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

1.5 Laboratory practices


Experiments with protists might or might not be carried out in sterile conditions, depending on what needs to be measured and/or kept under control; regardless, a clean and tidy laboratory can make the difference between success and failure. This can be obtained by defining clear rules about how to operate common equipment, where to find and replace materials, how to access and handle cultures of protists and bacteria safely; in this appendix we outline these “rules of etiquette”, that should be notified to all the personnel with access to the laboratory and be displayed in form of checklists.

It is recommended to follow general laboratory protocols and safety rules (wearing lab-coats, cleaning benches with Ethanol before and after work, etc.). The following biosafety rules should be followed: glassware should be washed at 80 °C, and protist cultures should only be disposed after killing all protists (e.g., collecting all culture waste and autoclave it or add 2% bleach; only thereafter dispose into the waste water system).

Specific members of the personnel should be explicitly in charge for taking care of recurrent tasks, such as maintaining organism stock cultures (see section 1.6), preparing commonly used media (see section 1.2), and refurnishing the laboratory with chemicals and consumables of common use.



For general use:

  • Autoclave for sterilizing medium, pieces of equipment (glass containers, pipettes, consumables, etc.), and biohazardous waste.

  • Sterile bench for dealing with axenic cultures.

  • Pipettes.

  • Disposable gloves.

  • Some paper towels.

  • Plastic bags for biohazardous waste.

For microcosm set-up:

  • Adequate volume of sterile protist medium (see section 1.2).

  • Bacterial culture(s) (agar slant or plate).

  • Loop for getting bacterial sample off agar.

  • Flame for sterilizing.

  • Adequate number of autoclaved microcosms vessels, and a few spare (e.g. jars, tubes, flasks).

  • 150 ml measuring cylinder.

  • Pipettes with teat, or Gilson-type pipette with tips.

  • Protist cultures, checked for the presence of unwanted organisms (e.g. microflagellates), and at appropriate density.

  • Fine permanent marker.

  • Medium permanent marker.

  • Stickers.

  • Sterile wheat seeds.


  • 70% Ethanol.


General laboratory care

  1. Provide initial training to personnel.

  2. Display checklists regarding general laboratory etiquette as well as instructions on how to use common instruments.

  3. Keep up-to-date journals regarding when and by whom instruments are used.

Rules of etiquette for the daily routine

  1. Do not allow food or beverages in the laboratory.

  2. Keep the laboratory doors closed.

  3. Wash hands when accessing the laboratory.

  4. Wear disposable gloves and sterilize them with ethanol 70% when working in sterile/axenic conditions.

  5. Clean the sterile bench with ethanol 70% before and after use; leave nothing in it aside from dedicated items.

  6. Wash hands when leaving the laboratory.

  7. At the end of the day:

  8. tidy and clean the benches with ethanol;

  9. remove, sterilize and dispose biohazardous waste;

  10. ensure that adequate supplies remain, if not arrange for more.

Periodic tasks

  1. Maintain a stock of commonly used media.

  2. Maintain a stock of commonly used consumables.

Microcosm setup

Setting up microcosms for bacterivore protists requires two main steps: 1) inoculating fresh, sterile medium (Protist Pellet Medium, hereafter PPM, see also section 1.2 and supplement thereof) with bacteria, and 2) adding protists to the bacterized PPM.

Step 1: adding bacteria to the sterile PPM

  1. You will probably have the sterile PPM in 1 litre volumes in one or more large flasks. Working under a sterile bench, pour about 100 ml into a small autoclaved vessel.

  2. Using sterile technique, take a loop of bacteria from the bacterial culture and dip and swirl it into the media in the small vessel.

  3. Put the small vessel in a warm (25°C or so; not critical) place for a couple of hours, to let the bacteria grow.

  4. Under the sterile bench, divide the now bacterized media in the small vessel into however many large flasks you have.

  5. Put the large flasks in a warm place overnight (see TIMING).

Step 2: adding the protists to the bacterized PPM

CRITICAL STEP: all the steps specified below need to be performed in a sterile environment if it is important to avoid the presence of bacteria other than those inoculated during step 1 (adding bacteria to the sterile PPM) from the microcosm vessels.

