We use a variety of methodologies to study biofilm formation.  One of the most common and simplest methods is using microtiter dishes. In microtiter assays, bacteria (in this case P. fluorescens) are inoculated into wells containing the appropriate growth medium. After growing 8-10 hrs, the wells are stained with the dye crystal violet, which stains the bacteria, but not the plastic wells. After rinsing, a biofilm ring can be seen at the air-medium interface (see top left). The dye can be solubilized in ethanol to get a semi-quantitative measure of biofilm formation over time. This same technique can be used to isolate mutants defective in biofilm formation (bottom left). We call these mutants sad for surface attachment defective.  

We have developed a number of variations on these assays, for example using 24 well plates to visualize early biofilm events by phase contrast microscopy.  Direct visualization of bacteria is an excellent complement to indirect dye-based assays.

A second important tool is the flow cell. A flow cell allows the continuous flow of fresh nutrients into a chamber with a window that allows direct viewing of the biofilm under the microscope. In this way, the biofilm can be viewed directly without disturbing the community.  We have also modified 6 well dishes for use in flow cell studies, which we refer to as the Kadouri Method, which allows biofilms to form in 6 parallel chambers few from a single medium reservoir.  This method is described in detail in the Current Protocols article cited below. 

Here is an example of such a set-up showing the media reservoir, bubble trap, and flow cell chamber along with a peristaltic pump and associated tubing.

Our thanks to Morton Hentzer and Matt Parsek for helping us get the system set-up and Wilbur Clark for building the chamber.

Relevant Publications:

O'Toole, G.A., L.A. Pratt, P.I.Watnick, D.K. Newman, V.B. Weaver, and R. Kolter. Genetic Approaches to the Study of Biofilms. In Doyle, R.J. (ed.), Biofilms. Methods in Enzymology, Academic Press, San Diego, CA. Invited Review. 310:91-109.

Bloemberg, G.V., G.A. O'Toole, B.J.J. Lugtenberg, R. Kolter. 1997. Green Fluorescent Protein as a Marker for Pseudomonas spp. Appl. Environ. Microbiol. 63:4543-4551.

Merritt, J.H., Daniel E. Kadouri and G.A. O’Toole.  2005. Growing and Analyzing Static Biofilms.  In: Current Protocols in Microbiology, J. Wiley & Sons, Hoboken, NJ, Vol. 1, 1B.1.1-17.