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

1.2 Culture medium

Introduction

All experimental protist-microcosm studies keep protists in a freshwater-based medium containing nutrients and sometimes bacteria. The composition of the medium (e.g., nutrient content, pH, presence/absence of bacteria) has far-reaching consequences on dynamics, performance, and evolution of protist populations. Comparability across studies in terms of species traits, population and community dynamics and general cultivability thus strongly depends on the use of common media types. Generally, stock cultures are kept in an optimal medium, which prevents local extinctions and facilitates the maintenance of species. During experiments, media composition might be adjusted to mimic specific conditions, such as low nutrients, shared or partitioned set of resources among species, or viscosity to modify movement behaviour of protists (Luckinbill 1973; Haddad et al. 2008; Altermatt & Holyoak 2012), and are described in detail under section 3.4.

There is a large number of culture media for protists in the wider sense. Extensive summaries and manuals for making media are commonly available (e.g., Cassidy-Hanley 2012), especially at web-pages of culture collections, and it is not our goal to cover all of these media types, but rather identify the most commonly used. Useful websites summarizing a wider range of media recipes include:

Generally, the water used for the medium is either deionized water, in which micro- and macronutrients are added to reach a reasonable osmolarity, or tap water or commercial well water. Deionized water has the advantage that the chemical composition of the final medium is well-known and highly reproducible. However, this approach is generally more laborious, and often less-defined media made of tap-water are used. Local tap-water should only be used when it is of constant quality and not chlorinated. Before use, the tap water can be aged (to gas-out any chlorine). Nutrients and carbon-sources are added to the water.

All media are autoclaved at 121 °C prior to the use. Autoclaving for 20 minutes is recommended for a volume of 2L, larger volumes may take longer. Before use, the medium must cool down to the temperature used in the experiment (usually around 20 °C) and bacteria may be added as food source (see section 1.3).

We describe five different and commonly used media: Bristol medium, Chalkley’s solution, Proteose peptone medium, Protozoan pellet medium, and wheat/hay (= wheat/lettuce) medium (Fig. 1). The former two are based on deionized water to which anorganic nutrients are added. These two media cannot be used per se for keeping protists, but need an additional carbon source. However, these two media are generally recommended to be used as a replacement of tap or well-water, in which the concentrations of inorganic nutrients is either not known or not standardized. The latter three medium types are common and simple approaches of media in which organic nutrients are added as a carbon source. Protists feed either directly on this carbon source, or indirectly through feeding on bacteria that grow in the medium. The use of bacteria, as well as the making of axenic or monoxenic media is described in section 1.3. The viscosity of the medium can be changed (e.g., for behavioural studies), by adding methyl-cellulose (e.g., Luckinbill 1973) (see section 3.4).

All media can be prepared by persons with basic laboratory skills (including technician and graduate students), and can be learnt within a few hours of instructions. Precaution needs to be taken during the handling of hot media (after autoclaving; only people that have been specifically instructed to the use of the autoclave at hand should use it) and during the handling of chemicals. Wearing lab coats and protective glasses is advised.

Fig. S1. Autoclaved bottle with protozoa pellet medium ready to use. Note the black stripes on the autoclave tape indicating that it was autoclaved, and also giving date and initials of when and by whom the medium was made. The sediments at the bottom are remains of dissolved protozoa pellets, and are generally discarded.

Materials

Equipment

For the making of all media the following equipment is needed:

  • Autoclave to sterilize the medium as well as beakers etc. used to handle the final medium.

  • Microbalance with a precision of at least 0.01 g to weigh the chemicals used for the different media.

  • Graduated beakers to measure different volumes of liquid. We recommend a set of graduated beakers with the following maximum volume: 10 mL, 20 mL, 100 mL, 500 mL, 1000 mL and 2000 mL.

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

  • Containers/flasks to make, autoclave and temporarily store the medium. We recommend using containers with a volumetric content about 50% larger than the actual medium volume to be made in order to avoid spilling during autoclaving. For making 1 L of medium, 1.5 L Erlenmeyer glass beakers have been proven highly suitable (or for 2 L medium, 3 L Erlenmeyer glass beakers).

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

  • Spatula to handle chemicals.

