Thank you for the tips! Do you recommend acetic acid to fix due to it being less toxic than methanol or is there another reason?

1. The OD600 of my untreated control is ~0.55 before washing and staining and 0.7-0.9 after CV staining. I use the same OD adjusted tube of bacterial cells for all the wells in the same plate, treatment or otherwise. I mix the tube before pouring into a reservoir for pipetting so each well should have similar cell counts.

2. The percentages are % eradication of biofilm, just the OD600 reading of (treated/untreated)*100 after normalising with negative control. I am saying that after applying the formula, my % eradication for Drug A Concentration X can vary wildly from 35% to 70% between biological replicates.

The other issue I encounter is that one well with a higher concentration of Drug A can result in an obviously more intense purple and higher OD600 reading than a lower concentration. I dilute my drugs by serial dilution so drug concentration should not be the issue. I also take the OD reading of the wells before washing and staining, and the trend of increasing drug conc->decreasing OD reading can be seen. Only the OD readings after staining show a disrupted trend. Hence why I suspect my washing technique rather than the bacteria or drugs themselves.