Chapter 19
Biofilm Formation and Quantification Using
the 96-Microtiter Plate
Roman Thibeaux, Malia Kainiu, and Cyrille Goarant
Abstract
Biofilm formation in microtiter plates is certainly the most commonly used method to grow and study
biofilm. This simple design is very popular due to its high-throughput screening capacities, low cost, and
easy handling. In the protocol described here, we focus on the use of 96-well optically clear, polystyrene flatbottom plate to study biofilm formation by Leptospira spp. and quantify the biofilm formation by crystal
violet (CV) staining. We also describe an alternative method, based on phase contrast image analysis that we
believe is more suitable for accurately quantifying biofilm growth by reducing handling of this fragile
structure.
Key words Biofilm, 96-well plate, Static culture, Crystal violet, Phase contrast images, Quantification
1 Introduction
Microbial biofilms are commonly defined as sessile microbial consortia established in a three-dimensional structure and consist of
multicellular communities composed of prokaryotic cells embedded in a matrix synthesized by the microbial community. Biofilm
formation is a multistage process that starts with microbial adhesion
with a subsequent production and accumulation of an extracellular
matrix mainly composed of polymeric substances such as polysaccharides, extracellular DNA, or proteins [1]. Microbial biofilm
constitutes a serious problem for public health because of the
increased resistance to antimicrobial action and their potential to
cause infections in patients with indwelling medical devices [2].
Over the last couple of decades, a number of model systems
have been tested for in vitro study of biofilm [3]; among them
microtiter plates are being widely used. Originally developed to
investigate bacteria attachment [4], it further proved to be compatible with the study of sessile development [5]. The advantages of
the microtiter plate assay are its simplicity, the use of basic laboratory materials, the adaptability to small or large numbers of
Nobuo Koizumi and Mathieu Picardeau (eds.), Leptospira spp.: Methods and Protocols, Methods in Molecular Biology, vol. 2134,
https://doi.org/10.1007/978-1-0716-0459-5_19, © Springer Science+Business Media, LLC, part of Springer Nature 2020
207
Biofilm Formation and Quantification Using
the 96-Microtiter Plate
Roman Thibeaux, Malia Kainiu, and Cyrille Goarant
Abstract
Biofilm formation in microtiter plates is certainly the most commonly used method to grow and study
biofilm. This simple design is very popular due to its high-throughput screening capacities, low cost, and
easy handling. In the protocol described here, we focus on the use of 96-well optically clear, polystyrene flatbottom plate to study biofilm formation by Leptospira spp. and quantify the biofilm formation by crystal
violet (CV) staining. We also describe an alternative method, based on phase contrast image analysis that we
believe is more suitable for accurately quantifying biofilm growth by reducing handling of this fragile
structure.
Key words Biofilm, 96-well plate, Static culture, Crystal violet, Phase contrast images, Quantification
1 Introduction
Microbial biofilms are commonly defined as sessile microbial consortia established in a three-dimensional structure and consist of
multicellular communities composed of prokaryotic cells embedded in a matrix synthesized by the microbial community. Biofilm
formation is a multistage process that starts with microbial adhesion
with a subsequent production and accumulation of an extracellular
matrix mainly composed of polymeric substances such as polysaccharides, extracellular DNA, or proteins [1]. Microbial biofilm
constitutes a serious problem for public health because of the
increased resistance to antimicrobial action and their potential to
cause infections in patients with indwelling medical devices [2].
Over the last couple of decades, a number of model systems
have been tested for in vitro study of biofilm [3]; among them
microtiter plates are being widely used. Originally developed to
investigate bacteria attachment [4], it further proved to be compatible with the study of sessile development [5]. The advantages of
the microtiter plate assay are its simplicity, the use of basic laboratory materials, the adaptability to small or large numbers of
Nobuo Koizumi and Mathieu Picardeau (eds.), Leptospira spp.: Methods and Protocols, Methods in Molecular Biology, vol. 2134,
https://doi.org/10.1007/978-1-0716-0459-5_19, © Springer Science+Business Media, LLC, part of Springer Nature 2020
207