Incubation dilution experiments

• Page authors: PRIMO
• Responsible curator: Hagen Buck-Wiese

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Microzooplankton grazing (dilution method)
Approach: dilution of whole seawater with 0.2 µm filtered seawater; Chl-a or FCM tracking
Context: incubation
Spatial scale: point sample
Temporal scale: hours (one diel cycle)
Units: d-1 (growth rate µ; grazing rate g)
Community captured: whole seawater (all phytoplankton)
Co-measurements: temperature, simulated or in situ PAR, depth of collection; Lagrangian sampling recommended; nutrient-amended bottles to delineate nutrient limitation

Method Overview

The dilution method estimates phytoplankton growth rates (µ) and microzooplankton grazing rates (g) simultaneously by exploiting the relationship between prey dilution and net growth rate. Whole seawater is diluted with particle-free (0.2 µm filtered) seawater to create a series of dilution levels (e.g., 100%, 75%, 50%, 25% of whole seawater). At lower dilutions, grazer-to-prey encounter rates are reduced proportionally while the intrinsic phytoplankton growth rate remains constant. By plotting the net phytoplankton growth rate — tracked by chlorophyll-a fluorometry or flow cytometry (FCM) — against the dilution factor, the y-intercept of the linear regression gives µ (d^-1) and the negative slope gives the grazing rate g (d^-1).

A simpler "two-point" variant uses only a single diluted and an undiluted treatment, sacrificing regression statistics for efficiency. Nutrient-amended parallel bottles allow the method to distinguish growth suppression by nutrient limitation from grazing effects.

Scale of measurement

Each dilution series provides a point measurement in space. Incubations are typically run for 24 h to integrate over the light cycle. Spatial context requires replicate sampling stations or Lagrangian sampling over multiple days.

Data generated

Phytoplankton growth rate µ (d^-1) and microzooplankton grazing rate g (d^-1) for the bulk chlorophyll community or, when FCM is used, for specific phytoplankton populations resolved by optical properties.

Units & currency

Units are d^-1. The currency is Chl-a or cell abundance.

Sample size

Typical samples are 1–2 L per dilution treatment.

Limitations

The method assumes that grazing rates scale linearly with dilution and that grazer community composition does not change during the incubation. Bottle effects can alter trophic interactions relative to in situ conditions. Nutrient depletion in unamended dilution bottles may suppress phytoplankton growth and bias µ estimates downward; paired nutrient-amended treatments address this but may introduce their own artifacts. The two-point variant can produce biased estimates when the growth–dilution relationship is non-linear.

Example Applications & Protocols

Classic examples

• Landry et al. (2009) Lagrangian studies of phytoplankton growth and grazing relationships in a coastal upwelling ecosystem off Southern California
• Chen (2015) Assessing the accuracy of the "two-point" dilution technique
• Morison & Menden-Deuer (2017) Doing more with less? Balancing sampling resolution and effort in measurements of protistan growth and grazing rates

Recent applications

• Marrec & Menden-Deuer (2024) Changes in phytoplankton size-structure alter trophic transfer in a temperate, coastal planktonic food web

Common calculations/conversions

• Net growth rate k (d^-1) = ln(C_final/C_initial) / t; regression of k vs. dilution factor gives µ (y-intercept) and g (negative slope).
• Grazing in carbon units: g × [C_phyto] gives µg C grazed L^-1 d^-1.