optical forward scattering for bacterial colony
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Optical forward scattering for bacterial Optical forward scattering for bacterial colony differentiation and identification colony differentiation and identification ……..……..an interdisciplinary approachan interdisciplinary approach
Light scatteringProf. A. Bhunia (bhunia@purdue.edu)
l
Prof. D. HirlemanDr. Euiwon BaeNan BaiS. GuoDr. Nebeker, Ben BucknerSchool of Mechanical Engineering
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Microbiology
Data analysisand processing
Dr. A. AroonnualAmanda BettassoDr. P. BanadaKarleigh HuffDr. Seung OhkAbrar Adil (UG)Amanda LathropDept of Food Science
Engineering
Prof. J. P. RobinsonDr. B. RajwaValery PatsekinBulent BayraktarDept of Basic MedicalSciences, Bindley Bioscience Center
USDA-ARSSupport/
collaboration
Collaborator: Gary Richards, USDA-ARS, DE
Oct 27-28, 2009
BARDOT BARDOT –– ReportingReporting BARDOT based analysis of serovars of E. coli,
Salmonella and Vibrio Detection of Salmonella from spiked peanut
butter Roadmap for multiRoadmap for multi--scale Forward Scattering scale Forward Scattering
Ph iPh i (FSP)(FSP)
22
PhenomicsPhenomics (FSP)(FSP) Generate scatter patterns for molds and yeast Determine scatter patterns for microcolonies for
early detection/identification System parameter optimization for best
performance (interfacing image analysis software)
BaBacterial cterial RRapid apid DDetection using etection using OOptical ptical Scattering Scattering TTechnology (BARDOT)echnology (BARDOT)
Laserλ: 635 nmBeam dia: 1 mm
Power: 0.1-0.2 mWCCD Pixel: 640x480
LaserPetri
Petri dish with bacterial colonies
CCD camera Cable to computer
CCD Pixel: 640x480
ComputerComputer
dish with
colonies
CCD chip
Scatter image
Banada et al. 2009. Biosens. Bioelectron. 24:1685-1692
Specifications
EnUrgaEnUrga
Second generation
pDimension: 24x20x17 inchWeight: 75 lbsOperationColony counts: line scan: 40 secScatter image: 5 sec/colony
3rd Generation BARDOT
4th generation!!!Advanced Bioimaging Systems(advancedbioimagingsystems.com)
•Analyzed scatter patterns of 10 different O-antigenic E coli strains
Analysis of scatter patterns of different O-antigenic E. coli strains
with selective and non-selective media(A. Aroonnual et .al.)
antigenic E. coli strains
•Media examined: Non-selective media (BHI) and selective media (SMAC and CT-SMAC)
•Growth conditions: 37oC for 12-13 h
2
Cryo-SEM photographs of coloniesL. monocytogenes L. innocua
L. mononocytogenes L. innocua
Banada et al. 2007. Biosens. Bioelectron. 22:1664
Banada et al. 2009. Biosens. Bioelectron. 24:1685
0
5
10
15
20
EPS Proteins
L. monocytogenes
L. innocua
0
100
200
300
400
500
600 L. mono L. innocua
Pentosesand uronic
acids
Hexoses
mg/m
l
µg/m
l
• Part of lipopolysaccharide (LPS) in gram-negative
bacteria (Lipid A, Core polysaccharide, and O-
polysaccharide)
• O-antigen specific chains determine specificity
O-antigen
http://bs.kaist.ac.kr/~mbtlab/Gram_Neg.jpg
http://www.mbl.edu/marine_org/images/animals/Limulus/blood/lpstyl01.gif
Gram-negative cell wall Lipopolysaccharide of Gram-negative bacteria
BARDOT images of E. coli colonies on BHI
O157:H7 DEL933
O157:H7 SEA13A53
12
12
E. coli strain Time (h) Images on BHI
O127:H6 EPEC
O142:H6 EPEC
O25:K98 NM ETEC
O78:H11 ETEC
12
12
12
12
O5 NM
O46:H38
12
12
E. coli strain Time (h) Images on BHI
BARDOT images of E. coli colonies on BHI
O111:H11
O91:H21
12
12
O157:H7 EDL933
O157:H7 SEA13A53
12
13
grey
grey
E. coli strain Time (h) Color Images on SMAC
BARDOT images of E. coli colonies on SMAC
O127:H6 EPEC
O142:H6 EPEC
O25:K98 NM ETEC
O78:H11 ETEC
12
13
12
13
grey
grey
pink
pink
O5 NM
O46:H38
13
13
grey
pink
E. coli strain Time (h) Color Images on SMAC
BARDOT images of E. coli colonies on SMAC
O46:H38
O111:H11
O91:H21
13
13
13
pink
grey
pink
3
BARDOT images of E. coli colonies on SMAC and CT-SMAC
O157:H7 EDL933
12
14
SMAC
CT SMAC
E. coli strain Time (h) Media BARDOT Images
O127:H6 EPEC
14
12
18
CT-SMAC
SMAC
CT-SMAC
BARDOT analysis of BARDOT analysis of SalmonellaSalmonella serovars serovars with selective and nonwith selective and non--selective mediaselective media
(A. Bettasso et .al.)(A. Bettasso et .al.)
