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Chapter 2 and the biofilm formed therefore comparison of biofilm density across five surfaces would be difficult. Moreover, despite the introduction of computer based programmes for measuring biofilm volume based on confocal images limitations such as miscalculation of density still remain unresolved [179]. Therefore CLSM and SEM still remain largely qualitative methods for assessing biofilm density. Grid counting of SEM images involves dividing the surface area into small grids within which the cells are then counted to allow for an estimate of the total population. This method allows enumeration of all cells attached to a surface, therefore eliminating the need for sonication and culturing the bacteria and plate counting. The main disincentive to using grid counting is that it does not give any indication of the volume of live cells contained in the total biomass. Moreover, based on the dense structure of the biofilm cells and the presence of an EPS outer layer, it is not always possible to differentiate single cells for enumeration as depicted in the SEM images provided in this thesis. In light of the advantages and disadvantages associated with the use of microscopy, enumeration via the plate count method remains the most frequently used method for biofilm measurement [68]. The plate count method involves removal of the biofilm (usually performed via scraping, sonicating or vortexing) followed by re-suspension and disaggregation of the biofilm in a liquid before spreading onto solid media and cultured over night at optimum temperature. 2.3.4. Sonication as a method of removal for enumeration Ultrasonication (sonication) was chosen as an appropriate method for biofilm removal in this thesis. Under the optimum conditions, sonication should remove biofilm cells from a surface without disrupting or damaging the cells. The use of sonication has previously been demonstrated to be an Page 51
Chapter 2 effective method for removal of biofilm cells from surfaces [72, 114, 118, 120, 122, 180-182]. The surfaces under examination are placed in sealed containers of sterile liquid. During the procedure the ultrasonic vibration leads to the formation of small bubbles in the liquid containing the surfaces. As a result the bubbles interact and hit off the surface resulting in the biofilm becoming dislodged from the surface without disrupting the integrity of the biofilm cells. The placement of the water-bath within the sonication unit allows the frequency of the sound to travel at a faster pace. However the challenge of sonication is to optimize removal of the biofilm matrix while simultaneously preventing cell damage [105]. The sonication water-bath controls the intensity of ultrasonic waves emitted per second (kiloHetz - kHz) by increasing the pitch and the frequency of the resonance sound. The degas function on the water-bath reduces the volume of gas naturally contained in the liquid solution before the process begins. The temperature of the water-bath can also be optimized by use of the heating block built into the bath. Room temperature can be maintained using a thermometer positioned in the bath with the addition of warm water or ice to alter the temperature. Monson and colleagues highlighted that that in order to sufficiently remove biofilm cells from a surface whilst detecting and preventing cell damage quality checks must be performed to optimize conditions for the organism and surfaces tested [181]. Quality checks can be verified quantitatively by performing viable plate counts on the organism when in planktonic form (normal bacterial suspension) and also by comparing the sonication method for removal of bacteria to other methods. Other methods used for biofilm removal for cell enumeration include scraping the surface to dislodge the biofilm matrix [98] or swabbing [71]. However all removal methods should also be verified preferable through microscopy to confirm level of removal from the surface [183]. Page 52
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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
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 64 and 65: Chapter 2 2.1.4. CDC Biofilm Reacto
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 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
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
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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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