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Chapter 5
curli and cellulose may be subject to environmental stress, such as low
nutrient concentration.
5.1.3. Biofilm formation assessed using the microtitre plate method
The microtitre plate system is commonly used for the assessment of
biofilm formation [32, 56, 82, 86, 96, 227]. This high throughput method is
reported as rapid and reproducible for assessing the biofilm forming ability
of a number of test organisms simultaneously. The multi-well structure
allows multiple strains, and or multiple concentrations of nutrients or
biocides to be assessed within one plate. However it is unclear to what
extent results based on microtitre tray may be generalised to biofilm
formed under other conditions [73, 102].
As described in chapter 1, the microtitre plate method is a static-flow
biofilm model that allows biofilm development on the base of the multiwell
microtitre plate made of plastic such as polystyrene. After incubation
for a defined period of time (generally 24-48 hours), the inoculated media
is extracted and the wells of the plate are stained with a bacterial staining
agent such as crystal violet. Unbound crystal violet is removed through
washing. The assumption is that the quantity of bound crystal violet is
related to the density of biofilm formed on the microtitre plate. The bound
stain is then resolubilized using an alcohol: acetone solution [56, 228] or
glacial acetic acid solution [112, 165]. The optical density of the remaining
crystal violet is then measured to determine the amount of biomass or
biofilm in the wells of the plate.
Numerous authors have reported the use of Salmonella colony morphology
on CR agar as a preliminary indication of strain biofilm forming capability
before performing biofilm development studies using more conventional
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