  • Labelling tape and pen to label the medium bottle.

  • All glassware and tools used in the making of the medium should be rinsed with deionised ultrapure (or equivalent) water to ensure that no soap or acide residue remains on the surface of the glassware after it has been washed.

Reagents

All media are made of either deionized or well water, and chemicals and nutrients that are added either as solutions or solid particles. For media in which different stock solutions are prepared, we give the components of the stock solutions and concentrations therein, for all other media we only list the reagents needed.

Bristol medium

  • Deionized water (dH2O)

  • Stock solutions described in Table S1

Table S1. Stock solutions needed for Bristol medium.

Component Amount Concentration stock solution
NaNO3 10 mL/L 10 g/400mL dH2O
CaCl2*2H2O 10 mL/L 1 g/400mL dH2O
MgSO4*7H2O 10 mL/L 3 g/400mL dH2O
K2HPO4 10 mL/L 3 g/400mL dH2O
KH2PO4 10 mL/L 7 g/400mL dH2O
NaCl 10 mL/L 1 g/400mL dH2O

Chalkley’s solution

  • Deionized water (dH2O)

  • Stock solutions described in Table S2

Table S2. Stock solutions needed for Chalkley’s medium,

Component Amount Concentration stock solution
NaCl 5 mL/L 2 g/100mL dH2O
KCl 5 mL/L 0.08 g/100mL dH2O
CaCl2 5 mL/L 0.12 g/100mL dH2O

*Proteose peptone medium: *

  • Bristol medium

  • proteose peptone, e.g. from BD Diagnostic Systems No.: 211684 or BD Diagnostic Systems No.: 212750. Available through retailers like Fisher Scientific.

  • FeCl3 Solution at a concentration of 270 mg FeCl3.6H20 per 10 ml (10 µM FeCl3)

  • Facultativly: 0.2% yeast extract (e.g., Becton Dickinson or Oxoid L21).

*Protozoan pellet medium: *

  • tap/well water or Chalkley’s solution

  • Protozoan Pellet (provided by Carolina™ Biological Supply Company, Burlington NC)

Wheat/hay-wheat/lettuce medium

  • tap/well water or Chalkley’s solution

  • organic wheat seeds or dry organic hay/straw or dried/baked organic lettuce

Procedure

Bristol medium

To get 1 L of total medium, the following procedure is advised:

  1. Fill about 900 mL of deionized water (dH2O) into an autoclavable beaker with a minimum volume of 1.5 L.

  2. Add each of the components of table S1 in the order specified while stirring continuously.

  3. Bring total volume to 1 L by adding dH2O.

  4. Cover the beaker and autoclave the medium at 121 °C for 15–20 minutes.

  5. Before use, the medium must cool down to the temperature used in the experiment (usually around 20 °C).

  6. Label the medium bottle with the name of the medium type, the initials of the person who made it, and the date when it was made.

  7. The medium can be stored at 4 °C for a few weeks, it should be discarded when contaminations with bacteria are observed (i.e., when medium gets cloudy).

Timing: Preparation of medium: 1–2 h, autoclaving 0.5 h, cooling down 12 h.

Chalkley’s solution

To get 1 L of total medium, the following procedure is advised:

  1. Fill about 900 mL of deionized water (dH2O) into an autoclavable beaker with a minimum volume of 1.5 L.

  2. Add 5 mL each of the stock solutions of table S2 in the order specified while stirring continuously.

  3. Bring total volume to 1 L by adding dH2O.

  4. Cover the beaker and autoclave the medium at 121 °C for 15–20 minutes.

  5. Before use, the medium must cool down to the temperature used in the experiment (usually around 20 °C).

  6. Label the medium bottle with the name of the medium type, the initials of the person who made it, and the date when it was made.

  7. The medium can be stored at 4 °C for a few weeks, it should be discarded when contaminations with bacteria are observed (i.e., when medium gets cloudy).

Timing: Preparation of medium: 1–2 h, autoclaving 0.5 h, cooling down 12 h.

