Results of a biofilm assay using the strains listed in Table 1 demonstrates the range of phenotypes of individual strains and the stimulatory effect of glucose. Biofilm cultures were grown in 0.5X TSB supplemented with 0.25% or 1% glucose (gluc) at 37 °C for 18 h. A) Photographs of two representative rows from each tissue culture treated 96-well assay that was stained with crystal violet. B) The amount of crystal violet bound was quantified by measuring absorbance at 600 nm and plotted for each strain. Each bar represents the average of 8 individual assay wells, and the error bars represent the standard deviation.

Optimization of assay conditions. A) A biofilm growth curve of S. aureus ATCC 35556 grown in 0.5X TSB supplemented with 1% glucose. B) Biofilms were grown in varying media conditions to identify optimal growth conditions and two methods of biofilm detection are compared. Biofilms were grown at 37 °C for 18 h. Biofilms were stained as described above and the amount of crystal violet bound to the bottom of each assay well was measured directly as absorbance at 600 nm, or crystal violet was eluted from each well, diluted 1:10, and the absorbance at 600 nm was measured. C) Comparison of absorbance at 600 nm readings of neat vs. a 1:10 dilution of eluted crystal violet demonstrates the linear range of the assay.

An example of a Minimal Biofilm Inhibitory Concentration (MBIC) assay for eight anti-biofilm compounds that were tested against S. epidermidis 18972. The assay plate in which biofilm cultures have been stained with crystal violet is shown in the photograph. The concentrations of each compound tested are indicated above the plate, and the compound tested in each row is indicated on the left side of the plate. The MBIC and MIC are defined as the lowest compound concentration that produces a ≥ 80% inhibition of biofilm growth or planktonic growth, respectively. The compounds used in this assay (Compounds 1–8), their chemical names and structures, and the MBIC and MIC values obtained as a result of the assay are also shown. Note that Gentian violet produced an MIC that was equal to the MBIC. Because this compound strongly absorbs light at 600 nm, the antibacterial activity of this compound became apparent only when it was diluted in the dose response assay. The compounds used in this assay are commercially available from MicroSource Discovery Systems, Gaylordsville, CT). Alternatively, three of these compounds (Tomatidine, Actinonin, and Emodin) can be purchased from Sigma-Aldrich (St. Louis, MO). MBX-1240 can be purchased from ChemBridge Corp. (San Diego, CA).

Streaking bacteria on solid agar to obtain single, well-isolated colonies. A) Streaking plates. Plates can be streaked using a standard wire inoculating loop, a disposable plastic loop, a toothpick, or a long “picking stick”. The wire loop can be sterilized in a flame, but it must be cooled by touching the agar surface prior to making contact with bacteria. Single colonies can be isolated by repeated streaking as illustrated in the figure. B) An example of the results obtained using this method. Single, well-isolated colonies are indicated.

Setting up assay plates for biofilm formation on alternative substrates

An example of the screening results obtained using S. epidermidis 18972 and a mock screening plate. A) A photograph of the crystal violet stained mock screening plate that contained nine compounds that are inhibitors of biofilm formation and two antibiotics. The remainder of the wells in columns 2–11 contained DMSO. Columns 1 and 12 contain the 0% INH (DMSO only) and 100% INH (100 µg chloramphenicol/ml; Cm100) controls, respectively. The wells containing anti-biofilm and antibacterial compounds exhibited significant reductions in the intensity of crystal violet staining. B) The OD₆₀₀ values obtained for the planktonic cultures. Compounds that produced ≥ 40% inhibition planktonic growth are highlighted in orange. The compound in well number C07, highlighted in blue, produced an OD600 of 2.64, which is significantly higher than those produced by the 0% INH (DMSO only) controls. This well contained gentian violet, an intensely colored compound that absorbs light at 600 nm. Beware of colored compounds that absorb at 600 nm, as they can mask antibacterial activity. C) The OD₆₀₀ values obtained for the crystal violet stained biofilm cultures. Compounds that produced ≥ 80% inhibition planktonic growth are highlighted in orange. D) A table listing the compounds used in the mock screening plate, their location on the plate (row, column), and the screening call (HIT or antibacterial). A compound that produced % ≥80% biofilm inhibition and ≤ 40% planktonic growth inhibition were designated anti-biofilm “HITS”. Compounds that produced ≥ 40% planktonic growth inhibition were designated as “Antibacterial” compounds. Seven of the antibiofilm compounds used in the mock screening plate were identified previously in a pilot screen using a compound library comprised of 2000 known bioactive chemicals (Spectrum library; MicroSource Discovery Systems, Gaylordsville, CT). MBX-1240 and MBX-1246 were identified in screen of compounds purchased from Chembridge Corp. (San Diego, CA) (Opperman et al., 2009)