Strategies to avoid biofilm formation during bacterial experiments

Experiments
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Are there best practices to avoid formation and buildup of bacterial biofilm during an eVOLVER experiment? I have tried adding 0.01% tween 20 to the media (as suggested here), but I still got significant buildup in a handful of E. coli strains.

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The most effective way is to transfer to different vials, and its not too difficult to pause the experiment and switch out vials periodically. This does become annoying however, especially in longer term experiments where selection for wall growth may drive rapid biofilm accumulation.

For E. coli, we had someone test out Tween and PEG media additives, and found that Tween-20 at 0.1% did significantly reduce the biofilm formation. So I might recommend increasing the amount you’re adding up to that concentration.

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I’ll also note that stirring may have an effect, and increasing the rate, or using larger stir bars may help.

I also have tried doing surface treatments on the glass vials, in order to make them super-hydrophobic, but this proved to be inconsistent.

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I can confirm that tween20 0.1%, stir rate 10 and the bigger stir bars seem to inhibit buildup of any visible biofilm after ~70 hours of chemostat growth. Vial on the left had 0.01% tween20, the one on the right 0.1%.

image

Thanks again for the help!

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Hey I’m new to the forum, but this echoes my previous results, where 0.1% tween-20 abolished my biofilm issues with mutagenic e.coli.

I also tried minimal medium, a different undefined broth (MHB), and PEG-2000. Those last two actually made the problem worse! (I think my preparation of minimal medium had some solids in it, which nucleated biofilm formation… probably a preventable problem)

I may perform a quick test to see if 0.1% can be tamped down to 0.05% or so, as in the paper you linked, 0.1% has a significant effect on growth.

Question: what are these bigger stir-bars you are using?
results

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^ biofilm was measured by resuspending the crud on the glass at the end of 72 hours in 20mL of more concentrated tween-water and taking the OD.

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The larger stir bars are: 20mm x 3mm SBM-2003-MIC
We used these originally, but found that the lack of a center ring makes them jump more frequently than the smaller ones from fisher, and since they rub on the vial more, we worried about vial-vial differences in stirring due to friction.

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Thanks, it could be interesting to know what is the optimal concentration. Still, I am not too worried about the effect on growth, since I’m planning to use the same concentration throughout the whole experiment.