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Chapter 2
and the biofilm formed therefore comparison of biofilm density across five
surfaces would be difficult. Moreover, despite the introduction of
computer based programmes for measuring biofilm volume based on
confocal images limitations such as miscalculation of density still remain
unresolved [179]. Therefore CLSM and SEM still remain largely qualitative
methods for assessing biofilm density.
Grid counting of SEM images involves dividing the surface area into small
grids within which the cells are then counted to allow for an estimate of
the total population. This method allows enumeration of all cells attached
to a surface, therefore eliminating the need for sonication and culturing
the bacteria and plate counting. The main disincentive to using grid
counting is that it does not give any indication of the volume of live cells
contained in the total biomass. Moreover, based on the dense structure of
the biofilm cells and the presence of an EPS outer layer, it is not always
possible to differentiate single cells for enumeration as depicted in the SEM
images provided in this thesis.
In light of the advantages and disadvantages associated with the use of
microscopy, enumeration via the plate count method remains the most
frequently used method for biofilm measurement [68]. The plate count
method involves removal of the biofilm (usually performed via scraping,
sonicating or vortexing) followed by re-suspension and disaggregation of
the biofilm in a liquid before spreading onto solid media and cultured over
night at optimum temperature.
2.3.4. Sonication as a method of removal for enumeration
Ultrasonication (sonication) was chosen as an appropriate method for
biofilm removal in this thesis. Under the optimum conditions, sonication
should remove biofilm cells from a surface without disrupting or damaging
the cells. The use of sonication has previously been demonstrated to be an
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