Salmonella surface characterization and adhesion to food and other surfaces Samantha Begnoche...
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Transcript of Salmonella surface characterization and adhesion to food and other surfaces Samantha Begnoche...
Salmonella surface characterization and adhesion to food and other surfaces
Samantha BegnocheAdvisor: Dr. Sharon WalkerBioengineering Research Institute for Technical ExcellenceAugust 20, 2009
Background From 1988 – 1995 reported cases varied
between 40,000 and 50,000 Previous projects limited to genotypic nature Objective: Understand surface chemistries and
transport kinetics of Salmonella to keep future outbreaks to a minimum.
Some Facts Still, approximately 40,000 cases of Salmonella
poisoning reported annually ~400 deaths annually are result of Salmonella Children, elderly, and immune diseased are
most vulnerable
Goals Short-term work:
Characterize strains’ surface chemistries
Three strains focused on: SGSC 4910 (newport) SGSC 2377 (enteritidis) SA 5983 (typhimurium)
S. typhimurium respresentationProvided by Berat Haznedaroglu
Methods Bacteria Preparation- Inoculate bacteria night before- Preculture in LB media for specified time- Harvest – centrifuge, wash in KCl twice,
resuspend for stock solution
Incubation CentrifugeInoculation
Characterization methods Viability
Mix stock solution and dye. Vortex and wait 15 minutes. Count live (green) and dead (red).
Size measurement Measure length and width using images taken with
phase contrast microscope. Calculate spherical radii.
Characterization methods Hydrophobicity measurement
Using MATH test with n-dodecane, measure the percentage of cells that choose the hydrocarbon versus the electrolyte condition. <40% is hydrophilic
Electrophoretic mobility measurement Using ZetaPALS, calculate the electrophoretic
mobility and zeta potential from a solution diluted to an optical density of .200 to .225 to result in a concentration of about 107 cells.
Parallel plate flow chamber methods Parallel plate flow chamber
Take picture of cells flowing through at 20 second intervals for set amount of time
Number of cells depositing on the surface is counted and plotted as a function of time
Adhesion rate is calculated from slope
Fluorescent light
Fluorescent light
Parallel plate flow chamber system
Viability Results SA 5983:
1 mM KCl: 81.5 ± 1.7% 10 mM KCl: 87.4 ± 2.1% 100 mM KCl: 89.7 ± 11.8%
Size Measurement Results SA 5983: 0.326 ± 0.113 µm SGSC 4910: 0.368 ± 0.145 µm SGSC 2377: 0.317 ± 0.067 µm
Hydrophobicity ResultsHydrophobicity of SGSC 4910 at different ionic strength
0
10
20
30
40
50
60
70
80
90
100
1 10 100
Ionic Strength (mM)
% P
arti
tio
nin
g
SGSC 4910
GFP SGSC 4910
Hydrophobicity ResultsHydrophobicity of SGSC 2377 at different ionic strengths
0
10
20
30
40
50
60
70
80
90
100
1 10 100Ionic Strength (mM)
% P
art
itio
nin
g
SGSC 2377
GFP SGSC 2377
Hydrophobicity ResultsHydrobicity of SA 5983 at different ionic strength
0
10
20
30
40
50
60
70
80
90
100
1 10 100Ionic Strength (mM)
% P
art
itio
nin
g
Sa 5983
GFP Sa 5983
Electrophoretic Mobility ResultsElectrophoretic Mobility by Ionic Strength
SGSC 4910
-2.5 -2 -1.5 -1 -0.5 0
1
10
30
100
Ion
ic S
tren
gth
(m
M)
Electrophoretic Mobility
GFP SGSC 4910
SGSC 4910
Electrophoretic Mobility ResultsElectrophoretic Mobility by Ionic Strength
SGSC 2377
-2.5 -2 -1.5 -1 -0.5 0
1
10
30
100
Ion
ic S
tre
ng
th (
mM
)
Electrophoretic Mobility
GFP SGSC 2377
SGSC 2377
Electrophoretic Mobility ResultsElectrophoretic Mobility by Ionic Strength
SA 5983
-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5
1
10
30
100
Ion
ic S
tre
ng
th (
mM
)
Electrophoretic Mobility
Gfp Sa 5983
Sa 5983
Parallel Plate Results SA 5983 exhibits no attachment for 1 mM KCl
solution. Amount of deposition is expected to increase
with increasing ionic strength Also expected to increase at lower rates
Parallel Plate Flow Chamber ResultsFor 100 mM KCl 1.5 ml/min 2.0 ml/min
Length (um) 211 211
Width (um) 168 168
Microscope viewing area (m2) 3.5448E-08 3.5448E-08
average particle diameter (nm) 338 338
No of particles in inflow (particles/L) 500000000 500000000
Slope (particles/sec) 0.118 0.0487
Flux Jz (particles/sec-m2) 3328819.68 1373843.376
Kpp (m/s) 6.60E-06 2.75E-06
* Data is representative as work is ongoing
Closing Three strains are very different As of yet, no inferences can be drawn
connecting phenotypic nature and adhesion.
Further work 96 well plate coated to
mimic surface layer of foods
Ultimately: identify properties that inhibit adhesion and a solution to foodborne outbreaks
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References1. http://www.clker.com/search/cartoon+food/32. http://drjean.org/html/monthly_act/
act_2005/09_Sep/pg04.html3. http://www.steveklotz.com/blog/?m=2006094. Yates, Walker, Bianchi. Ensuring Food Safety
from Pathogens: Farm to Fork.
Acknowledgments Thank you, especially, Dr. Sharon Walker and Olgun Zorlu
for teaching me everything that you have and making it run so smoothly.
Thank you very much, Dr. Sharon Walker’s lab group, including Gexin Chen, Indranil Chowdury, Amy Gong, Berat Haznedaroglu, Ian Marcus, Brian Perez, and Chad Thomsen for all the help in the lab.
Thank you, Jun Wang, for all the work done to make this a great summer for all of us.
Thank you, National Science Foundation for funding opportunities like this one for college students like myself.