Need help in counting colonies in colony forming assay

[nado]

I am doing colony forming assay but my cells aren't attached type of cells, I tried adding 4%PFA and methanol was added to crystal violet but they're still getting removed while washing any help? (A)
I am doing a colony forming assay for different cells, but the cells formed different colonies with different sizes that I can't distinguish if it's a colony or artifact, any help? (B)

[grablegend]

Hi,

For (A) – cells getting removed during washing:

If your cells are loosely attached or semi-adherent, they can detach easily during fixation and washing. A few things you might try:

Make sure fixation is done gently and long enough (e.g., 4% PFA for Doodle Baseball 15–20 min at RT before washing).

Wash very gently by slowly adding/removing solution along the wall of the well instead of directly onto the cells.

Reduce the number of washes.

Some people fix with methanol alone (100% cold methanol for ~10 min) instead of adding it to crystal violet — depending on the cell type, this sometimes improves retention.

Avoid vigorous pipetting during staining steps.

What cell type are you using?