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Chapter 4 the surface the temperature is allowed to equilibrate to room temperature after which a small aliquot of the test disinfectant liquid (100µl) is placed on the face of the surface for 5 minutes. The surfaces are then placed in 10ml of neutralizing agent with 5g of glass beads. The suspensions are shaken for 1 minute at 150 rpm and then used for serial dilutions (10 -3 — 10 -6 ) followed by performing the pour plate technique with tryptone soy agar. Plates are incubated at 37°C for 24 hours. However the current CEN method EN13697:2001 [133] does not address the efficacy of disinfectants against biofilm. Due to the short contact time there is little opportunity for cells to initiate biofilm formation before disinfectant is applied. Therefore there are no guidelines to demonstrate the efficacy of disinfectant products against an established biofilm. 4.1.3. The ASTM standards for evaluating disinfectant efficacy The American Society for Testing and Materials (ASTM) provides guidance and protocols for assessing the efficacy of disinfectants against established bacterial biofilms. There are currently two active standards (published in 2012) in use for examining the efficacy of disinfectants against an established biofilm. The standard designated E2871-12 describes the method for evaluating efficacy of disinfectants against a P. aeruginosa biofilm grown in the CDC biofilm reactor (CBR) using a single tube method [125]. The method uses a similar procedure for cultivating a biofilm on surfaces as outlined in this thesis. The previously published ASTM method for growing the biofilm using this reactor (E2562) has also been published elsewhere [72, 204]. The method used for assessing the disinfectants differs from the method described in this thesis in the volume of disinfectants and neutralizing agents used. The standard protocol (E2871-12)[125] does not outline what Page 119
Chapter 4 contact time should be used with the disinfectants. Therefore this component should be adjusted based on the specification of the particular disinfectant in use. The standard designated E2799-12 outlines a method of testing disinfectant efficacy against an established P. aeruginosa biofilm using the MBEC assay [126]. The MBEC assay consists of a 96 well microtiter plate with appendages (pegs) attached to the lid. The biofilm is grown on the surface of the pegs in a closed batch system and each well in the plate may contain different test strains or concentration of nutrients. After the biofilm is established, the lid (with pegs attached) can be attached to a new plate with disinfectant agents contained in each well. After a specific contact time, the pegs can be broken off the top of the lid and placed in broth. The broth can be used for serial dilutions and enumeration via the plate count method. The biofilm development time using this method is 24 hours which may be too short to test the disinfectant efficacy against a developed biofilm as depending on the conditions within the system. The time allowed for biofilm development was also discussed previously in more detail in chapter 3. The sonication time was also longer (30±5minutes) than recommended using other systems including the CBR, which may reflect the organisms under examination. There are also no recommendations for specific contact times with disinfectants using the MBEC method. 4.1.4. The influence of biofilm models on the efficacy of disinfectant agents As discussed, there are a number of different models for examining the efficacy of disinfectant agents against bacterial suspensions, surface adherent bacterial cells and cells associated with a biofilm matrix. The Page 120
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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
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Summary of Content Summary of Conte
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“To show your true ability is alw
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Acknowledgements ever met, Fiona th
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Acknowledgements This Ph.D. thesis
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Chapter 1 1.1. Salmonella Salmonell
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Chapter 1 may impose enormous costs
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Chapter 1 cracks or penetration of
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Chapter 1 detected by agglutination
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Chapter 1 become colonized with >10
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Chapter 1 antigens [z27],[z45] [1].
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Chapter 1 S. Agona has also been im
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Chapter 1 isolated from undercooked
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Chapter 1 The S. Typhimurium strain
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Chapter 1 ecosystem with the use of
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Chapter 1 [82]. Previous research h
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Chapter 1 to genes involved in flag
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Chapter 1 observation has also been
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Chapter 1 [119] and flow cell react
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Chapter 1 based disinfectant) the s
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Chapter 1 formation of two strains
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Chapter 1 shield the S. Agona cells
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Chapter 1 Key Objectives of this st
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Chapter 2 2. Abstract Food-borne pa
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Chapter 2 indicated that there may
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Chapter 2 taken from industrial set
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Chapter 2 2.1.4. CDC Biofilm Reacto
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Chapter 2 can be performed without
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Chapter 2 To summarise, previous au
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Chapter 2 2.3. Methods for examinin
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Chapter 2 and the biofilm formed th
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Chapter 2 2.4. Statistical Analysis
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Chapter 2 Table 2.1: Strain charact
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Chapter 2 XII. The gas port and med
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Chapter 2 II. Primary fixative cons
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Chapter 2 IX. Once the conditions w
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Chapter 2 Figure 2.1: Image of CBR
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Chapter 2 2.6. Results 2.6.1. Asses
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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
Page 114 and 115: Chapter 3 was plated onto TSA for e
Page 116 and 117: Chapter 3 Figure 3.1: The mean log
Page 118 and 119: Chapter 3 Figure 3.3: SEM image of
Page 120 and 121: Chapter 3 Table 3.2: Difference bet
Page 122 and 123: Chapter 3 3.4.3. Intra-serovar vari
Page 124 and 125: Chapter 3 3.4.4. Strain variation i
Page 126 and 127: Chapter 3 3.4.5. The density of bio
Page 128 and 129: Chapter 3 3.5. Discussion As illust
Page 130 and 131: Chapter 3 As discussed in section 3
Page 132 and 133: Chapter 3 increased biofilm appeare
Page 134 and 135: Chapter 4 Examining the efficacy of
Page 136 and 137: Chapter 4 through laboratory based
Page 138 and 139: Chapter 4 acid and quaternary ammon
Page 142 and 143: Chapter 4 availability of multiple
Page 144 and 145: Chapter 4 peracetic acid (100, 200,
Page 146 and 147: Chapter 4 As discussed previously,
Page 148 and 149: Chapter 4 Dey/Engley (Difco) neutra
Page 150 and 151: Chapter 4 sterilisation was achieve
Page 152 and 153: Chapter 4 IX. Aliquots of 100µl of
Page 154 and 155: Chapter 4 XV. XVI. XVII. XVIII. XIX
Page 156 and 157: Chapter 4 4.3. Results 4.3.1. Suspe
Page 158 and 159: Chapter 4 Table 4.3: Mean log 10 de
Page 160 and 161: Chapter 4 Benzalkonium chloride was
Page 162 and 163: Chapter 4 4.4. Discussion Suspensio
Page 164 and 165: Chapter 4 effective at eliminating
Page 166 and 167: Chapter 4 Surprisingly, Nguyen et a
Page 168 and 169: Chapter 4 though a contact time of
Page 170 and 171: Chapter 4 provide a more standardis
Page 172 and 173: Chapter 4 hours instead of the stan
Page 174 and 175: Chapter 5 5. Abstract Examining bio
Page 176 and 177: Chapter 5 dry and rough (bdar) morp
Page 178 and 179: Chapter 5 absence of curli and cell
Page 180 and 181: Chapter 5 studies such as the micro
Page 182 and 183: Chapter 5 discussed in chapter 4. T
Page 184 and 185: Chapter 5 5.2. Methods 5.2.1. Colon
Page 186 and 187: Chapter 5 XIII. XIV. XV. XVI. XVII.
Page 188 and 189: 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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