title: A method for rapid quantitative assessment of biofilms with biomolecular staining and image analysis.
abstract: The accumulation of bacteria in surface-attached biofilms can be detrimental to human health, dental hygiene, and many industrial processes. Natural biofilms are soft and often transparent, and they have heterogeneous biological composition and structure over micro- and macroscales. As a result, it is challenging to quantify the spatial distribution and overall intensity of biofilms. In this work, a new method was developed to enhance the visibility and quantification of bacterial biofilms. First, broad-spectrum biomolecular staining was used to enhance the visibility of the cells, nucleic acids, and proteins that make up biofilms. Then, an image analysis algorithm was developed to objectively and quantitatively measure biofilm accumulation from digital photographs and results were compared to independent measurements of cell density. This new method was used to quantify the growth intensity of Pseudomonas putida biofilms as they grew over time. This method is simple and fast, and can quantify biofilm growth over a large area with approximately the same precision as the more laborious cell counting method. Stained and processed images facilitate assessment of spatial heterogeneity of a biofilm across a surface. This new approach to biofilm analysis could be applied in studies of natural, industrial, and environmental biofilms.
authors.affiliation: Pacific Northwest National Laboratory, Battelle for the USDOE, PO Box 999, MSIN P7-50, Richland, WA, 99352, USA.
authors.forename: Curtis
authors.initials: C
authors.lastname: Larimer
authors.affiliation: Marine Sciences Laboratory, Pacific Northwest National Laboratory, 1529 W. Sequim Bay Road, Sequim, WA, 98382, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Winder
authors.affiliation: Marine Sciences Laboratory, Pacific Northwest National Laboratory, 1529 W. Sequim Bay Road, Sequim, WA, 98382, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Jeters
authors.affiliation: Pacific Northwest National Laboratory, Battelle for the USDOE, PO Box 999, MSIN P7-50, Richland, WA, 99352, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Prowant
authors.affiliation: Swanson School of Engineering, University of Pittsburgh, Benedum Hall, 3700 O'Hara Street, Pittsburgh, PA, 15261, USA.
authors.forename: Ian
authors.initials: I
authors.lastname: Nettleship
authors.affiliation: Pacific Northwest National Laboratory, Battelle for the USDOE, PO Box 999, MSIN P7-50, Richland, WA, 99352, USA. Raymond.Addleman@pnnl.gov.
authors.forename: Raymond Shane
authors.initials: RS
authors.lastname: Addleman
authors.affiliation: Marine Sciences Laboratory, Pacific Northwest National Laboratory, 1529 W. Sequim Bay Road, Sequim, WA, 98382, USA. George.Bonheyo@pnnl.gov.
authors.forename: George T
authors.identifier: http://orcid.org/0000-0001-8853-5744
authors.initials: GT
authors.lastname: Bonheyo
mesh_terms: D000465:Algorithms; D018441:Biofilms; D006801:Humans; D007091:Image Processing, Computer-Assisted; D011552:Pseudomonas Infections / Q000382:microbiology; D016958:Pseudomonas putida / Q000737:chemistry* / Q000254:growth & development / Q000502:physiology*; D013194:Staining and Labeling / Q000379:methods*
keywords: Biofilm; Biofilm growth intensity; Biofouling; Biomolecular stain; Image analysis