Edge Effects in Plate-Based Assays#

Colonies on the outer rows and columns of an agar plate consistently grow differently from those in the interior. This systematic bias — the “edge effect” — can confound quantitative comparisons between strains or conditions.

Causes#

  • Temperature gradients — the plate edge loses heat faster than the center, creating a radial temperature gradient that affects growth rate

  • Humidity gradients — agar near the plate edge dries faster, concentrating nutrients and changing water activity

  • Gas exchange — outer wells have greater access to atmospheric oxygen

  • Nutrient diffusion — edge colonies have fewer neighbors competing for nutrients

The magnitude of the edge effect depends on incubator design, plate type, and organism growth rate. It is typically strongest in 384-well high-density plates.

Detection#

Edge-affected colonies are usually larger or more intensely pigmented than interior colonies of the same strain. This is visible in the raw measurements as a ring of elevated values around the plate perimeter.

PhenoTypic’s EdgeCorrector identifies edge-affected wells using a permutation test against interior wells at matching grid positions.

Correction#

EdgeCorrector operates on measurement DataFrames (not images). It:

  1. Identifies interior colonies (not on the outer rows/columns)

  2. Selects the top-n interior values as a reference baseline

  3. Tests each edge colony against the interior distribution

  4. Adjusts values that are statistically elevated

from phenotypic.analysis import EdgeCorrector

corrector = EdgeCorrector(
    on="Area",
    groupby=["Strain"],
    nrows=8, ncols=12,
    top_n=3, pvalue=0.05,
)
corrected_df = corrector.correct(measurements_df)

Experimental Mitigation#

Statistical correction is a post-hoc remedy. For critical experiments, consider:

  • Randomized plate layout — distribute strains randomly across the plate so edge effects average out across conditions

  • Border wells as controls — inoculate border wells with a reference strain and exclude them from analysis

  • Humidified incubation — reduces evaporation gradients

  • Multiple replicates — average over several plates with different strain positions