and colonies were transferred to fresh Omnitray
plates and grown for an additional 48 h. A 96 well master microtiter
plate (Nunc) was prepared containing 150 lL of MRS in each
well. The master plate was inoculated with colonies from the
Omnitray using a replicator lid and the plate was incubated for
48 h under anaerobic conditions at 37 C. After 48 h, cells were agitated
and a new lid was dipped into the cells and transferred to a
new plate which was incubated for 48 h, corresponding to the static
attachment phase of a conventional biofilm reactor. Lids were
transferred to fresh plates (time point zero) and subsequently
transferred every 48 h until the assay endpoint at 144 h. Analysis
of the biofilm formation was performed as previously described
(Rich et al., 2011). Replicator lid pins were stained with a 0.1% crystal
violet solution for 30 min at room temperature, and destained
with 95% ethanol. The amount of crystal violet staining, representing
biofilm growth, was measured at 600 nm using a SpectraMax
M5 microtiter plate reader (Molecular Devices, Sunnyvale, CA).