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

1.3 Bacteria

Introduction

Bacteria to add

For non-axenic cultures, it is advised to add a set of known bacteria as food source. Generally, this happens in a two-step process. In a first step, bacteria cultures are individually grown to carrying capacity in medium to be used in the experiment. From these stock cultures, a small inoculum is then transferred to the actual medium used in the experiment, where bacteria are allowed to grow for a short time (e.g., 12 to 24 hours), before the medium is then used to cultivate protists. We recommend individually growing an extensive volume (e.g., 1 L) of each bacterium species to carrying capacity, and then make 1 mL aliquots of inocula. These can then be frozen in glycerol, and be used across experiments for a standardized set-up of bacteria populations. While different non-pathogenic bacteria species have been successfully added and used in protist microcosm experiments, the control of the bacterial community is often not very extensive. Thus, while the experimenter usually incoulates the microcosms with a few known bacteria species, there may be other species present in the protist stock cultures or subsequentely invade the experiment. A better control of the bacterial communities in protist microcosm experiments would thus be a desired improvement for future work.

Commonly used freshwater bacteria species include Bacillus subtilis, B. brevis (=parabrevis), B. cereus, Enterobacter aerogenes, Proteus vulgaris, Serratia fonticola, or S. marcescens. Generally, two to three species are used in a mixture. Please be aware that even the non-pathogenic strains of some of these species are only allowed to be used in “Biohazard level 2” labs in some countries. It is advised to use non-pathogenic and Biohazard level 1 strains/species only.

Adding bacteria in standardized aliquots

For standardized experiments, and to allow a consistency in bacterial resources, it is advised to add the same set of bacteria to the experiments/cultures over time. Thereby, bacteria species are initially grown in isolation to high densities, then split into aliquots and stored at –80 °C. Subsequently, the same set of bacteria can be used from these stocks to start experiments with protists.

Removing bacteria

The advantage of axenic cultures is the higher level of standardization and reproducibility. To maintain axenic cultures, or to transform non-axenic cultures into axenic ones, the medium needs to be treated with antibiotics, and subsequently sterile techniques need to be used continuously. To remove bacteria, a combination of 250 μg/ml penicillin G, streptomycin sulfate and 1.25 μg /ml amphotericin B (Fungizone-GIBCO) is added to cultures kept in any type of media. If this is not successful, the addition of 2 μl/ml Normocin™ (InvivoGen) has been reported to successfully eliminate bacteria (Asai & Forney 2000). Axenic cultures are often used for single species experiments (especially Tetrahymena sp.) (e.g., Asai & Forney 2000; Fjerdingstad et al. 2007; Pennekamp & Schtickzelle 2013), while almost all experiments containing multiple species of protists are done under non-axenic conditions (e.g., Petchey et al. 1999; Haddad et al. 2008; Altermatt, Schreiber & Holyoak 2011). Importantly, an often diverse but undocumented diversity of bacteria and “microflagellates” may persist in non-axenic conditions. It is not uncommon to notice that many species often thrive much better under non-axenic cultures, and that it is much more difficult to maintain these species under axenic conditions.

Materials

Equipment

For the handling of bacteria (addition or removal to protist cultures), the following equipment is needed:

• Sterile working bench.

• Bunsen burner (or other flame source)

• Spatula or wire loop to transfer bacteria.

• Micropipettes to handle solutions in the range of 0.1 to 10 mL.

• Sterile beakers and jars.

• Aluminium foil to cover the lid of the medium container and maintain it sterile after autoclaving.

• Labelling tape and pen to label cultures.

• Stock cultures of the respective bacteria species (includes Bacillus subtilis, B. brevis (=parabrevis), B. cereus, Enterobacter aerogenes, Proteus vulgaris, Serratia fonticola, or S. marcescens), ordered at bacteria stock centres.

Reagents

• Protist culture medium (see section 1.2).

• Penicillin G.

• Streptomycin sulfate.

• Amphotericin B (Fungizone-GIBCO).

• 2 μl/ml Normocin™.

