Written and maintained by CASRAI Editorial Board
Last updated
Short answer
A colony formation (clonogenic) assay tests whether a single cell can divide enough to form a colony of at least 50 cells. It measures reproductive survival, not metabolic activity. The surviving fraction it yields is only as reliable as three choices: the plating-efficiency assumption, the colony-size cutoff and the counting method. Each can shift results substantially.
Definition and scope
The clonogenic assay is an in vitro cell survival test. In the Nature Protocols method by Franken and colleagues, it asks whether a single cell keeps its capacity for unlimited division, scored as a colony of at least 50 cells. The authors describe it as the preferred method for determining reproductive cell death after ionizing radiation, and it is also used for other cytotoxic agents. “Colony formation assay” and “clonogenic assay” name the same readout.
That definition separates it from viability readouts. A cell can stay alive and metabolically active yet never divide again; the clonogenic assay counts it as a loss, whereas a metabolic assay such as the MTT assay may not.
How the assay is used in practice
Franken et al. describe the sequence in outline: cells are seeded at appropriate dilutions, colonies form over roughly one to three weeks, and the colonies are then fixed (glutaraldehyde in that protocol), stained (crystal violet) and counted. The protocol also covers analysis of radiation dose-survival curves. Other published studies use different fixatives and stains, for example ethanol with methylene blue in the 50-cell-line study by Brix et al., so the chemistry is a lab choice rather than part of the definition.
Two quantities come out of the counts, as defined in Brix et al. (2020):
- Plating efficiency (PE): colonies obtained divided by cells seeded, in untreated cultures.
- Surviving fraction (SF): colonies obtained divided by cells seeded at a given treatment dose, normalized by the PE.
Brix et al. seeded a geometric series of twelve cell densities for each radiation dose, aiming for wells running from 15 or fewer colonies up to uncountable. Accurate seeding therefore starts with an accurate cell count; see the guides on the hemocytometer, automated cell counters and serial dilution.
Related concepts: where the numbers can go wrong
Three peer-reviewed studies each examine one weak point of the assay. The table lists only what those papers report.
| Weak point | What the study found | Source |
|---|---|---|
| Constant plating efficiency assumed | Across 50 cancer cell lines, seeding density often changed colony growth, so a constant PE failed for many lines. Cellular cooperation (auto- and paracrine stimulation) was moderate to high in 28 of 50 lines. In a seven-line panel, the PE of four lines rose by one to two orders of magnitude as more cells were seeded. | Brix et al., Radiation Oncology, 2020 |
| Fixed colony-size threshold | Size distributions of abortive and clonogenic colonies overlapped at every readout time, so no size cutoff separated them cleanly. Across the size thresholds and incubation times compared, cell survival estimates varied between 3% and 105%. A growth-curve classifier using time-lapse imaging reached about 95% accuracy on average. | Koch et al., 2023 (PMC10412889) |
| Counting method | Counting by manual pen showed significant bias against microscope and semiautomated counts, which were interchangeable. One publicly available automated algorithm showed a lower correlation with microscope counts (R² 0.384). | Buryska et al., 2023 (PMC10668582) |
Where it matters for labs and supply decisions
Brix et al. conclude that PE-based survival results carry false precision unless linearity between cells seeded and colonies obtained is shown for both control and treated cultures, and that assay-intrinsic errors can exceed one order of magnitude when cooperation is ignored. For cell lines with high cooperation they call such results at best semiquantitative. They propose power regression with interpolation of matched colony numbers across doses as an approach that accounts for cooperation.
A practical checklist follows from the three studies, and each item traces to one of them (these are the page’s reading of the papers, not rules from a handout):
- Seed a range of densities per condition rather than a single one, as Brix et al. did, so that linearity can be checked (Brix et al.).
- Check that colony number scales with cells seeded in both control and treated plates before using PE normalization (Brix et al.).
- State the size threshold and readout (incubation) time, because Koch et al. compared survival estimates across both and found large variability (Koch et al.).
- Use one counting method per experiment and say which; do not mix pen counts with microscope counts (Buryska et al.).
Cultures that are contaminated or mis-passaged undermine every step above; the cell culture contamination and cell passaging guides cover that groundwork, and cell culture reference numbers lists vessel surface areas and seeding densities.
Common misunderstandings
- “A colony is any visible clump.” In the Franken definition a colony is at least 50 cells, because the test asks about sustained division. Koch et al. report that size distributions of delayed abortive and clonogenic colonies overlap.
- “PE is a fixed property of a cell line.” Brix et al. found that for many lines it moves with seeding density.
- “The assay is the same as a viability assay.” It scores reproductive capacity, not whether cells are alive or metabolically active.
Frequently asked questions
What is a colony formation assay?
An in vitro cell survival test based on the ability of a single cell to grow into a colony, defined as at least 50 cells (Franken et al.).
Is a colony formation assay the same as a clonogenic assay?
Yes. Franken et al. use the two names for the same assay, and Brix et al. list both as keywords.
What is plating efficiency and surviving fraction?
Plating efficiency is colonies obtained divided by cells seeded in untreated cultures. The surviving fraction is colonies obtained divided by cells seeded at a given dose, normalized by the plating efficiency (Brix et al.).
How long does it take for colonies to form?
Franken et al. describe colonies forming in 1 to 3 weeks; Brix et al. incubated cells for 8 to 33 days depending on proliferation rate.
Why can clonogenic survival results be unreliable?
Plating efficiency may change with seeding density because of cellular cooperation, size thresholds can mix abortive and clonogenic colonies, and manual pen counting can be biased (see the table above).
Sources
All links checked 2026-10-29.
- Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of cells in vitro. Nature Protocols 2006;1(5):2315-2319. doi:10.1038/nprot.2006.339 (PMID 17406473).
- Brix N, Samaga D, Hennel R, Gehr K, Zitzelsberger H, Lauber K. The clonogenic assay: robustness of plating efficiency-based analysis is strongly compromised by cellular cooperation. Radiation Oncology 2020. pmc.ncbi.nlm.nih.gov/articles/PMC7597001.
- Buryska S, Arji S, Wuertz B, Ondrey F. Using Bland-Altman analysis to identify appropriate clonogenic assay colony counting techniques. Technol Cancer Res Treat 2023. pmc.ncbi.nlm.nih.gov/articles/PMC10668582.
- Koch RA, Boucsein M, Brons S, Alber M, Bahn E. A time-resolved clonogenic assay for improved cell survival and RBE measurements. Clin Transl Radiat Oncol 2023. pmc.ncbi.nlm.nih.gov/articles/PMC10412889.
