Un-inoculated broth can be used as medium control.... Inoculated broth without an a biofilm inhibitor or biofilm disruptor can act as positive control because detection of biofilm is mostly by the OD value obtained by the staining assays. first subtract the OD value of the inoculated broth from that of medium control OD. Then subtract your test value (usually it will be less than the positive control because u see inhibition) from that of the OD value of the inoculated broth without an a biofilm inhibitor you can get the correct value of the test....

I am investigating whether a substance can inhibit/disrupt biofilm formation. So, my test wells would include biofilm forming bacteria added with the substance I'm studying. The negative control wells should consist of inoculated bacteria with the solvent used to dissolve the substance (but without the substance itself). For the positive control wells, it should consist of inoculated bacteria added with known substance that inhibits biofilm formation/disrupts preformed biofilm.

In a Water Science and Technology-paper, we did more or less the same on pilot scale.

Your control should be a biofilm, formed by the mentioned species, but untreated. In that way, you can compare the treated with the untreated biofilm and assess the efficacy of the inhibition/treatment method.

Keep in mind that treatment and inhibition of biofilm requires a totally different experimental set-up.

your study shows you want to evaluate the capacity of biofilm inhibiting substance . your experiment should have 1. media only with biofilm inhibiting substance,2. media plus biofilm inhibiting substance plus biofilm forming organism, 3. media plus biofilm inhibiting substance plus biofilm forming organism plus solvent dissolving biofilm forming substance, 4. media plus biofilm inhibiting substance plus solvent dissolving biofilm forming substance.

As your main focous is on the substance that dissolves biofilm you can name the control with substance positive and without substance negative.

Here is a general suggestion. Isothermal microcalorimetry is proving to be an excellent tool for continuous real-time evaluation of biofilm formation and subsequent microbial dynamics. See attached 2012 paper.

Addition to previous answer. For an introduction to biofilm and general bioscience usage of isothermal microcalorimetry (with ca. 65 references) see

https://en.wikipedia.org/wiki/Isothermal_microcalorimetry

I have gotten the answer that I was looking for. According to a literature that I read, high iron concentration represses the genes that are involved in biofilm formation of S. aureus. So, I conducted an experiment to evaluate the effect of iron chloride on biofilm formation of S. aureus ATCC6538P. My results showed that at 100 micromolar concentration, the inhibition effect of iron chloride on the biofilm formation was statistically significant (approximately 70 % inhibition, with distilled water as negative control).

This means that 100 micromolar of iron chloride solution can act as positive control for my current experiment, which is to evaluate the anti-biofilm effect of my plant extracts. Therefore, I can proceed with the following:

Sample wells: plant extracts + bacteria suspension

Negative control wells: solvents used to prepare plant extracts + bacteria suspension

Positive control wells: 100 micromolar of iron chloride + bacteria suspension

Media control wells: media used to prepare bacteria suspension

Thank you everyone for your outputs. They are greatly appreciated.

hi, can you please tell me..was it iron (II) chloride or iron (III) chloride?

I have more or less the same concern for the antibiofilm test, the control sets. In my opinion, we also need the background control for antibiofilm test involving plant extract because the extract also contains its own color that will be read by the multireader machine. 

Secondly, does anyone knows the equation to calculate the % of biofilm inhibition that will substract the background control?

Thank you :-)