  1. Clear and wipe down an appropriately large amount of desk space.

  2. Put the flasks of bacterized medium at hand. If you’re being very careful, and have multiple large flasks of bacterized media, mix these up, so to minimize any existing difference between flasks.

  3. Pour the appropriate volume of PPM in each of the microcosm vessels (MV). This can be done in two ways:

A. By means of a precision scale.

  1. Take one empty MV and put it on the scale.

  2. Tare the scale so that it reports zero weight with the empty MV on it.

  3. Pour the exact volume of PPM required, by means of a pipette.

  4. Write down the weight shown by the scale (as distilled water has a density of 1 g/ml, the number of grams shown should be very close to the number of ml poured).

  5. For all other MVs, put them on the scale, tare the scale and pour PPM until the scale shows the same value noted at step iv. CRITICAL STEP: tare the scale for each and every MV used.

B. Using a MV as a reference for all the others. This method is less precise but faster to execute than the one at point A.

  1. Take one MV and put in the appropriate volume of tap water (say 100 ml). Mark on the outside of the vessel the level of the liquid using a fine permanent marker. Pour away the liquid.

  2. Use this reference MV to put similar lines on all other MVs (without removing their lids).

  3. Pour in the bacterized PPM to the line on a MV, or better add this in two steps (first half of the large flask, then second half).

CRITICAL STEP: at step 3, the large flask containing the bacterized PPM needs to be well swirled before each pour, otherwise the bits of PP will remain in the bottom, and be poured only into the last few MVs.

  1. If needed, place the required number of wheat seeds in each MV.

  2. Now randomly assign MVs to treatments and label them (with permanent marker on the MV, or on a sticker stuck to the MV).

  3. Estimate the density in the source cultures of each species of protist in the experiment.

  4. Put in the appropriate volume / number of each species of protist in the appropriate MVs.

  5. Record the number / volume you put in, and the density of the source culture, for each species. From this you can calculate the initial population density in the MVs.

  6. Put the MVs into the appropriate incubator.

Correct handling of the microcosms

  1. Ensure that microcosms are out of the experimental environment for as short a time as possible.

  2. You may find it useful to remove samples from multiple microcosms in the room with the incubator, and then count these wherever. This avoids lots of going back and forth, or removing multiple microcosms from incubators for prolonged periods.

  3. Lids should be off for as little time as possible. Best practice is to never put a lid down. I.e., take it off, keep it in your hand, and put it back on. Don’t put lids down on the bench.

  4. Don’t attempt to carry more than one microcosm/sample in either hand. Don’t attempt to carry three or more at once.

  5. If you have to move microcosms between rooms, either carry only one (you need your other hand to open doors), or move them on a trolley or a tray.

  6. During an experiment, ensure that the volume of medium in each microcosm is correct. This may mean topping up, perhaps during any removal and replacement of media that may be occurring. The top-up can be done with fresh medium to deal with medium removal, or with sterile, deionized water to deal with evaporation.

Troubleshooting (Tips and Tricks)

Put one person in charge of dealing with emergencies such as power failures, instrument faults and equipment breakdown; keep the contact details of repair technicians at hand.

When setting up microcosms:

  • How to avoid errors adding the correct species to the correct MV? Add one species at a time. Separate on the desk all the MVs that require this species, then double check this, even triple checking is worth it, since this is critical. Add the species to these MVs. Do this for each species separately.

  • Adding prey and predator protist species? Add the prey as described, wait a day or two, then add the predator, to allow for time for prey to increase in density somewhat.

  • Adding species from a mixed stock culture? You might need, for example, to add a predator without putting in the prey from the stock culture. You need to use a micropipette to count out individual predators. It really helps to have a stock culture where the predators are as numerous as possible, and the prey as rare as possible; this can be obtained by simply giving time to the predator to reduce the prey density before collecting it.

Anticipated results

A laboratory running smoothly; microcosms accurately set up.