I am trying to grow biofilms of a bacterial isolate library on a 384 well flat bottom plate for fluorescent imaging. In order to get the biofilms within a reproducible focal range, I would like to get them to grow on the bottom of the wells rather than in the middle of the wells were the liquid/air interface usually is. Does anyone have any advice on how I can push the biofilm further down the well without compromising integrity and structure? I have found some ideas in the literature, including 1) using low inoculum volumes, 2) tilting the plate at an angle (not sure how reproducible this is), or 3) adding wash steps during biofilm development to isolate adherent cells only.
Does anyone have any ideas? Any tips or tricks to test would be appreciated.
Dear Danielle,
You certainly have a practical problem in visualising your biofilms by fluorescent microscopy using microtitre well plates. However, I would not be comfortable about trying to 'force' the growth of meniscus-associated or air-liquid interface biofilms onto the wall bottoms, as this would clearly present the bacteria with a different growth environment, which might therefore effect physiology and gene expression. Our model pseudomonad clearly prefers growth at the meniscus and out across the A-L interface, and I would presume that any mutants growing as submerged surface-liquid interface biofilms down the walls or along the bottom have some significant physiological or genetic alteration.
Rather than trying to change the growth conditions, how about investigating alternative ways of harvesting A-L biofilm samples for microscopy? 384 well plates might be a good way to screen mutants, but it is almost impossible to visually verify biofilm-formation let alone to assess the relative growth on different interfaces. You may need to move to 24 well plates which would allow you to readily isolate A-L interface biofilm samples using a wire-loop or a small plastic test strip while still maintaining the correct orientation (I prefer 30 ml vials and we can pour out our robust biofilms into a petri dish, and then sample from these and place directly on microscope slides). Any vigorous removal from the meniscus or from the well sides or bottom is likely to result in a highly damaged structure, but at high magnification it should still be possible to make some inferences about cell physiology and gene expression.
If you have substantial A-L biofilms, it might be possible to remove a lot of the liquid using a pipette until the biofilm is sitting on the well bottom, and this may allow good visualisation at least of the bottom surface of the surface. Imaging the top surface of the biofilm will be more tricky!
Andrew
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