Colony Formation Assay — Step-by-Step Protocol (Bio-Techne)

critical

No colonies at all — total failure

After 7 – 14 days of culture, the entire dish shows no visible colonies after staining.

💡 5 · ✓ 5

severe

Plated too dense — colonies merge into one another

Many colonies fuse together so individual colonies cannot be counted reliably; counts will be over- or under-estimated.

💡 2 · ✓ 3

severe

Replicate dishes vary widely — conclusion is unsupported

Three replicates of the same group give very different colony counts. Showing only a representative image without statistics is misleading.

💡 4 · ✓ 4

severe

Too few colonies — sparse, hard to count statistics

Only a handful of visible colonies per dish; counts are too low to give meaningful statistics.

💡 4 · ✓ 4

moderate

Uneven colony sizes (large mixed with small)

Some colonies are very large while others are tiny — suggests cells were not properly singularized at seeding.

💡 3 · ✓ 3

moderate

Dirty background or uneven crystal violet staining

Staining shows blotches, smears, or uneven intensity across the dish — technical artifacts can be mistaken for real biological differences.

💡 4 · ✓ 4

moderate

"Halo" or edge-ring artifact around dishes

A ring of stain appears around the dish edge; cells aggregate at the periphery due to evaporation / meniscus effects.

💡 3 · ✓ 3

minor

Crystal violet stain is too light — colonies hard to see

Stained dish shows only very faint purple; colonies are hard to distinguish from background.

💡 3 · ✓ 3

minor

Plate scratches or impurities interfere with imaging

Imaging shows linear scratches, dust specks, or crystal violet precipitate that look like colonies or obscure them.

💡 3 · ✓ 3

minor

Crystal violet stain is too dark — high background

Entire dish is deeply purple; individual colony boundaries are blurred by background staining.

💡 3 · ✓ 3