Biofilm localization in the vertical wall of shaking 96-well plates

Abstract

Microtiter plates with 96 wells are being increasingly used for biofilm studies due to their high throughput, low cost, easy handling, and easy application of several analytical methods to evaluate different biofilm parameters. These methods provide bulk information about the biofilm formed in each well but lack in detail, namely, regarding the spatial location of the biofilms. This location can be obtained by microscopy observation using optical and electron microscopes, but these techniques have lower throughput and higher cost and are subjected to equipment availability. This work describes a differential crystal violet (CV) staining method that enabled the determination of the spatial location of Escherichia coli biofilms formed in the vertical wall of shaking 96-well plates. It was shown that the biofilms were unevenly distributed on the wall with denser cell accumulation near the air-liquid interface. The results were corroborated by scanning electron microscopy and a correlation was found between biofilm accumulation and the wall shear strain rates determined by computational fluid dynamics. The developed method is quicker and less expensive and has a higher throughput than the existing methods available for spatial location of biofilms in microtiter plates.

Figure 1

Biofilm localization in shaking 96-well microtiter plates placed in a 50 mm incubator at 150 rpm. (a) Photograph of a well stained with crystal violet; (b) schematic representation of a well where the dark grey area corresponds to the wetted area without shaking, the light grey area represents the area increase upon shaking, and the dotted line depicts the inclination of the air-liquid interface; (c) time averaged shear strain rates (values below 20 s⁻¹ are not represented); (d) illustration of the biofilm distribution on the vertical wall assayed by the differential CV staining; (e) representative scanning electron micrographs of the wall sections defined in image (d); (5000x magnification bar = 10 μm).