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Chapter 5<br />

cells peaks [165, 191]. Diez-Garcia et al. suggested that although the<br />

number <strong>of</strong> planktonic cells may increase over time, the number <strong>of</strong> cells<br />

attached may not increase accordingly and be subject to the surface area<br />

provided [165]. However when the strains were incubated at 37°C bi<strong>of</strong>ilm<br />

produced by ten strains increased in density from 48 hours and 168 hours<br />

(although in most cases the increase was not significant p>0.05). The<br />

changes in density <strong>of</strong> bi<strong>of</strong>ilm over time, may suggest that the increased<br />

temperature may support bacterial bi<strong>of</strong>ilm formation over the extended<br />

period <strong>of</strong> time. Similarly Vestby et al. found that incubation <strong>of</strong> 9 strains <strong>of</strong><br />

S. enterica over 2-, 3- and 4- days at room temperature 20±1°C in a<br />

microtitre plate based system only resulted in an increase in the density <strong>of</strong><br />

bi<strong>of</strong>ilm formed only for S. Senftenberg strains, but no increase in the<br />

density <strong>of</strong> bi<strong>of</strong>ilm formed by the S. Agona and S. Typhimurium strains [56].<br />

The results <strong>of</strong> this chapter contradict what was reported by Wong et al.<br />

using a MBEC method [149]. As previously discussed in chapter 4, Wong et<br />

al. also indicated that Salmonella bi<strong>of</strong>ilm density did not increase over an<br />

extended period <strong>of</strong> time (7 days) at 37°C [149]. The difference in bi<strong>of</strong>ilm<br />

development and measurement methods may have been responsible for<br />

the difference in findings.<br />

It is also necessary to note that the amount <strong>of</strong> crystal violet dye bound is<br />

dependent on the quantity and composition <strong>of</strong> the extracellular polymeric<br />

substances (EPS) present in the bi<strong>of</strong>ilm. Therefore, it is possible that the<br />

change in optical density measurements <strong>of</strong> bi<strong>of</strong>ilm attached after 48 and<br />

168 hours may be a reflection on this change in EPS and not relating<br />

exclusively to a change in the number <strong>of</strong> viable cells in the bi<strong>of</strong>ilm. This<br />

may impact on correlation <strong>of</strong> bi<strong>of</strong>ilm density assessed by viable counts.<br />

The results <strong>of</strong> this chapter indicated that neither sodium hydroxide (1M)<br />

sodium hypochlorite (500mg/L) nor benzalkonium chloride (0.02%) were<br />

Page 189

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