I tried with Crystal violet but was not able to stain the colonies. It seemed that the stain could not penetrate the semisolid agar.
I'm surprised that crystal violet didn't work for you. Check out protocol in attached paper.
The colonies photographed in Fig. 3 were stained for c. 12hours with 0.5 ml of 1mglmlp-iodonitrotetrazolium violet (Sigma Chemical Co.) in water, followed by fixation with 0.5 ml of 3% acetic acid.
Article The in vitro growth of murine high proliferative potential-c...
Dr. Muench,
I stained the colonies for 30 min (according to some protocol), can that be the reason?
Satej....what percentage of crystal violet you have used? 0.1% can do the desire job in 20-30 mins.
We see crystal violet in many protocols, yes. Maybe, you can try a different dye. Prepare nitro blue tetrazolium chloride solution by making a 1 mg/ml stock solution in 1x PBS (8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 in H2O to final volume of 1,000 ml).
There is always the alternative of an automated colony counter where stains are generally not required to see and count colonies. The most often used system is the GelCount. https://www.oxford-optronix.com/en/GelCount
(Similar as Gülsah Torkay says):
1. Remove carefully the media over soft-agar.
2. Wash with PBS and remove again.
3. Add 200 µl (p24 wells) of MTT 1 mg/ml in PBS.
4. Incubate overnight at 37°C
5. Remove carefully the MTT staining solution, wash with PBS, remove again and take photografs of wells.
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