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Chapter 2
2.3. Methods for examining biofilm density and substrata
2.3.1. Atomic Force Microscopy (AFM)
Atomic force microscopy uses a scanning cantilever or probe that taps the
surface of a specimen on contact. The probe then gently scans the surface
and based on tapping of the surface encountered. A laser beam is aimed at
the outer area of the probe. The electrons emitted from the laser are
deflected back to a photo-detector which deciphers the distance of the
probe from the surface at each scanned point to measure surface
roughness of the overall sample [172] .
2.3.2. Scanning Electron Microscopy (SEM)
SEM is a widely used method for examining biofilm on surfaces [114, 122,
173-174]. SEM has previously been used and standardised by Williams and
colleagues to confirm biofilm growth of Staphylococcus epidermidis
established using the CBR [174]. Gilmore et al. also used SEM analysis to
confirm dense biofilm formation by Proteus mirabilis using the CBR [122].
The basic principle of SEM is that a beam of electrons are emitted from an
electrode gun and accelerated at high voltage (e.g. 20 kiloVolts - kV) onto a
specimen. Once the beam of electrons hits the specimen, the electrons
rebound off the surface and are collected through the secondary electron
imaging (SEI) collector [175]. The SEI collector deciphers the electrons
collected and coverts them to digital analogs or signals. The signals are
then converted to grey-scale pixilation using computer programming [175].
In order to optimize conditions for SEM visualization, the specimens are
fixed (primary and secondary fixatives) which preserve the cellular details
of the biofilm [174]. After fixation the specimens are dehydrated in a series
of ethanol to remove excess water from the sample [175]. Any water
remaining may destroy the sample if overheated in the SEM chamber. The
ethanol series is followed by the addition of a critical point dryer to
eliminate surface tension from liquids on the surface of the specimen.
Hexamethyldisilizane (HMDS) is now recognised as a safer option for
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