For me, I'd look for inhibition of biofilm formation as well as biofilm biomass reduction through crystal violet assay and also cell viability reduction through BacLight Live Dead staining method. So, basically I utilize two main assays: crystal violet assay and cell viability determination. The reason why I look at these two aspects is due to (1) the ability of some phytochemicals to inhibit bacteria from initiating the formation of biofilm, (2) some phytochemicals are able to directly act on the exopolymeric substance (EPS), thereby reducing the integrity of the whole biofilm thus easily washed away and (3) some phytochemicals can enter the EPS layer and kill the bacteria within the biofilm. What I've found so far are (a) phytochemicals that can inhibit the formation of biofilms but do not kill bacteria that resides within biofilms, (b) phytochemicals that can kill bacteria within biofilms but do not disrupt the biofilm and (c) phytochemicals that can kill bacteria within biofilms as well as disrupt the EPS layer. So what happens in (a) is that crystal violet assay showed positive for antibiofilm but cell viability test showed no significant difference compared to controls. However, in (b), crystal violet assay showed no significant difference compared to controls while the cell viability test revealed significant reduction in bacteria viability. Finally, in (c), both assays showed significant reduction. These are my criteria when I'm screening for antibiofilm activity of phytochemicals. To everyone out there, do you have any thoughts/comments/critics on these ideas? I'd be pleased to know :)
Important activity of phytochemicals are action on exopolymeric substance (EPS), thereby reducing the integrity of the whole biofilm. phytochemicals can enter the EPS layer and kill the bacteria within the biofilm. There is a possibility that phytochemicals can inhibit the formation of biofilms but do not kill bacteria that resides within biofilms, (b) phytochemicals that can kill bacteria within biofilms but do not disrupt the biofilm and (c) phytochemicals that can kill bacteria within biofilms as well as disrupt the EPS layer.Surfaces coated with the naturally-occurring polysaccharide chitosan (partially deacetylated poly N-acetyl glucosamine) resisted biofilm formation by bacteria . More importantly anti-biofilm properties of chitosan-coated surfaces are best examples.
Important activity of phytochemicals are action on exopolymeric substance (EPS), thereby reducing the integrity of the whole biofilm. phytochemicals can enter the EPS layer and kill the bacteria within the biofilm. There is a possibility that phytochemicals can inhibit the formation of biofilms but do not kill bacteria that resides within biofilms, (b) phytochemicals that can kill bacteria within biofilms but do not disrupt the biofilm and (c) phytochemicals that can kill bacteria within biofilms as well as disrupt the EPS layer.Surfaces coated with the naturally-occurring polysaccharide chitosan (partially deacetylated poly N-acetyl glucosamine) resisted biofilm formation by bacteria . More importantly anti-biofilm properties of chitosan-coated surfaces are best examples.
To complete the previous answers, the adhesion step is also to be explored. So in our lab we look at the global kinetic of biofilm formation using parallel and different approaches (culture, qPCR, confocal observations,...) considering that no one is perfect! Crystal violet method has to be considered as a screening method (coloration detection only at a very high level of microorganisms : about 1E8). Initial inoculum, incubation time and overall medium composition have a great impact on the biofilm growth and structuration and you have to take them into account : i.e. if you use in a minimum medium able to promote only the growth of adherent cells, the planktonic one can be considered as a release from the biofilm and has to be measured; if you are in current medium, planktonic cell growth is rapid and the biofilm formation is rather a deposition of these cells than an adherent cell growth.
I think Dr. Ravi Kant and Dr. Christine nicely elaborated your query.
Agree with all of you. the important aspects are:
a. adherent biomass (normally 2 hours after inoculation)
b.biofilm biomass
c. biofilm viability
d .EPS composition
these measurements could be done in formation and detachment assay
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