*Proteose peptone medium: *

Proteose peptone medium is a modified Bristol's medium, and generally 1% or 2% proteose peptone medium is used. This medium is generally used for axenic cultures, and especially well-suited to grow Tetrahymena sp. under axenic conditions (Cassidy-Hanley 2012). 1%–2% Proteose peptone medium is rich enough to promote high cell densities. The medium must be autoclaved and not filtered for sterilization, as some particulate matter is required to induce formation of food vacuoles in Tetrahymena (Cassidy-Hanley 2012). Sterilized medium can be frozen in aliquots at –20 °C for storage. To get 1 L of total medium at pH ~6.8, the following procedure is advised (Asai & Forney 2000; Cassidy-Hanley 2012):

  1. Fill 950 mL of ready-made Bristol medium into an autoclavable beaker with a minimum volume of 1.5 L.

  2. For a 1% Proteose Peptone medium, add 10 mL proteose peptone. For a 2% Proteose Peptone medium, add 20 mL proteose peptone.

  3. Add 100 µl FeCl3-solution.

  4. Facultative: add 0.2% yeast extract (e.g., Becton Dickinson).

  5. Bring total volume to 1 L by adding Bristol medium.

  6. Cover the beaker and autoclave the medium at 121 °C for 15–20 minutes.

  7. Before use, the medium must cool down to the temperature used in the experiment (usually around 20 °C).

  8. Label the medium bottle with the name of the medium type, the initials of the person who made it, and the date when it was made.

  9. The medium can be stored at 4 °C for a few weeks, it should be discarded when contaminations with bacteria are observed (i.e., when medium gets cloudy).

Timing: Preparation of medium: 1–2 h, autoclaving 0.5 h, cooling down 12 h.

*Protozoan pellet medium: *

This medium is among the less-defined media, but very commonly used due to its simple preparation and suitability for relatively many species. This medium is generally only used for bacterized cultures. It can be used for a very wide range of protozoa cultures. For long-term or stock cultures, heterotrophic cultures can additionally receive two autoclaved wheat seed per 100 ml medium. The content of the Protozoan pellet medium (and Protozoan pellets themselves) is not very well defined. Protozoan pellets are supposedly made of dried, compressed organic material (alfalfa). The chemical composition with respect to nutrients of Protozoan Pellet medium is described in table S3. To get 1 L of total medium, the following procedure is advised:

  1. Fill 1 L of deionized tap water or ready-made Chalkley’s medium into an autoclavable beaker with a minimum volume of 1.5 L.

  2. Add 0.44 g/L–1 ground up Protozoan pellets.

  3. Cover the beaker and autoclave the medium at 121 °C for 15–20 minutes.

  4. Before use, the medium must cool down to the temperature used in the experiment (usually around 20 °C).

  5. Label the medium bottle with the name of the medium type, the initials of the person who made it, and the date when it was made.

  6. The medium can be stored at 4 °C for a few weeks, it should be discarded when contaminations with bacteria are observed (i.e., when medium gets cloudy).

Timing: Preparation of medium: 1–2 h, autoclaving 0.5 h, cooling down 12 h.

Table S3. Physio-chemical description of Protozoan Pellet medium made with local, nutrient-poor well-water. Mean and standard deviation (sd) values of 4 replicates are given.

Component Value (mean±sd)
DOC (mg C/L) 259.6±7.4
TOC (mg C/L) 407±6
DN (mg N/L) 24.9±0.2
TN (mg N/L) 33.7±0.4
Chloride (mg/L) 72.4±0.4
Nitrate (mg N/L) 10.8±0.1
Sulfate (mg/L) 101.2±0.1
Conductivity (µS/cm 20 °C) 1424±3.5
pH 34.4±0.1
Alcalinity (mmol/L) 10.8±0
Total hardnes (mmol/L) 6.9±0
Silicic Acid (mg/L) 137.4±1.6
o-P (µg P/L) 225±19.8
DP (µg P/L) 1216±48.1
TP (µg P/L) 2660±58.2
Na (mg/L) 42.4±0.3
K (mg/L) 54±0.1
Ca (mg/L) 189±0.6
Mg (mg/L) 45.8±0.2
Ammonium (µg/L) 1501±29.1
Nitrite (µg N/L) 7.8±0.1
Mn (µg/L) 8.7±0.4

