title: Quantification of confocal images of biofilms grown on irregular surfaces.
abstract: Bacterial biofilms grow on many types of surfaces, including flat surfaces such as glass and metal and irregular surfaces such as rocks, biological tissues and polymers. While laser scanning confocal microscopy can provide high-resolution images of biofilms grown on any surface, quantification of biofilm-associated bacteria is currently limited to bacteria grown on flat surfaces. This can limit researchers studying irregular surfaces to qualitative analysis or quantification of only the total bacteria in an image. In this work, we introduce a new algorithm called modified connected volume filtration (MCVF) to quantify bacteria grown on top of an irregular surface that is fluorescently labeled or reflective. Using the MCVF algorithm, two new quantification parameters are introduced. The modified substratum coverage parameter enables quantification of the connected-biofilm bacteria on top of the surface and on the imaging substratum. The utility of MCVF and the modified substratum coverage parameter were shown with Pseudomonas aeruginosa and Staphylococcus aureus biofilms grown on human airway epithelial cells. A second parameter, the percent association, provides quantified data on the colocalization of the bacteria with a labeled component, including bacteria within a labeled tissue. The utility of quantifying the bacteria associated with the cell cytoplasm was demonstrated with Neisseria gonorrhoeae biofilms grown on cervical epithelial cells. This algorithm provides more flexibility and quantitative ability to researchers studying biofilms grown on a variety of irregular substrata.
authors.affiliation: The University of Iowa, Department of Pharmaceutical Sciences and Experimental Therapeutics, S215 PHAR, 115 S. Grand Ave, Iowa City, IA 52242, United States.
authors.forename: Stacy
authors.initials: S
authors.lastname: Sommerfeld Ross
authors.affiliation: The University of Iowa, Department of Pharmaceutical Sciences and Experimental Therapeutics, S215 PHAR, 115 S. Grand Ave, Iowa City, IA 52242, United States.
authors.forename: Mai Han
authors.initials: MH
authors.lastname: Tu
authors.affiliation: The University of Iowa, Department of Microbiology, Iowa City, IA 52242, United States.
authors.forename: Megan L
authors.initials: ML
authors.lastname: Falsetta
authors.affiliation: The University of Iowa, Department of Microbiology, Iowa City, IA 52242, United States.
authors.forename: Margaret R
authors.initials: MR
authors.lastname: Ketterer
authors.affiliation: The University of Iowa, Department of Microbiology, Iowa City, IA 52242, United States.
authors.forename: Megan R
authors.initials: MR
authors.lastname: Kiedrowski
authors.affiliation: The University of Iowa, Department of Microbiology, Iowa City, IA 52242, United States.
authors.forename: Alexander R
authors.initials: AR
authors.lastname: Horswill
authors.affiliation: The University of Iowa, Department of Microbiology, Iowa City, IA 52242, United States.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Apicella
authors.affiliation: The University of Iowa, Department of Biomedical Engineering, Iowa City, IA 52242, United States.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Reinhardt
authors.affiliation: The University of Iowa, Department of Pharmaceutical Sciences and Experimental Therapeutics, S215 PHAR, 115 S. Grand Ave, Iowa City, IA 52242, United States; The University of Iowa, Department of Chemical and Biochemical Engineering, Iowa City, IA 52242, United States. Electronic address: jennifer-fiegel@uiowa.edu.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Fiegel
mesh_terms: D018441:Biofilms / Q000254:growth & development*; D002460:Cell Line; D004847:Epithelial Cells / Q000382:microbiology; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D009344:Neisseria gonorrhoeae / Q000502:physiology; D011550:Pseudomonas aeruginosa / Q000502:physiology; D013211:Staphylococcus aureus / Q000502:physiology; D013499:Surface Properties
keywords: Biofilm; COMSTAT; Confocal microscopy; Quantification; Surface; Tissue