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

3.3 Disturbance and perturbation manipulations


Disturbances can either be a temporary change in the environment that affects the community (i.e., a pulse perturbation), but where eventually the environmental conditions return to the initial state, or be a permanent change in the environment (i.e., a press perturbation), or somewhere on the continuum between pulse and press. Disturbances as persisting changes in the environmental conditions and possible species-specific resistance to the disturbance itself include change in temperature (e.g., to mimic global warming, Petchey et al. 1999; Scholes, Warren & Beckerman 2005) and change of the medium with respect to pH or chemical composition (e.g., Jin, Zhang & Yang 1991).

When studying disturbances/perturbations, most interest is on different aspects of the regime (e.g., pulse, press, frequency, magnitude) on population and community dynamics. In principle, disturbances (or perturbations in general) can be achieved through manipulations of many aspects of the abiotic environment (Sousa 1984). For example, this includes temperature, acidity, or toxins. However, manipulations of these are mostly general (e.g., manipulation pH) or not very commonly done with protists (e.g., effect of toxins), and so we do not cover each of them in detail.

The probably most commonly applied disturbance in microcosm experiments is density-independent mortality, where either a part of the community is replaced by autoclaved medium (e.g., Warren 1996; Haddad et al. 2008; Altermatt et al. 2011), or where a part of the community is killed (by heating or sonication), but the medium retained in the culture, such that chemical and nutritional conditions remain constant (e.g., Jiang & Patel 2008; Violle, Pu & Jiang 2010; Mächler & Altermatt 2012). This type of disturbance is easy to apply but does not allow species-specific resistance to disturbance, but rather reflects different recoveries from disturbances, strongly determined by a species growth rate, and we discuss the different types in the following.

Density-independent mortality via sonication works through a generator providing high voltage pulses of energy (at frequency of about 20 kHz), to piezoelectric converter. The converter transforms the electrical energy to mechanical vibration through the specific characteristics of internal piezoelectric crystals. The vibration is subsequently amplified and then transmitted to the horn (probe). The horn’s tip is subsequently expanding and contracting longitudinally. The amplitude is defined by the distance the tip expands and contracts, and can be set by the user. The energetic waves created by the vibration have disrupting effects on biological membranes and other biological structures (e.g., cell walls, proteins), such that they physically disintegrate.



Replacing medium:

  • Pipettes or measuring beakers.


  • Pipettes or measuring beakers.

  • Microwave.

  • Cooler or box with ice to cool medium after treatment.

  • Heat-protecting gloves to hold vessels after microwaving.


  • Pipettes or measuring beakers.

  • Sonicator system, composed of a generator, a converter and a horn (also known as probe).

  • Ice-bath (e.g., measuring beaker with crushed ice).


No specific reagents beyond what is described in sections 1.2 to 1.4 are needed.


Replacing medium:

Depending on the level of disturbance, a large part of the medium (50–99%) (Warren 1996; Fukami 2001; Scholes, Warren & Beckerman 2005; Haddad et al. 2008; Altermatt et al. 2011; Altermatt, Schreiber & Holyoak 2011; Altermatt & Holyoak 2012; Limberger & Wickham 2012) containing protists is replaced with freshly autoclaved medium. Replacing less than 30% of the medium has generally very little effects on the population and community dynamics of protists, and is sometimes even used as a standard procedure during long-term maintenance. It is very important that all handling procedure (e.g., mixing before disturbance) except the disturbance itself is also applied to the controls.

  1. Take the vessel with the protist community to be disturbed.

  2. Thoroughly mix it (shaking or with pipette).

  3. Remove the proportionate content that should be disturbed. Note: in case of very high disturbance levels (e.g., 99%), it may be easier to remove the content that should be maintained with a pipette, temporarily keep it in the pipette tip, discard all of the rest, and add it back to the vessel.

  4. The discarded medium including the protists should be safely disposed, to avoid that protists can escape into the natural environment (autoclaving the disposed medium or by adding bleach).

  5. Replace the discarded medium with freshly autoclaved (possibly bacterized, see section 1.2, 1.3) medium.


The procedure below is for applying density-dependent mortality equally to all species. However, it is possible to cause this mortality in a particular species (the one with the lowest temperature tolerance) only. This requires careful calibration of a temperature disturbance applied to the whole community, so that only this species suffers mortality (e.g., Worsfold, Warren & Petchey 2009).

  1. Take the vessel with the protist community to be disturbed.

  2. Thoroughly mix it (shaking or with pipette).

  3. Remove the proportionate content that should be disturbed. Note: in case of very high disturbance levels (e.g., 99%), it may be easier to remove the content that should be maintained with a pipette, temporarily keep it in the pipette tip, disturb all of the rest, and add it back to the vessel.

  4. Place a vessel with the proportion of the medium that should be disturbed in a microwave and heat it up to boiling temperature. The intensity and duration of microwaving needs to be adjusted to the chosen volume. Ideally, the medium is just quickly heated up to 80–90 °C, but does not boil. This kills all protists but minimize evaporation (cover lids, but do not use aluminium foil but glass cover lids) and chemical reactions in the medium due to heat.

  5. Let the disturbed (i.e., heated) medium cool down as quickly as possible (using an ice bath) to the exact same temperature as the remaining (i.e., undisturbed) part and put it back.

  6. The heating and cooling should be done as quickly as possible (ideally, in less than 1 h), to avoid time-lag effects. For the control treatments, also remove the same part of the medium as being disturbed, store it temporarily at room temperature/conditions the replicates are handled, and only put it back to the replicate after the same time as the disturbed ones are put back.


  1. Take the vessel with the protist community to be disturbed.

  2. Thoroughly mix it (shaking or with pipette).

  3. The intensity of disturbance can be set in two-ways: A) a proportion of the medium is sonicated such that all protists die; B) the duration of the sonication process can be varied, such that part of the protists can survive when sonicated for only short periods or at low intensities (usually a few seconds).

  4. Remove the content that should be disturbed. We recommend sonicating at maximum amplitude over a short time-span (e.g., 30 to 60 s for a sonicator with 700 W and 20 KHz maximum working power).

  5. During sonication, the medium can considerably warm and get hot. To avoid a temperature-effect (e.g., also compared to the control), the sample vial with the medium to be sonicated should be placed in an ice bath.

  6. Put the sonicated medium back to the undisturbed fraction of the sample.


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