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Chapter 2
IX.
Once the conditions were set and the microorganisms were visualised
and in focus the images were taken and saved.
2.5.3. AFM Analysis
I. AFM analysis was performed to determine the roughness of each of the
five surfaces.
II. The 5 surfaces (unused) were cleaned with a micro-90 detergent
solution to remove any debris from the surface.
III. The laser in the AFM was aligned after which the photo-detector was
adjusted using the settings on the screen.
IV. The sample was loaded into the chamber and brought into focus.
o Initial Scan Size: 1μm, Scan Rate: 1 Hz, Scan Angle: 0 X and Y
Offsets: 0
V. The tapping mode was commenced (cantilever tune) and the probe
was engaged.
VI. The surface roughness was measured through the use of the tapping
mode of AFM on three separate measurements of each surface which
are fed back to the computer programme attached.
VII. The images and measurements were saved after each reading.
VIII. The mean surface roughness (nanoscale roughness - Ra) was calculated
by determining the roughness of the surface spanning the diameter of
the coupon. This assessment was repeated in triplicate.
2.5.4. Validation of Removal Techniques
In order to optimize biofilm removal the use of alternative sonication
conditions were investigated.
I. Biofilm was developed using P. aeruginosa ATCC 27852 on the five
surfaces using the method described in section 2.5.1. After biofilm
formation for 48 hours the surfaces were sonicated at 50 kHz in 10ml
PBS. The quantity of viable cells was determined using the plate count
method as previously described.
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