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Chapter 2
XII. The gas port and media inlets and waste port were sealed and the
device was autoclaved prior to every use.
XIII. Any dislodged coupons were aseptically re-inserted into holders
and the device was filled with 350ml of TSB (30g/L).
XIV. 10 litres of diluted TSB (10g/L) were autoclaved in the carboy.
XV. All tubing was connected to fit through carboy cap, fluid break and
reactor attachment were assembled and autoclaved.
XVI. The reactor system was assembled (see figure 2.2).
XVII. The reactors were placed on the magnetic stirrers which rotate at
160rpm, maintained at room temperature.
XVIII. The flow rate of 1ml/minutes was confirmed after each assembly
before inoculating reactor.
XIX. The reactors were inoculated with overnight suspension of the test
strain.
XX. The density of the inoculum (CFU/ml) was confirmed using spread
plate onto TSA agar.
XXI. The reactor was run on batch phase for 24 hours.
XXII. After 24 hours the reactor system was operated under continuous
flow phase for an additional 24 hours.
XXIII. The reactor system was disassembled.
XXIV. All tubing and carboys were covered and autoclaved immediately.
XXV. Using a sterile forceps and set screw the coupons were extracted
from the reactor and placed within the confines of a bio assay tray
in the safely cabinet.
XXVI. Using a sterile syringe the coupons were washed with 20ml sterile
H 2 0 to remove any planktonic cells from the surface and placed in
the wells of a sterile 24-well plate.
XXVII. During each run care was taken to ensure the face of the coupon
directly exposed to the shear stress of the reactor was facing
upwards during the handling and sonication of coupons
(particularly important for steel, polycarbonate and glass as both
sides are identical).
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