2.2. Detection of Biofilm Formation by Crystal Violet Staining

Biofilm formation for the six heat-resistant E. coli isolates was detected using an in-house, two-component apparatus (Figure 1A). A single colony from each BAP culture was inoculated into 10 mL of Luria Bertani (LB) broth (Becton Dickinson, Mississauga, ON, Canada) followed by incubation at 37 °C for 24 h with agitation. Stationary phase cultures of the isolates were adjusted to an optical density (OD) at 600 nm of 0.5 (Microscan Turbidity Meter, Siemens, Oakville, ON, Canada) with phosphate buffered saline (pH 7.0) from which 1 mL aliquots were washed by centrifugation at 20,000× g for 2 min and re-suspended with 1 mL of saline. For each isolate that biofilm formation was to be determined, the wells of a 96 well flat bottom clear polystyrene microplate (Corning; Millipore Sigma, Milwaukee, WI, USA) were filled with 140 µL of LB broth in triplicate. To these wells, 10 µL of the aliquots of bacterial cells were added; 10 µL of saline was added to 140 µL of LB broth to serve as a blank. Sterile sticks (Puritan 6” wooden applicator stick; Puritan Medical Products, Guilford, ME, USA) were taped onto the longitudinal sides of the microplate to ensure that the pegs of the 96 well PCR plate (MicroAmp; Thermo Fisher Scientific, Waltham, MA, USA) that was laid on top of the microplate would not come in direct contact with it. The pegs of the PCR plate were submerged into the wells of the flat bottom microplate inoculated with bacterial culture or saline and the top and bottom components of the apparatus were sealed together with additional tape. The apparatus was stored in an air-tight plastic container lined with damp paper towels to prevent evaporation over a 24 h incubation period at 4 °C. Unlike conventional biofilm assays that typically assess static biofilm formation from the wells of the flat bottom microplate, biofilms formed by motile bacterial cells were detected from the pegs of the PCR plate. Following incubation, the PCR plate was disassembled from the apparatus and washed in 200 mL of Milli-Q water for 30 s with light agitation by hand (4x). The washed PCR plate was then turned with the pegs facing upwards to remove excess Milli-Q water and dried for 10 min. To a 96 well round bottom clear polystyrene microplate (Corning; Millipore Sigma), 200 µL of crystal violet (Millipore Sigma) diluted to 1% with Milli-Q water was added to each well corresponding with the wells of the flat bottom microplate. The PCR plate was laid on top of the round bottom microplate so that the pegs were submerged in the crystal violet solution for 30 min at 24 °C. Following staining, the PCR plate was again washed in 200 mL of Milli-Q water with light agitation for 30 s (4x) and dried for 10 min (Figure 1B). Lastly, a second round bottom microplate was prepared with 150 µL of 95% ethanol per well for de-staining of the pegs. The PCR plate was immersed in the round bottom microplate for 30 min at 4 °C during which any crystal violet adhering to the pegs dissolved into the ethanol. Absorbance of crystal violet was measured at 595 nm using a SpectraMax 190 Microplate Reader (Molecular Devices LLC, San Jose, CA, USA) and SoftMax Pro software. Screening for biofilm formation in LB broth was repeated for all isolates at incubation temperatures of 24 °C and 37 °C.

Two-component apparatus for detecting biofilm formation (A). The pegs of the PCR plate are submerged into wells of the flat bottom microplate containing bacterial cells. The PCR plate rests on top of 2 sterile sticks to prevent the pegs from direct contact with the bottom of the microplate. Biofilms form on the pegs that are subsequently stained with 1% crystal violet (B).