View/Open - ARAN - National University of Ireland, Galway

Chapter 3
formation on multiple surfaces by a number of strains over short (≤48
hours) and long periods of time (≥72 hours). In previously published work
in this area authors have examined biofilm formation of a single strain over
an extended period of time [102, 106, 148-149, 188] or examined the use
of two strains [71] or four strains of the same serovar (S. Enteritidis) [67]
after biofilm formation after a specific duration of time. This is a significant
gap in knowledge as it may be that there are important differences
between early biofilm and late biofilm. It is likely that late biofilm is more
relevant to the situation that can arise in food processing environments
when a specific strain is repeatedly isolated over a period of months or
years.
3.2. Method – 168 hour biofilm
This section describes investigation of biofilm density when biofilm was
established over an extended period of time (168 hours / 7days) and a
comparison of density at 48 and 168 hours. Essentially, the method for the
168 hour biofilm was similar to the standardised method (method
described in chapter 2) except the period of continuous flow was extended
from 24 hours to 144 hours. The strains studied were an S. Agona from the
outbreak (S09-0494), S. Agona reference strain (SL483), S. Typhimurium
(S09-0419), S. Typhimurium reference strains SL1344 and a S. Enteritidis
(S09-0717). The characteristics of these strains were previously described
in Table 2.1 in chapter 2.
3.3. Validation of techniques
Following SEM analysis (see Figure 3.1 and 3.2) it became clear that the
entire biofilm was not removed from the surface using the conditions (20
kHz for 7 minutes) previously optimized in chapter 2 (see section 2.3 in
chapter 2). In order to improve removal alternative sonication conditions
were examined. Following the conditions described in chapter 2, sonication
was performed at 20 kHz for 7, 10 and 14 minutes. The suspended biofilm
Page 92