Hello community.

I am trying to perform a test on fungal biofilms (dermatophytes) and, after incubating the fungal inoculum (1 McFarland unit) with RPMI medium (pH 7) at 37ºC for 72h, I observe the following:

1.- A fungal layer (I am not sure if it can be considered biofilm or not) is formed covering the surface of the RPMI medium in the well.

2.- Evidently, when I remove the medium, this layer is removed along with the medium. I have tried different ways: removing the medium by overturning the plate, pipetting and aspirating the medium. Always the same result.

3.- At the bottom of the well, there are cloudy remains that do not stain with crystal violet, safranin or give rise to a colour change in the XTT assay.

4.- When on some occasions that layer that I mentioned before does remain adhered to one of the walls of the well (although not at the bottom of the well) and I do the XTT test, there is a clear change in colour.

After all this, my hypothesis is that the fungal layer is viable, but for some reason it is not able to adhere to the bottom of the well.

Any solutions or recommendations?

Many thanks in advance.