Salmonella Salmonella serovarsserovars cultured overnight in cultured overnight in RappaportRappaport VassiliadisVassiliadis RR10 10 brothbroth
Spread plated on BHI (general) and XLD Spread plated on BHI (general) and XLD (selective) and Brilliant Green agars(selective) and Brilliant Green agars
Incubated at Incubated at 3737°°C for C for 1212hh Screened with BARDOTScreened with BARDOT
1414
Salmonella serovar scatter patterns Salmonella serovar scatter patterns on BHIon BHI
S. Typhimurium var copenhagen
S. Litchfield
BHI, 12 h, 37°C
S. Indiana
S. Typhi
S. Enteriditis PT14b
Salmonella Typhimurium on BHI
Salmonella serovar scatter patterns on BHISalmonella serovar scatter patterns on BHI
S. Agona
S. Cholerasuis
BHI/12 h/ 37°C
S. Heidelberg
S. Poona
S. Schottmuelleri
S. Seftenberg
S. Typhimurium var copenhagen
S. Litchfield
SalmonellaSalmonella on selective media (XLD)on selective media (XLD)
Salmonella
XLD/12h/37°C
S. Indiana
S. Typhi
S. Enteriditis PT14b
Salmonella Typhimurium on XLD
Salmonella on selective media (XLD)Salmonella on selective media (XLD)
S. Agona
S. Cholerasuis
S. Heidelberg
12h /XLD/ 37°C
S. Poona
S. Schottmuelleri
S. Seftenberg
4
Application of BARDOT for Application of BARDOT for SalmonellaSalmonelladetection/identification from peanut detection/identification from peanut
butter butter (A. Bettasso et al.) (A. Bettasso et al.) Peanut butter samples were inoculated with Peanut butter samples were inoculated with
100 100 CFU/CFU/2525g with g with SalmonellaSalmonella Typhimurium Typhimurium varvar CopenhagenCopenhagen
AnalysedAnalysed on Days on Days 00,,77,,1414,,2121, and , and 2828 Enriched for Enriched for 0 0 or or 1212h in RV brothh in RV broth Spread plated on XLD and BHI Spread plated on XLD and BHI Incubated for Incubated for 1212h at h at 3737°°CC Screened with BARDOTScreened with BARDOT
SalmonellaSalmonella detection from peanut butterdetection from peanut butterXLD BHI
0h enrich 12h enrich 0h enrich 12h enrich
Day 0
Day 7
Day 14
Day 21
Day 28
Vibrio cincinnatiensis
Vibrio
Vibrio alginolyticusVibrio campbelli
Vib i i i
V. cholerae O1 (6707)
V. cholerae non O1 (140-58)
Vibrio cincinnatiensis
Vibrio fluvialis
Vibrio mimicus
Vibrio parahaemolyticus
Vibrio anguillarumVibrio holisae
Vibrio orientalis
Vibrio metchinovic
V. cholerae O139 (CDC
V. cholerae O1E 16
V. cholerae non O1 140-58 18
V. cholerae O1 6707 24
Vibrio sp. BARDOT images on BHI1%NaClh
V. cholerae O139 CDC 14
V. parahemolyticus ATCC 17803 14
V. parahemolyticus
SPRC 10290 tdh+/trh+ 15
Vibrio sp. BARDOT images on BHI1%NaClh
Vp DIE 12052499 tdh+/trh+ O1:Kuk 16
Vp CPA 7081699 tdh+/trh-, O4:K8 13p ,
Vp BAC 983547 tdh-/trh-, O4:K55 11
Vp AQ4037 tdh-/trh+, O3:K6 12
Aroonnual et al.
Detection of Detection of Vibrio choleraeVibrio cholerae in presence in presence of other Vibrio of other Vibrio sppspp
2424
V. orientaris V. fluviaris V. cholerae nonO1
Mixture of Vibrio cultures (V. orientaris, V. fluviaris, and V. cholerae nonO1 on BHI+1%NaCl at 30oC for 18h.
Aroonnual et al.