Wheat/hay-wheat/lettuce/Cerophyll medium

This is the least standardized type of medium, consisting of an organic nutrient source (dried plant material) suspended in water. The amount, type and origin of the plant material may vary, and includes wheat seeds (e.g., Haddad et al. 2008; Altermatt, Schreiber & Holyoak 2011), straw/hay, dried/baked lettuce (e.g., Sonneborn 1950; Fellous et al. 2012a; Fellous et al. 2012b) or rye leaves (Cerophyll) (Cassidy-Hanley 2012). Only use plant material grown without pesticide (i.e., from organic farming). This medium is generally only used for bacterized cultures. To get 1 L of total medium, the following procedure is advised:

  1. Fill 1 L of deionized tap water or ready-made Chalkley’s medium into an autoclavable beaker with a minimum volume of 1.5 L.

  2. Add carbon sources, there are 3 options to add carbon sources:

  1. Add 20 wheat seeds.

  2. Alternatively: add 20 wheat seeds and 1 g of dry straw.

  3. Alternatively: add 1 g of dried/baked lettuce (dried/baked at 110 °C for multiple hours, discard dark brown/black portions).

  1. Cover the beaker and autoclave the medium at 121 °C for 15–20 minutes. This step can be skipped for hay or dried lettuce, and is even common practice to revive dormant stages of protists. Wheat seeds need to be autoclaved, as they otherwise germinate in the medium.

  2. Before use, the medium must cool down to the temperature used in the experiment (usually around 20 °C).

  3. Label the medium bottle with the name of the medium type, the initials of the person who made it, and the date when it was made.

  4. Generally, the wheat seeds or hay/lettuce particles remain in the medium/vessel.

  5. The medium can be stored at 4 °C for a few weeks, it should be discarded when contaminations with bacteria are observed (i.e., when medium gets cloudy).

Timing: Preparation of medium: 1–2 h, autoclaving 0.5 h, cooling down 12 h.

Troubleshooting (Tips and Tricks)

In some protist microcosm studies, vitamin powder (e.g., 0.06 g/L Herpetivite powdered vitamin supplement, Research Labs, Los Gatos, California, USA) has been added to the medium to improve performance and well-being of the cultures (Donahue, Holyoak & Feng 2003; Fukami 2004). Also, in several studies soil or soil-extracts have been added to the medium (McGrady-Steed & Morin 2000; Scholes, Warren & Beckerman 2005; Altermatt et al. 2011). However, even when autoclaving the medium thoroughly, contaminations by microbes from this soil (from dormant and often very persistent spores) is a problem, and soil-additions are hard to standardize.

References

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Altermatt, F. & Holyoak, M. (2012) Spatial clustering of habitat structure effects patterns of community composition and diversity. Ecology, 93, 1125-1133.

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.

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.

Donahue, M.J., Holyoak, M. & Feng, C. (2003) Patterns of Dispersal and Dynamics among Habitat Patches Varying in Quality. The American Naturalist, 162, 302-317.

Fellous, S., Duncan, A., Coulon, A.l. & Kaltz, O. (2012a) Quorum Sensing and Density-Dependent Dispersal in an Aquatic Model System. PLoS ONE, 7, e48436.

Fellous, S., Duncan, A.B., Quillery, E., Vale, P.F. & Kaltz, O. (2012b) Genetic influence on disease spread following arrival of infected carriers. Ecology Letters, 15, 186-192.

Fukami, T. (2004) Assembly history interacts with ecosystem size to influence species diversity. Ecology, 85, 3234-3242.

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.

Luckinbill, L.S. (1973) Coexistence in Laboratory Populations of Paramecium Aurelia and Its Predator Didinium Nasutum. Ecology, 54, 1320-1327.

McGrady-Steed, J. & Morin, P.J. (2000) Biodiversity, density compensation, and the dynamics of populations and functional groups. Ecology, 81, 361-373.

Scholes, L., Warren, P.H. & Beckerman, A.P. (2005) The combined effects of energy and disturbance on species richness in protist microcosms. Ecology Letters, 8, 730-738.

Sonneborn, T.M. (1950) Methods in the general biology and genetics of paramecium aurelia. Journal of Experimental Zoology, 113, 87-147.