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Chapter 2 2.1.4. CDC Biofilm Reactor The CDC Biofilm Reactor (CBR) is a one litre vessel with an effluent spout positioned to hold approximately 350ml of media once assembled. Continuous mixing of the reactors bulk fluid is provided by a magnetised baffled stir bar, which is controlled by a digital stir plate to determine the rates per minute (rpm). The top of the reactor consists of polyethylene top with three inlet ports (media, gas exchange and inoculation port) the top also supports eight independent rods. Each rod accommodates three removable coupons (biofilm growth surfaces) for a total of 24 sampling opportunities. The reactor is connected to two carboys (large autoclavable re-usable plastic bottles). The fresh media carboy and waste media carboy are connected to the CBR using silicone tubing. The culture was circulated into the reactor via sterile tubing which is fed through a peristaltic pump which controls the flow rate through the reactor. An advantage of the CBR is that the mixing and feed rate are optimum (the inflow rate is identical to the outflow rate) which is designed to flush out planktonic cells whilst only leaving the sessile cells in the reactor. This method also ensures that the conditions are as near as possible to identical in all locations within the reactor [171]. The inlet spout is connected to a glass flow-breaker which prevents contamination from the inoculated media from entering the clean media if there is a reflux in the piping. The CBR reactor is set-up to run within the confined area of a fume hood, the tubing is secured in position (above the inlet spout) to the wall using heavy duty duct tape. All components of the CBR are autoclaved before and after use. Images of the CBR and equipment are provided in figures 2.1 and 2.2 on page 63. 2.1.5. Advantages and disadvantages of the CBR The main advantage of the CBR is the ability to examine up to 24 individual coupons simultaneously. This means that research such as investigating biofilm density or protein development at different time points during biofilm development and on different substrata can be assessed simultaneously under uniform conditions using one reactor [121]. It also Page 43
Chapter 2 facilitates testing of different disinfectants at varied concentrations on coupons that carry biofilm which has developed under uniform conditions [117]. Previous authors have also demonstrated that the CBR results in a more dense biofilm and a smaller log 10 reduction after contact with disinfectant agents than other methods such as drip flow reactor or the current standard of assessing the efficacy of a disinfectant against a microbial suspension attached to a surface (EN13697:2001) [117]. The European Committee for Standardisation (CEN-Comité Européen de Normalisation) EN13697:2001 and other current standards are described in greater detail in chapter 4. It is possible that the larger surface area provided by the coupons in the CBR in comparison to other biofilm reactor models such as the microtitre plate based system may be responsible for a more dense biofilm formed on the surface over the allocated period of time [165]. The use of multiple surfaces and provision of a larger surface area exposed to nutrient flow and mixing or shear stress may also be more representative of the conditions found in food processing. However there are also a number of disadvantages to the CBR. Firstly, the CBR is designed for batch-flow and continuous flow provision of sterile media to optimize biofilm development. Therefore, given the size of the reactor and the flow rates chosen (0.5-11ml minute -1 ) a large quantity of media is required to achieve the biofilm development over the continuous flow period. For a flow rate of 1ml minute -1 over 7 days over 14 litres of fresh media is required. The process of replenishing supply of sterile media also allows opportunity for contamination unless strict aseptic techniques are applied. Therefore the CBR is costly and time consuming in comparison to other methods that can use a 10/100 fold less quantity of test chemical per experiment such as the MBEC method described in chapter 1. Performing disinfectant assays using the CBR requires more handling of the substrata and materials such as sterilized tweezers, glass containers, pipettes, syringes and plates whereas the use of a microtitre plate method Page 44
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Salmonella enterica - biofilm forma
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Table of Contents Page Number 1.16.
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Table of Contents Page Number 4.1.2
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Table of Contents Page Number 6.8.4
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List of Abbreviations Table Page Nu
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List of Abbreviations List of Abbre
Page 14 and 15: Summary of Content Summary of Conte
Page 16 and 17: “To show your true ability is alw
Page 18 and 19: Acknowledgements ever met, Fiona th
Page 20 and 21: Acknowledgements This Ph.D. thesis
Page 22 and 23: Chapter 1 1.1. Salmonella Salmonell
Page 24 and 25: Chapter 1 may impose enormous costs
Page 26 and 27: Chapter 1 cracks or penetration of
Page 28 and 29: Chapter 1 detected by agglutination
Page 30 and 31: Chapter 1 become colonized with >10
Page 32 and 33: Chapter 1 antigens [z27],[z45] [1].