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The extendable version of the BARDOT The extendable version of the BARDOT systemsystem
Research laboratory systemResearch laboratory system Dynamic learningDynamic learning, n, nonon--exhaustive exhaustive
learning (could be also paired learning (could be also paired with mathematical modelling of with mathematical modelling of colony growth)colony growth)
Multiple spectral bandsMultiple spectral bands, m, multiple ultiple S l i t g ti
Tunable Light source
Backscatter detector
Fluorescence detector
Polarizers
Interferprofilosc
ence
l i
mag
er
polarizationspolarizations Integrated microincubation Integrated microincubation
systemsystem
Colony localization using confocal Colony localization using confocal profilometryprofilometry
Sample interogation area
Forward scatter detector
Polarizers
rometric
ometerFl
uore
sco
nfoc
al
PatternPattern--matching using orthogonal radial polynomials (Zernike, matching using orthogonal radial polynomials (Zernike, pseudopseudo--Zernike, Zernike, ChebyshevChebyshev…)…)
A B C D
Example: unsupervised hierarchical clustering Example: unsupervised hierarchical clustering ((SalmonellaSalmonella))
Kentucky
Agona
Thomasville
Typhimurium (Copenhagen)
40
6040 60
LD2
5
105 10
-5
0
-5 0
LD32
4
6 2 4 6
-4
-2
0
-4 -2 0
Linear Discriminant AnalysisMixture V. cholerae V. parahaemolyticus V. vulnificus
Aver
age
true
pos
itiv
e ra
te0
0.2
0.4
0.6
0.8
1.0
V. cholerae
V. parahaemolyticus
V vulnificus
Example: classification of Example: classification of VibrioVibriopathogens (single colonies)pathogens (single colonies)
SVM classifier
Scatter Plot Matrix
LD1
0
20
0 20
Average false positive rate0.0 0.2 0.4 0.6 0.8 1.0
0.0 V. vulnificus
Class Classifier Sensitivity Specificity Accuracy AUC
MixtureFLD 0.93 0.99 0.97 1.00
SVM-L 0.93 0.98 0.97 0.99
SVM-RBF 0.96 0.99 0.98 1.00
V. choleraeFLD 0.77 0.98 0.94 0.97
SVM-L 0.88 0.98 0.96 0.99
SVM-RBF 0.94 0.99 0.98 1.00
V. parahaemolyticusFLD 0.95 0.90 0.93 0.98
SVM-L 0.95 0.94 0.95 0.99
SVM-RBF 0.97 0.96 0.97 1.00
V. vulnificusFLD 1.00 1.00 1.00 1.00
SVM-L 1.00 1.00 1.00 1.00
SVM-RBF 0.99 1.00 1.00 1.00
Classification of plates containing Classification of plates containing VibrioVibrio pathogenspathogens (multiple colonies)(multiple colonies)
The plate is considered “negative” (or clean) if ALL the colonies are non-pathogenic (or they do not belong to the pathogenic classes of interest)
The plate is considered “positive” (or contaminated) if AT LEAST ONE of the colonies belongs to a pathogenic class.
This classifier can be tuned selecting proper cut-off levels to obtain desired sensitivity and specificity
Species Sensitivity Specificity
V. cholerae 1.00 1.00
V. parahaemolyticus 0.96 1.00
V. vulnificus 1.00 1.00
GrowthTime
2 hr 8 hr 24 hr
Scalar diffraction theoryDiscrete Dipole Approximation
Roadmap for multi-scale Forward Scattering Phenomics (FSP)
Tra
nsm
issi
on T
ype
Th
eory
Exp
erim
ent
&
Inst
rum
ent
yp pp
BARDOT w/ micro incubatorμ-BARDOT BARDOT
Reaction –diffusion model
6
Micro-scale : colony dipole model constructorModel Parameters: Bacteria No. = 200Bacteria Size = 0.855 µm x 0.315µm x 0.315µmColony Size = 13.5 µm x 1.35 µmDipole Spacing = 0.045 µmWavelength = 0.6328 µm (p-polarized plane wave with normal incidence)Bacteria Refractive Index = 1.38Extracellular Refractive Index = 1.40
Modeling Results from DDSCAT
FS is favored against BS with 1000 times stronger signal
Wavefront and Scattering Pattern Generated by Ray Optics Model (I)
Incoming wave(laser)
Bacterial colony as a amp/phase modulator
Scattered lightAt image plane
Wavefront and Scattering Pattern Generated by Ray Optics Model (II)
-2
Wavefront emerging from the colony imprinted with its 3D phase structure
information
Intensity field distribution of the propagating wave calculated by
Fresnel Diffraction
-2 -1.5 -1 -0.5 0 0.5 1 1.5 2
-1.5
-1
-0.5
0
0.5
1
1.5
2
ConclusionsConclusionsBARDOT show differential scatter signatures for BARDOT show differential scatter signatures for
different different E. coli E. coli serovars primarily on serovars primarily on nonselective medianonselective media
BARDOT show differential scatter signatures for BARDOT show differential scatter signatures for different different SalmonellaSalmonella serovars primarily on serovars primarily on
l i dil i diselective mediaselective mediaSalmonella was successfully detected from spiked Salmonella was successfully detected from spiked
peanut butter samples with or without peanut butter samples with or without enrichment from samples stored for enrichment from samples stored for 00, , 77, , 1414, , 21 21 and and 28 28 days at RT.days at RT.
Vibrio cholerae, V. parahemolyticus, V. vulnificus Vibrio cholerae, V. parahemolyticus, V. vulnificus can be detected from a mixture of other Vibriocan be detected from a mixture of other Vibrio
3535
The new version of the BARDOT systemThe new version of the BARDOT system
Sample interogation area
Red-laser light source
Forward scatter
Whi
te li
ght
imag
er
• Developed at Purdue University and funded by USDA
• Commercialized by Advanced Bioimaging Systems
• Fully customizable for any workflow/requirements
• Deployed at USDA July, 2009
scatter detector
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