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.