Page 34 and 35: Chapter 1 S. Agona has also been im
Page 36 and 37: Chapter 1 isolated from undercooked
Page 38 and 39: Chapter 1 The S. Typhimurium strain
Page 40 and 41: Chapter 1 ecosystem with the use of
Page 42 and 43: Chapter 1 [82]. Previous research h
Page 44 and 45: Chapter 1 to genes involved in flag
Page 46 and 47: Chapter 1 observation has also been
Page 48 and 49: Chapter 1 [119] and flow cell react
Page 50 and 51: Chapter 1 based disinfectant) the s
Page 52 and 53: Chapter 1 formation of two strains
Page 54 and 55: Chapter 1 shield the S. Agona cells
Page 56 and 57: Chapter 1 Key Objectives of this st
Page 58 and 59: Chapter 2 2. Abstract Food-borne pa
Page 60 and 61: Chapter 2 indicated that there may
Page 62 and 63: Chapter 2 taken from industrial set
Page 66 and 67: Chapter 2 can be performed without
Page 68 and 69: Chapter 2 To summarise, previous au
Page 70 and 71: Chapter 2 2.3. Methods for examinin
Page 72 and 73: Chapter 2 and the biofilm formed th
Page 74 and 75: Chapter 2 2.4. Statistical Analysis
Page 76 and 77: Chapter 2 Table 2.1: Strain charact
Page 78 and 79: Chapter 2 XII. The gas port and med
Page 80 and 81: Chapter 2 II. Primary fixative cons
Page 82 and 83: Chapter 2 IX. Once the conditions w
Page 84 and 85: Chapter 2 Figure 2.1: Image of CBR
Page 86 and 87: Chapter 2 2.6. Results 2.6.1. Asses
Page 88 and 89: Chapter 2 2.6.3. SEM Analysis of su
Page 90 and 91: Chapter 2 Figure 2.6: Complete remo
Page 92 and 93: Chapter 2 2.6.5. Mean log 10 densit
Page 94 and 95: Chapter 2 2.6.6. Mean log 10 densit
Page 96 and 97: Chapter 2 Table 2.5: Mean log 10 de
Page 98 and 99: Chapter 2 The results displayed in
Page 100 and 101: Chapter 2 Table 2.7: Difference bet
Page 102 and 103: Chapter 2 Table 2.8: The mean log 1
Page 104 and 105: Chapter 2 Figure 2.7: Graph of the
Page 106 and 107: Chapter 2 also used “high” soni
Page 108 and 109: Chapter 2 2.8. Summary All 13 strai
Page 110 and 111: Chapter 3 3. Abstract It has been e
Page 112 and 113: Chapter 3 classified as persistent
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Chapter 3 was plated onto TSA for e
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Chapter 3 Figure 3.1: The mean log
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Chapter 3 Figure 3.3: SEM image of
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Chapter 3 Table 3.2: Difference bet
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Chapter 3 3.4.3. Intra-serovar vari
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Chapter 3 3.4.4. Strain variation i
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Chapter 3 3.4.5. The density of bio
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Chapter 3 3.5. Discussion As illust
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Chapter 3 As discussed in section 3
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Chapter 3 increased biofilm appeare
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Chapter 4 Examining the efficacy of
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Chapter 4 through laboratory based
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Chapter 4 acid and quaternary ammon
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Chapter 4 the surface the temperatu
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Chapter 4 availability of multiple
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Chapter 4 peracetic acid (100, 200,
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Chapter 4 As discussed previously,
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Chapter 4 Dey/Engley (Difco) neutra
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Chapter 4 sterilisation was achieve
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Chapter 4 IX. Aliquots of 100µl of
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Chapter 4 XV. XVI. XVII. XVIII. XIX
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Chapter 4 4.3. Results 4.3.1. Suspe
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Chapter 4 Table 4.3: Mean log 10 de
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Chapter 4 Benzalkonium chloride was
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Chapter 4 4.4. Discussion Suspensio
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Chapter 4 effective at eliminating
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Chapter 4 Surprisingly, Nguyen et a
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Chapter 4 though a contact time of
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Chapter 4 provide a more standardis
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Chapter 4 hours instead of the stan
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Chapter 5 5. Abstract Examining bio
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Chapter 5 dry and rough (bdar) morp
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Chapter 5 absence of curli and cell
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Chapter 5 studies such as the micro
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Chapter 5 discussed in chapter 4. T
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Chapter 5 5.2. Methods 5.2.1. Colon
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Chapter 5 XIII. XIV. XV. XVI. XVII.
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Chapter 5 XVI. XVII. XVIII. XIX. XX
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Chapter 5 5.3.2. Repeatability of m
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Chapter 5 Figure 5.2: Stained biofi
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Chapter 5 was formed at room temper
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Chapter 5 Table 5.3: The density of
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Chapter 5 5.3.4. The density of bio
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Chapter 5 Table 5.6: The difference
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Chapter 5 Table 5.7: The difference
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Chapter 5 Table 5.8: Summary of ass
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Chapter 5 interpretation therefore
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Chapter 5 measures of S. enterica b
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Chapter 5 cells peaks [165, 191]. D
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Chapter 5 5.5. Limitations of the s
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Chapter 6 Discussion of S. enterica
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Chapter 6 However, a number of auth
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Chapter 6 It is of interest to exam
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Chapter 6 were similar at 48 hours.
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Chapter 6 attributable to a small n
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Chapter 6 chosen as an appropriate
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Chapter 6 undetermined. However, th
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Chapter 6 reason for this may be du
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Chapter 6 Moreover, based on the re
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Chapter 6 responsible for increased
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Chapter 6 not fully capture the ran
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Chapter 6 However, the extent of bi
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Chapter 6 6.10. Conclusions and rec
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Chapter 6 studies may solve some of
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Bibliography Bibliography Page 221
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Bibliography 12. T. Takata, J.L., H
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Bibliography 29. Barker, J., M. Nae
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Bibliography 47. Threlfall, E.J., H
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Bibliography 65. CDC. Investigation
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Bibliography 83. Ledeboer, N., Frye
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Bibliography 100. Chia, T., Goulter
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Bibliography 117. Buckingham-Meyer,
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Bibliography 133. European Committe
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Bibliography Supernatant of a Hafni
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Bibliography 165. Díez-García, M.
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Bibliography cultivation of Staphyl
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Bibliography Compounds. 2012. Appli
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Bibliography 215. Buck, et al., A q
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Bibliography Canadian Journal of Ps
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Appendix 1 Appendix 1— List of Re
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Appendix 1 Appendix 1 - List of Equ
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Appendix 2 Colour index High outlie
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Appendix 3 Appendix 3-Figure 2: PFG
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Appendix 4 Poster Presentation Envi
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