Background

Bacterial adhesion is the first step of many important processes such as pathogen tissue invasion, symbiont recruitment and biofilm formation. Adhesion is a dynamic process, changing on a time scale of seconds (Fletcher, 1977; Vadillo-Rodríguez et al., 2004), therefore continuous, kinetic measurements are needed to study these events. Sampling a large surface area is also helpful, as adhesion tends to be heterogeneous and stochastic at a microscopic scale. In addition to having an assay that can make kinetic measurements, it is desirable to have an assay that is simple, cost-effective, and high throughput because it makes it possible to have multiple replicates and to test a wide range of experimental parameters.

While many methods for the measurement of adhesion have been reported - each one with advantages and disadvantages - none of the methods capture every aspect of this dynamic process. Microscopy allows detailed and direct measurement of adhesion but the main drawback of microscopy is a small field size. As a result, sampling of many different fields is needed to obtain statistical validity but sampling in this way makes it difficult to get high resolution kinetic data. This contradiction is highlighted in early time points of adhesion kinetics, when the number of cells per surface unit is low, causing a large stochastic effect due to small sample sizes (Vadillo-Rodríguez et al., 2004). Another popular method for bacterial attachment measurement is using crystal violet dye in microtiter plates (O'Toole, 2011). While this method is high throughput and inexpensive, it does not allow repeated measurements, making kinetics complicated and unreliable. Furthermore, sample preparation is not fast which makes this assay inadequate for monitoring events that occur at seconds-minutes timescales. Nano plasmonics is an effective way to make real time, kinetic measurements without the need for fluorescent labeling, but this method is expensive and requires highly specialized equipment, fabrication capabilities and expertise (Funari et al., 2018, Abadian and Goluch, 2015). The protocol presented here uses fluorescent labeling and is able to provide real time, kinetic measurements of bacterial adhesion in different culture densities, salt concentrations and different surfaces, using a variety of bacterial species (Shteindel et al., 2019), as well as changes in bacterial adhesion behavior under the effect of quorum sensing signals and in the different bacterivores (in preparation). All graphics in this protocol are adapted from Shteindel et al., 2019.