Bacterial cell culture
Actinomyces graevenitzii was cultured on Chacollate II agar (GC II Agar with hemoglobin [10 g/L] and IsoVltalexTM[1% v/v]) (BD, USA) at 37°C in an anaerobic incubator. Single colonies from agar plates were picked and separately suspended in 10 mL of brain heart infusion (BHI) broth medium. Streptococcus cristatus was incubated at 37°C in the incubator with shaking overnight. Bacterial suspension concentrations were determined using a hemocytometer and the final concentration of bacteria was adjusted to 1 × 107 cells/mL by dilution in with Iscove’s Modified Dulbecco’s Medium IMDM supplemented with 20% Fetal Bovine Serum (FBS).
Staphylococccus aureus strain SH1000-GFP, which constitutively expresses green fluorescent protein (GFP), was received as a generous gift from the laboratory of Mary Mullins at the University of Sheffield (Sheffield, UK). Bacterial cultures were routinely cultivated in BHI Agar Plates with 5 µg/ml Tetracycline (Teknova, CA). Single colonies from agar plates were picked and suspended in 10 mL of BHI broth medium (Remel, Lenexa, KS, USA) with 5 µg/ml Tetracycline and then incubated at 37°C in aerobic incubator with shaking overnight. After overnight incubation, bacterial suspensions were sub-cultured by adding 1 mL of the overnight culture into 49 mL of BHI broth with Tetracycline for 4 hours. Bacterial concentrations were determined using a hemocytometer and the final concentration of bacteria was adjusted to 5 × 106 cells/mL and diluted with IMDM supplemented with 20% FBS.
Fabrication of microfluidic devices :
Devices were fabricated using standard soft -lithography techniques on four-inch wafers. Photoresist (SU-8, Michrochem, Newton, MA) was spin-coated onto a silicon wafer and exposed to ultraviolet (UV) light, through a photolithography mask. Briefly, two layers of negative photoresist, the first 3 μm thin, the second 50 μm thick were patterned on a silicon wafer by sequentially employing two photolithography masks and processing cycles according to the instructions from the manufacturer. The silicon master wafer with photo patterned structures was employed to mold microchamber that were 200 μm in diameter, 50 μm in depth (Fig. 1A,B). To test effect of different depth another silicon master wafer with photo patterned structures was used to mold microchamber that were 200 μm in diameter, 10, 30,50 and 100 μm in depth. The entrance for each microchamber was 100 µm in length, 10 µm in width and 5 µm in depth. Polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning, Midland, MI) was mixed with cross-linking agent in a ratio of 10:1 and poured onto wafers. The PDMS was cured overnight at 65°C, after which the PDMS layer was peeled off the wafer and the arrays of wells were cut using a scalpel and inlet, outlet was punched using 0.75 µm puncher. The microfluidic devices were bonded to glass-bottom 6-well plates after treating the bonding surface of PDMS and plate with oxygen plasma. The plates were heated to 70°C for 15 minutes to complete the PDMS-to-glass bonding. Each device consists of 99 chambers, uniformly distributed inside groups of three channels.