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Chapter 5
measurement of biofilm formed in replicate wells for the same strains. On
examining the full set of observations for each strain (all provided in
Appendix 2) it was clear that for a number of strains there were a number
of clear outlier OD 590 measurements much greater than the other OD 590
measurements for the same strains from replicate wells. These outliers
give an elevated mean for those strains. The inconsistency in repeated
measurements for a given strain is a major limitation of the method as
applied here.
The outliers are most likely representative of the uneven biofilm formation
and incomplete resuspension of crystal violet from the surface of the
microtitre plate when the optical density was measured. This finding is also
illustrated in Figures 5.2-5.4. An uneven biofilm matrix or clumping of
biofilm formed in a microtitre plate system has also been demonstrated
elsewhere using confocal microscopy by Bridier et al. [178]. The work
presented by Bridier et al. examined biofilm formation of 60 bacterial
strains following fluorescent staining of the biofilm and construction of 3
dimensional images based on stacked confocal micrographs. The research
demonstrated the uneven biofilm formation by S. Agona, S. Enteritidis and
S. Typhimurium strains on the surface of the microtitre plate. This uneven
distribution of biofilm formation was also visualised for other organisms.
Moreover Bridier et al. indicated that of the 10 S. enterica serovars, S.
Agona biofilm formed a distinctive macro-colony structure, which may also
contribute to the clumping appearance when examined. Bridier et al. also
demonstrated that to a lesser extent all S. Enteritidis and S. Typhimurium
strains also formed an uneven biofilm [178].
The large range of optical density readings for each individual strain may
be a reason why previous authors have chosen to use the standard error
(dividend of standard deviation) to display the differences in repeated
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