In my antibiofilm test (quantification) using the 96-well plates and CV staining method, i found out that at lower extract concentrations, the production of biofilm was higher than the control (inoculum without extract). As the extract concentration increased, the inhibitory activity started to increase gradually. However the inhibitory activity started to reduce again at much higher concentration. Is this kind of enhancing and inhibiting trend related to the multiphasic dose-response relationship (i am not sure whether the term is correct or not) and mechanisms of biofilm induction things? I have subtracted the sample OD (extract with inoculum) with the blank OD (extract without inoculum).
The biofilm production was quantified at OD 570 nm using microtiter plate reader.
Hope you guys can share the idea for my outcomes. Thanks! :-)
I hope, you have kept an abiotic control in your experiment i.e. everything else except the inoculum. There needs to be a separate abiotic control for each concentration of the extract.
If yes, then your results may be described as 'dose-independent response'.
Dr Vijay, yes I did include the abiotic control such as background control of the extract (without inoculum) and the broth alone.
Dear Afifah, It is entirely possible that the biofilm response to your inhibitory extract may differ at low and high concentrations for a number of reasons (for example, at a low concentration of inhibitor, a second chemical present in the extract may act as a stimulant for growth; the response to the inhibitor may not work until a critical threshold is reached, etc.). However, another concern is whether you are replicating your measurements, as biofilm assays can be highly variable. If you do have replicate measurements (within a 96 well plate or between different plates), you should use a different approach to determining change : do an ANOVA and a post hoc test of the differences between means (such as Dunnett's test with a control) to see if statistically the control (biofilm without extract) and biofilms with extract differ significantly. If they do, then you can calculate the difference (biofilms with extract minus the control) if you want to display your results in that way (and remember, you need to keep tract of the errors in your measurements when you do that too - in this case, you add the Control error to the biofilms with extract errors to get the final error measurements). Given the problems in making individual measurements of biofilm growth in each well of a 96 well plate, I think that taking a statistical approach in analysing your data (before trying to find an explanation) is a better way forward : if you just do the subtraction, then errors in your measurements may mean that the results are unfounded. Regards, Andrew.
Thank you Dr Andrew for the very detail explanation and suggestion. I should be taking the statistical approach first before proceed with finding explanations and reasons to the results.
We observed enhancement of biofilm in the case of studies on the antifouling potential of commercial enzymes. Some significantly increased adhesion whereas others inhibited it (C. Leroy et al. Biofouling. 2008;24(1):11-22.)
Biofilm accumulation has already been observed when an insufficient amount of biocide was applied (Grant & Bott, 2005). This increase in adhered bacteria could be related to sedimentation, the accumulation of planktonic bacteria, or to cell growth within the biofilm whereas specific enzyme in addition to its enzymatic activity could be a substrate supporting bacteria growth. Dead cells in clusters may also be a nutrient source, allowing other cells to develop.
In some conditions of enzyme concentrations and depending upon the protocol used (prevention or detachment of bacterial adhesion) some enzymatic activities were capable of increasing bacterial adhesion
Hello, we found out that at sublethal concentrations of biocides the biofilm formation increases. Here is the link of the publication.
http://www.sciencedirect.com/science/article/pii/S0261219415301101
I am so thankful to all of you for sharing the knowledge and articles with me :-)
Will read and understand all of them :-) Really appreciate the help :-)
Dear Afifah,
I think as mentioned by Andrew J Spiers, Chantal Compere, Marta Sena-Vélez and others increased biofilm formation is possible at sub-lethal concentrations. I would relate this finding to reports on using sub-inhibitory concentrations of antibiotics and above MIC also. So this could be looking into resistance and growth (where at higher concentrations selection for resistant strains is higher, so clearance of susceptible strains promotes growth of minimally susceptible strains).
BTW, someone should invent statistics specifically oriented for biofilm assays!
Its possible and I have obtained same type of observation.
Please read my published conference paper attached herewith.
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