[New method for spatial structure quantification of biofilm with GFP tagged bacteria]

Green fluorescent protein (GFP) gene was applied to mark E. coli JM109 by the method of CaCl2 transformation. The GFP-tagged cells was inoculated into the LB medium containing 50 microg/mL of Ampicillin to develop biofilm on the surface of lava by mix-culture with lava particles in flask under 37 degrees C, 120 r/min for 16 h. The fluorescent images stack of different layer at specific area (250 microm x 250 microm) on lava covered with biofilm was obtained by confocal scanning laser microscopy. The quantifying parameters of biofilm spatial structure could be acquired after the image information was calculated by COMSTAT program, i. e., the average thickness of specific area of biofilm after 16 h was 0.120 844 microm, the maximum thickness was 10.5 microm, the volume was 0.136 986 microm3 /microm2, the surface area was 21 338.1 microm2, and the surface area per volume biofilm was 3.36 854 microm2 /microm3. This method might be expanded to reflect the spatial structure of biofilm with any other GFP tagged bacteria.