for a specific sample inside the cryoconservator very difficult without an external inventory system. Finding a missing sample can rapidly turn into a nightmare, with non-negligible risks for the samples and the user.

A reliable inventory control system is based on three complementary subsystems: (1) an individual tube labelling system, (2) a database recording the position of each sample together with its associated important data, and (3) a system limiting errors, particularly preventing the possibility to deposit/move/withdraw a sample without updating its record in the database.

Such an inventory control system can in principle be developed on paper or on simple electronic supports provided extreme care is taken to label, position and record the fate (moving, thawing, etc.) of each cryosample. We, however, strongly recommend to use/develop a system specifically designed for it, combining the use of barcodes for individual error-proof cryotube labelling, and a database system allowing both to record all important information associated to cryosamples (date, content, exact position in the cryoconservator, etc.) and to ensure the integrity of the inventory.

Commercial systems exist to implement such a referencing solution from one hand to another, from barcoded tubes to specialized laboratory software for inventory database (e.g. Labcollector®, However, it is also possible to create a customized and cheaper solution based on a general database management (e.g., Microsoft Access®, or FileMaker Pro®) or spreadsheet software, connected to a printer to create custom “wrap around” LN2 resistant labels (e.g., Brady® # 800537), and a barcode scanner. Prefer 2D barcodes (e.g., matrix) over 1D barcodes, as they are smaller and fitted with error-correction preventing reading errors.

A key point for data integrity, whatever the system, is to develop a carefully thought set of practices and rules to limit human errors as much as possible by having the system enforcing/preventing specific actions. For example, letting the database system automatically allocate an empty space (vs. user chosen) for each new cryotube and print it on a label to be affixed on the tube allows for easier and less error-prone placement of the cryotube and recording of its associated data. Similarly, enforcing every cryotube, when thawed, is recorded as such in the database ensures the current content of the cryoconservator is correctly reflected, allowing for easy sample search and inventory in silico. Recording freezing success (yes or no) for each cryotube, once it is known whether a culture has successfully developed after thawing, also allows to accumulate some knowledge that may be helpful to troubleshoot reasons for freezing failure.


  • Standard growing culture medium, with possible addition of suitable antibiotics to prevent contamination, whose impact can be bigger on fragile cultures freshly thawed.

  • Starvation medium: 10 mM Tris (pH 7.5, adjusted by adding HCl), sterilized in the autoclave.

  • DMSO (Dimethyl Sulfoxide), ACS reagent grade (e.g. Fisher #D1281 or Sigma-Aldrich #472301). DMSO must be sterilized by filtration using a 0.2 micron syringe filter which has been pre-washed with alcohol and rinsed with DMSO. CAUTION: DMSO is readily absorbed through the skin and can penetrate some rubber gloves, leading to potential introduction of harmful agents into the body.


Freezing usually implies a phase of culture under specific conditions to prepare the cells and ensure the highest cell viability, the use of specific cryoprotectants, and a progressive and controlled cooling down. Thawing also requires specific precautions to limit the thermic shock and ensure cells can rapidly go back to normal reproduction. All solutions and material in contact with the cell cultures must be sterile.


This protocol has been optimized for Tetrahymena by Nicolas Schtickzelle, Linda Dhondt (both Université catholique de Louvain, Biodiversity Research Centre, Belgium) and Michèle Huet (Station d'Ecologie Expérimentale du CNRS, Moulis, France) on the basis of the protocol described by Cassidy-Hanley (2012) but is likely a good basis for many protists. It spans a period of 13 days; optimized weekday for each step is indicated to avoid working during weekends.

The quantities given allow the preparation of 8 cryotubes per culture sample. As revival success cannot be 100% guaranteed for each thawed tube, we strongly advise against decreasing the number of cryotubes per culture sample. If more cryotubes are desired, adapt the quantities but be sure to respect the filling amount per recipient for optimal cell survival; for example to make 16 cryotubes, perform two 50 mL cultures, each in a separate 500 mL Erlenmeyer, instead of one single 100 mL culture. To avoid variation between lots, these cultures can be mixed together to get one single homogeneous culture, and then divided back (at step 3, and again at step 5).

Timing information is indicative, given for one culture frozen as a set of 8 cryotubes, and does not include time needed to prepare the material and reagents.

1st day (Wednesday – 0.5 h): Preculture

  1. Put 400 µL of stock culture with 5 mL of culture medium in a 50 mL tube.

3rd day (Friday – 0.5 h): Culture

  1. Transfer each pre-culture in a 500 mL Erlenmeyer flask filled with 50 mL of culture medium; culture them at 30 °C to log phase (c. 500,000 cells/mL according to strain) with 150 rpm shaking. Temperature and good culture aeration are important to ensure optimal recovery.

6th day (Monday – 1 h): Starvation

  1. Measure cell density in the culture and adjust, if necessary, to c. 500,000 cells/mL. Transfer into a 50 mL tube that can be centrifuged.

  2. Centrifuge (1100 g for 3 min at room temperature) and remove the supernatant by aspiration.

  3. Dissolve the pellet in 10 mL of Tris, transfer into a 500 mL Erlenmeyer flask and complete with Tris to reach a final 50 mL volume.

  4. Culture them for 3 days at 30 °C with 150 rpm shaking.

9th day (Thursday – 1 h): Freezing

  1. Label the appropriate number of cryotubes, and enter their details in the inventory system. The label on each cryotube should include the exact position where it will go in the cryoconservator.

