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Chapter 2
2.5. Methods
2.5.1. 48 hour biofilm development method
I. All experiments were performed in accordance with the current
good laboratory practices and health and safety requirements
outlined by National University of Ireland, Galway.
II.
III.
IV.
The test organisms were stored at -20°C on glycerol protect beads
between experiments.
Before each experiment the test strain was defrosted on the bench
and streaked on Tryptic Soy Agar (TSA) and Xylose Lysine
Deoxychocolate (XLD) plates which were incubated for 24 hours at
37°C.Isolated colonies were picked off the TSA plate and made to a
0.5 McFarland suspension (~1.5x10 8 CFU/ml) in sterile saline.
1ml of the suspension was added to 9ml of sterile Tryptic Soy Broth
(TSB) and incubated in shaking incubator for 24 hours at 37°C.
V. Twenty coupons (four of each of the five coupon types - glass, steel,
polycarbonate, tile and concrete) were cleaned for use in each
reactor run.
VI. Coupons were sonicated at room temperature for 30 minutes in a
detergent solution (2% Micro-90).
VII. Coupons were then rinsed and sonicated for an additional 30
minutes in reagent-grade water.
VIII. Using a 24-well plate the coupons were immersed in 1ml of 100%
ethanol for 2 hours, ethanol was chosen as opposed to 2M HCl as
this may chemical may result in degradation of surfaces i.e. rusting
of stainless steel.
IX. The coupons were visually inspected to ensure there was no
degradation prior to use.
X. The reactor was cleaned with micro-90 and ethanol prior to use.
XI. The coupons were fixed into the reactor rods using a sterile forceps
and set-screw, with the test area of the coupon (face) was placed
inward exposed to the sheer force provided by the baffler rotator
(see Figure 2.1).
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