• Glycerol.

Procedure

Adding bacteria

Bacteria are added from high-density cultures to the respective culture medium, where they are usually allowed to grow before protists are added. In many past studies, three different bacteria species have been added, but the procedure is identical for single species. Thereby, the following procedure is advised:

1. Using a sterile workbench, add each bacteria species received from the stock centre individually to 500 mL sterile culture medium. To transfer bacteria, sterilize the tube cap and spatula used for the transfer using a Bunsen burner. Maintain sterile working conditions throughout all subsequent working steps.

2. Grow the bacteria monocultures to carrying capacity (about 2–4 days) at 20 °C.

3. Make as many 1 mL aliquots of the bacteria-culture as desired (for long-term comparisons, this is ideally hundreds of aliquots). Therefore, 1/n mL of each bacteria monoculture (with n being the total number of bacteria monocultures) are added individually to 3 mL micro test tubes (e.g., Eppendorf^®).

4. Mix the bacteria culture with 50% glycerol (50% glycerol, 50% bacteria inoculum, i.e., 1 mL glycerol to 1 mL total bacteria inoculum).

5. Store at –80 °C.

6. For use in experiment, slowly defrost one mixed bacteria culture, and add to 100 mL of sterile culture medium.

7. Let the bacteria grow for 24 h.

8. Mix this bacteria culture with the respective total amount of culture medium needed for the experiment. We recommend adding 5% of this bacteria inoculum to the total medium volume.

9. Start experiment immediately.

Timing: 1–2 h for step 1. 2–4 days for step 2 (culture growing). 1–2 h for steps 3 to 5. 24 h for step 6 and 7 (growing phase).

Removing bacteria

To get axenic cultures, the following procedure is advised:

1. Add a combination of 250 μg/ml penicillin G, 250 μg/ml streptomycin sulfate and 1.25 μg /ml amphotericin B (Fungizone-GIBCO) to the focal protist culture (kept in any type of media).

2. Subsequently maintain sterile working procedures (all work done in a sterile bench and cultures only opened after sterilizing caps with a Bunsen burner), only use sterile equipment (pipette tips, jars, etc.)

3. Let the culture grow at general maintenance conditions (section 1.6) for four days.

4. Check in a subsample for the presence of bacteria with a confocal microscope at 500- to 1000-fold magnification.

5. Additionally, plate a subsample onto sterile agar-plates to check for the formation of bacteria colonies.

6. If there are still bacteria found in the culture, add 2 μl/ml Normocin™ (InvivoGen) to successfully eliminate bacteria (Asai & Forney 2000) and repeat steps 2 to 5.

Timing: 1–2 h for step 1. 2–4 days for steps 2 and 3 (culture growing). 1–2 h for steps 4. 24 h for step 5 (growing phase). 1 h for step 6.

References

Altermatt, F., Schreiber, S. & Holyoak, M. (2011) Interactive effects of disturbance and dispersal directionality on species richness and composition in metacommunities. Ecology, 92, 859-870.

Asai, D.L. & Forney, J.D. (2000) Tetrahymena thermophila. Academic Press, San Diego.

Fjerdingstad, E., Schtickzelle, N., Manhes, P., Gutierrez, A. & Clobert, J. (2007) Evolution of dispersal and life history strategies - Tetrahymena ciliates. BMC Evolutionary Biology, 7, 133.

Haddad, N.M., Holyoak, M., Mata, T.M., Davies, K.F., Melbourne, B.A. & Preston, K. (2008) Species’ traits predict the effects of disturbance and productivity on diversity. Ecology Letters, 11, 348-356.

Pennekamp, F. & Schtickzelle, N. (2013) Implementing image analysis in laboratory-based experimental systems for ecology and evolution: a hands-on guide. Methods in Ecology and Evolution, 4, 483-492.

Petchey, O.L., McPhearson, P.T., Casey, T.M. & Morin, P.J. (1999) Environmental warming alters food-web structure and ecosystem function. Nature, 402, 69-72.