  2. Transfer the content of each Erlenmeyer into a 50 mL tube.

  3. Centrifuge (1100 g for 3 min at room temperature) and remove the supernatant by aspiration, leaving 500 µL of Tris to dissolve the pellet.

  4. Add carefully 2 mL of DMSO (final DMSO concentration 8%), stir gently (cells become fragile by DMSO, so avoid shocks).

  5. Put immediately 300 µL in each cryotube and incubate at room temperature for 30min to allow DMSO to penetrate the cells (this is the so-called equilibration period).

  6. Cool down at –1 °C/min, overnight. Whatever the device used for this controlled cooling down, group cryotubes together according to the position they will occupy in the cryoconservator, to ease their transfer (see below).

10th day (Friday – 0.5 h): Transfer in LN2

  1. Fill 2 expanded polystyrene boxes with a few centimetres LN2: one will receive the cryotubes out of the -80°C freezer (or cooling unit), the other will receive the cryobox extracted from the cryoconservator. This allows keeping all cryotubes (new or existing) deeply frozen during manipulation. Be sure to regularly check the LN2 level in the two boxes and refill if necessary to maintain a level allowing cryotubes to be fully submerged in LN2.

  2. Rapidly move the cryotubes from the freezer to LN2, using tweezers or if possible by overturning the box in which they are and let cryotubes drop into LN2. Do not let any cryotube/cryobox outside LN2 for more than 30 seconds. Once they are in the expanded polystyrene box, soaked/floating in LN2, they are safe and you can take the necessary time to carefully select the appropriate cryotube for placement in the cryobox. No hurry means no mistake.

  3. Put each cryotube in the cryobox, at the exact position indicated in the label.

  4. When all tubes are placed into the cryobox, put the cryobox back into the cryoconservator, and proceed by loading remaining cryotubes into the next cryobox, until all are placed.

13th day (Monday – 0.5 h): Viability check

  1. Take out one tube per series and thaw it (see procedure below) to check the success of the freezing procedure, i.e. a viable culture is obtained.

Thawing (0.5 h)

  1. Use the inventory system to locate tubes to be thawed, and plan in which order they will be removed from the cryoconservator so as to minimize the time frozen cryosamples are out of the LN2.

  2. If cryotubes are conserved in the liquid phase, move them into the vapour phase during 24 h to minimize risks of explosion (see safety note above). Use procedure with two expanded polystyrene boxes (described at step 15 of freezing protocol) if cryotubes from several cryoboxes need to be gathered and placed into a single cryobox to be stored in vapour phase, ensuring no cryotube/cryobox is left out of LN2 for more than 30 seconds.

  3. Prepare all the material (pipettes, tweezers…) to ensure no delay will subsequently happen during the thawing procedure.

  4. Prepare a set of 50 mL tubes (one for each cryotube to be thawed), each containing 50 mL of standard culture medium at room temperature, and label them. Antibiotics should be added to minimize potential contamination.

  5. Preheat the water bath at 42 °C and place in it one or several tubes (e.g., 50 mL conical) containing an appropriate quantity of standard culture medium (1.5 mL * number of cryotubes to be thawed); be sure the top of the tube does not touch the water to avoid contamination. Once at 42 °C, take the tube out of the water bath and wipe it with an alcohol-soaked tissue prior to opening under the hood to minimize the risk of contamination.

  6. Fill 2 expanded polystyrene boxes with a few centimeters LN2: one will receive the cryotubes to be thawed, the other will receive the cryobox extracted from the cryoconservator. Do not let any cryotube/cryobox outside LN2 for more than 30 seconds. Be sure to regularly check the LN2 level and refill if necessary to maintain a level allowing cryotubes to be fully submerged in LN2.

  7. Take out the first cryobox from the cryoconservator, put it in one of the LN2-filled expanded polystyrene box, and extract the selected cryotube(s). Repeat, one cryobox at a time, until all cryotubes to be thawed are extracted and grouped in the other LN2-filled expanded polystyrene box.

  8. Place the first cryotube into the 42 °C water bath, and shake gently for c. 30 s.

  9. Take the cryotube out of the water bath and wipe it with an alcohol-soaked tissue prior to opening under the hood to minimize the risk of contamination.

  10. Add 1.5 mL of culture medium from the 42 °C prewarmed tube and shake gently to ensure the pellet is fully dissolved.

  11. Transfer the content of the tube into the appropriate labelled 50 mL tube containing 5 mL of culture medium, and culture at 30 °C.

  12. Repeat steps 6 to 10 for each cryotube to be thawed.

  13. After 24 to 48h, check the presence of live cells.

  14. Update the inventory system, indicating the tube(s) that were thawed and whether thawing was successful or not.


Cassidy-Hanley, D.M. (2012) Tetrahymena in the Laboratory: Strain Resources, Methods for Culture, Maintenance, and Storage. Methods in Cell Biology: Tetrahymena thermophila (ed. K. Collins), pp. 239-276. Academic Press, Amsterdam.

Day, J.G. & Stacey, G.N. (2007) Cryopreservation and freeze-dyring protocols. Sprinegr, Berlin.

Kawecki, T.J., Lenski, R.E., Ebert, D., Hollis, B., Olivieri, I. & Whitlock, M.C. (2012) Experimental evolution. Trends in Ecology & Evolution, 27, 547-560.

McAterr, J.A. & Davis, J.M. (2002) Basic cell culture and the maintenance of cell lines. Basic cell culture: a practical approach (ed. J.M. Davis), pp. 135–189. Oxford University Press, Oxford.