I am planning my protocol for my dissertation for an experiment quantifying bacterial (E. coli) biofilm formation on micro-plastic (5mm discs of polyethylene bags) by conducting a crystal violet endpoint assay, using a 96-well plate, where I take an optical density reading using a spectrophotometer. I am planning on reading the ODs at 0, 24, and 48 hours. I will use a different 96-well plate for each of these readings.

I am going to incubate the bacteria in growth medium with different amounts of micro-plastics included (0 to 3 or 4) and am not sure how to wash the plates without causing contamination or losing the micro-plastic and thus the biofilm. So far the only methods I have read involve washing the plate as a whole in different containers of distilled water, I am concerned that this would cause the micro-plastic to fall out and I would mix up the wells.

I was thinking I would need to use forceps sterilised under a blue flame to move the micro-plastic pieces from one plate into the corresponding well on a second plate, sterilising the forceps between each use. I would then add CV, then move the micro-plastic to a third plate in the same way to add the ethanol to release the stain, again without dislodging the plastic or risking contamination. I would then add the solution in the well to a cuvette, add more ethanol to create a sufficient volume and read OD in the spectrophotometer. This would be repeated for each well.

However, my problem appears in the wells where I would have 0 micro-plastics as I would have nothing to move and they will have a very low OD reading. I am also aware this will be quite time consuming and could risk contamination or losing parts of the biofilm when I pick the micro-plastic up with the forceps.

Does anyone have any references with methods that I could look at? Or any alterations to my protocol that would improve it?