S. mutans-GFP and C. albicans single and two-species biofilms were grown in an 8 well microscope slide (ibidi) under 5% CO2 at 37°C for 16 h 15 (link),20 . The biofilms were gently washed with PBS three times to remove any unattached cells and stained with calcofluor white to label C. albicans (Sigma-Aldrich). Biofilms were grown with either 1μM dextran-conjugated pHRodo red or dextran-conjugated Cascade Blue (Molecular Probes, Invitrogen) to monitor pH changes or glucan production, respectively21 (link). The stained biofilms were examined using fluorescence microscopy and CLSM as reported 22 (link). Three independent experiments were performed, and the images displayed are representative of all studies. Bio-volume of biofilms were quantified by the program COMSTAT23 (link),24 . ImageJ was used to quantify fluorescence of acid (pHRodo red) and glucan (cascade blue) production.