Congo red and Gram's iodine for plate screening of cellulase production by bacteria.
1. Some of the colonies wash off after applying the stain, so how do I measure the zone? Did you have this kind of experience?
2. Do I need to discard the stain using Pasteur pipette after the completion of the time (5 minutes for Gram's iodine/15-20 mins for Congo red) before visualizing the zones or can I just leave the stain on the plate while visualizing (taking pictures of halo zones? For Congo red staining, do I need to remove the Congo red before applying the 1M NaCl solution or I should just add it to the CR in the plate already? When I tried removing the stains using Pasteur pipette, the colonies were washing off and some run off with the reagent into the pipette.
3. If the colony washes off and the spot underneath the colony is clear, can that be taken as a sign of CMC hydrolysis. In other words, must the hydrolysis be outside the edge of the colony or it can also be underneath the colony?
4. When I incorporated Congo red dye into the CMC agar (0.1% Congo red), there were no visible halo zones around the isolates while a particular isolate had halo zone with the stain. Do I need to apply any reagent to the agar in order to visualize the zones?
I would be grateful for your useful advise.
NB: attached pic of colonies washing off.