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Chapter 2
XXVIII. Three sets of coupons (a set containing one of each of the 5 coupon
types) were placed in individual capped universals with 10ml of
sterile Phosphate Buffered Saline (PBS) and sonicated for 7 minutes
at 20 kHz at room temperature
XXIX. The fourth set of coupons were placed in a 24 well microtiter plate
and used for SEM analysis (see section 2.1.1).
XXX. After sonication the coupons were aseptically removed from the
universals using a sterile forceps.
XXXI. Three sets of coupons were vortexed for 30 seconds (within the
capped universals) to break down biofilm clumps and serial
dilutions were made of the PBS solution.
XXXII. Serial dilutions of 1x 10 -4 and 1x10 -5 were made.
XXXIII. 100µl of the serial dilution was placed onto TSA and spread evenly
over the surface of the plate using a spreader.
XXXIV. The TSA plates were incubated inverted for 24 hours at 37°C.
XXXV. A suspension from the reactor was plated onto XLD and CLED
following serial dilution to confirm the broth only contained the test
organism.
XXXVI. The number of colonies formed on each plate (CFU/plate) was
counted after 24 hour incubation.
XXXVII. The CFU/plate were converted to Mean Log 10 Density values for
statistical analysis using the formula adapted by Goeres et al. 2005
[72].
XXXVIII. Mean Log 10 density (CFU/coupon) = log 10 (mean CFU per plate) +
log 10 (vol. sonicated into) + log 10 (dilution factor) - log 10 (vol. plated)
- log 10 (surface area of the entire coupon).
2.5.2. Scanning Electron Microscopy
2.5.2.1. Primary and Secondary
I. The coupons were immersed in primary fixative overnight (12 hours).
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