Experimental reproducibility in flow-chamber biofilms.
The structural organization of microbial communities is influenced by many factors, e.g. nutrient composition, shear stress and temperature. This paper presents a general method for quantitative comparison of biofilm structures and assessment of experimental reproducibility between independent biofilm experiments. By using a novel computer program, COMSTAT, biofilm structures of Pseudomonas aeruginosa and an isogenic rpoS mutant were quantified. The strains were tagged with the green fluorescent protein (GFP) and grown in flow chambers with a defined minimal medium as substrate. Three independent rounds of biofilm experiments were performed and in each round, each of the two variants was grown in two separate channels. Nine image stacks were acquired in each channel 146 h after inoculation. An analysis of variance model incorporating the factors experiment round, bacterial strain, channel number and image stack number was used to analyse the data calculated by COMSTAT. Experimental reproducibility was verified by estimating the magnitude of the variance of the effects round (sigma(2)R) and the interaction between bacterial strain and round (sigma(2)BR). Mean thickness of the wild-type and rpoS mutant biofilms was estimated at 6.31 microm (SE 0.81 microm) and 16.85 microm (SE 0.87 microm), respectively.
Heydorn
Arne
A
Molecular Microbial Ecology Group, Department of Microbiology, The Technical University of Denmark block 301,DK-2800 Lyngby, Denmark1.
Ersbøll
Bjarne Kjær
BK
Department of Mathematical Modelling, The Technical University of Denmark, DK-2800 Lyngby, Denmark2.
Hentzer
Morten
M
Molecular Microbial Ecology Group, Department of Microbiology, The Technical University of Denmark block 301,DK-2800 Lyngby, Denmark1.
Parsek
Matthew R
MR
Environmental Engineering Group, Department of Civil Engineering, NorthWestern University, Evanston, Illinois, USA3.
Givskov
Michael
M
Molecular Microbial Ecology Group, Department of Microbiology, The Technical University of Denmark block 301,DK-2800 Lyngby, Denmark1.
Molin
Søren
S
Molecular Microbial Ecology Group, Department of Microbiology, The Technical University of Denmark block 301,DK-2800 Lyngby, Denmark1.
D000704:Analysis of Variance; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018441:Biofilms / Q000254:growth & development; D003470:Culture Media; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000254:growth & development*; D015203:Reproducibility of Results; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism; D012984:Software
D001426:Bacterial Proteins; D003470:Culture Media; D008164:Luminescent Proteins; D012808:Sigma Factor; C069567:sigma factor KatF protein, Bacteria; D049452:Green Fluorescent Proteins