After inoculation with 5 mLs obtained from the overnight culture of each isolate, the medium was incubated under static conditions (no shaking) at temperature 30 °C and pH 7.
Euglena gracilis (strain Z) was obtained from S.H. Hutner, Haskins Laboratory, Pace University, New York, NY, USA and maintained on Cramer-Myers (CM) medium [ 21] under static conditions at 27°C and with permanent lighting (16.4 W/m).
After two weeks, both the fungi were grown separately in 250 ml flasks containing 100 ml of Potato dextrose broth (PDB medium) under dark and static condition at room temperature for three weeks.
It was cultured in MRS medium for 12 14 h under static conditions at 35 °C in an incubator before being used as seed inoculum.
Second (days 8 14 or longer) coverslips were placed either: (i) in MCmB chambers, medium was renewed and cells were submitted to a flow rate of 250 500 μL/min, without further medium change (dynamic conditions), or (ii) in a new petri dish, medium was renewed and culture was continued under static conditions without further medium change (static conditions).
In order to modulate the properties of BC and expand its use as substrate for tissue engineering mainly in the form of biomembranes, glucose or dextrin were added into a BC fermentation mannitol-based medium (BCGl and BCDe, respectively) under static culture conditions.
First (days 1 7), coverslips were placed in petri dishes under static conditions without medium change.
For the induction of early EPCs, the ASCs were seeding onto fibronectin (30 mg/mL, Sigma) coated glass slide to reach 80 90% confluence and then cultured in endothelial cell growth medium (EGM, Lonza) for 3 days under static condition.
Bacteria were grown under static condition, either in a medium with very low sugar concentration (C medium, see Materials and Methods) or in the same medium supplemented with 30 mM glucose, and surface-attached bacteria were quantified after 12 hours.
