IMAGING BIOFILMS
	Department of Biology	Technology Facility
I & C Technical Guides

E. coli
The bacterial strains used here were chosen to facilitate the technology development. Clearly, the species/strain of interest can be used as long as it will attach to a glass surface. With the strain described here, we were able to use untreated glass.
All biofilms consisted of derivatives of the E. coli strain TG1 18, which constitutively produces the F pilus (Ghigo, 2001). This strain was transformed with plasmids pZEtetR21-GFP (Da Re et al., 2007) or pRSETB-tdTomato (Shaner et al., 2004) resulting in strains MV1257 and MV1264, respectively.
The strains were grown in M9 minimal medium with 0.4% glucose at 37˚C (Ghigo, 2001; Reisner et al., 2003). 30mg/ml kanamycin and 100mg/ml ampicillin were added to maintain the plasmids pZE21-gfp or pRSETB-tdTomato respectively. Antibiotics were omitted from the medium during biofilm growth with no evidence of plasmid loss. Gravity facilitated adhesion to the coverslip occurred over 30-60 minutes prior to initiating flow. A biofilm up to 180µm thick was obtained reproducibly within 48 hours after seeding with approximately 1x108 log-phase cells.

References:
Da Re, S., Le Quere, B., Ghigo, J.M. and Beloin, C. (2007) Tight modulation of Escherichia coli bacterial biofilm formation through controlled expression of adhesion factors. Appl Environ Microbiol, 73, 3391-3403.

Ghigo, J.M. (2001) Natural conjugative plasmids induce bacterial biofilm development. Nature,412, 442-445.

Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N., Palmer, A.E. and Tsien, R.Y. (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol, 22, 1567-1572.

Stains
The fluorescence from GFP or tdTomato expressing strains was analyzed directly or alternatively, the BACLight LIVE/DEAD stain (Invitrogen) (1ml of 1:1000 in media) was added just prior to imaging.  This stain consists of SYTO9 (green) that is taken up by all cells, and Propidium Iodide (red) that is taken up by cells with a compromised membrane. With the strain and growth conditions used here the fluorochromes completely penetrated the biofilm biomass.

Media
As well as affecting growth rates we noticed that within the closed system of the flow cell rich media caused the production and accumulation of gas in the chamber. This resulted in a flow cell void of biofilm and/or media but entirely filled with froth. LB had this effect, whereas growth on minimal media supplemented with glucose eliminated this problem altogether.

Minimal growth medium also has the advantage of minimal autofluorescence where as cells grown in LB often show autofluoresence.

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