BARDOT - Center for Food Safety Engineering - Purdue University

Optical forward scattering for bacterialcolony differentiation and identification……..an interdisciplinary approachLight scatteringProf. A. Bhunia (bhunia@purdue.edu)Dr. A. AroonnualAmanda BettassoDr. P. Banada MicrobiologyKarleigh HuffDr. Seung OhkAbrar Adil (UG)Amanda LathropData analysisDept of Food Scienceand processingCollaborator: Gary Richards, USDA-ARS, DEOct 27-28, 2009Prof. D. HirlemanDr. Euiwon BaeNan BaiS. GuoDr. Nebeker, Ben BucknerSchool of MechanicalEngineeringUSDA-ARSSupport/collaborationProf. J. P. RobinsonDr. B. RajwaValery PatsekinBulent BayraktarDept of Basic MedicalSciences, BindleyBioscience Center1BARDOT – Reporting• BARDOT based analysis of serovars of E. coli,Salmonella and Vibrio• Detection of Salmonella from spiked peanutbutter• Roadmap for multi-scale Forward ScatteringPhenomics (FSP)• Generate scatter patterns for molds and yeast• Determine scatter patterns for microcolonies forearly detection/identification• System parameter optimization for bestperformance (interfacing image analysissoftware)2BacterialRapidDetection using OpticalScattering Technology (BARDOT)Second generationPetridishwithcoloniesCCD chipλ: 635 nmBeam dia: 1 mmLaser Power: 0.1-0.2 mWCCD Pixel: 640x480Scatter imageComputerCCD cameraLaserPetri dishwithbacterialcoloniesCable tocomputerSpecificationsDimension: 24x20x17inchWeight: 75 lbsOperationColony counts: linescan: 40 secScatter image: 5sec/colonyEnUrgaBanada et al. 2009. Biosens. Bioelectron. 24:1685-16923 rd Generation BARDOTAdvanced Bioimaging Systems(advancedbioimagingsystems.com)4 th generation!!!Analysis of scatter patterns of differentO-antigenic E. coli strainswith selective and non-selective media(A. Aroonnual et .al.)•Analyzed scatter patterns of 10 different O-antigenic E. colistrains•Media examined: Non-selective media (BHI)and selective media (SMAC and CT-SMAC)•Growth conditions: 37 o C for 12-13 h1

Cryo-SEM photographs of coloniesL. mononocytogenes L. innocuaL. monocytogenes L. innocuaO-antigen• Part of lipopolysaccharide (LPS) in gram-negativebacteria (Lipid A, Core polysaccharide, and O-polysaccharide)• O-antigen specific chains determine specificity2015L. monocytogenesL. innocua600500400L. mono L. innocuaBanada et al. 2007. Biosens.Bioelectron. 22:1664Banada et al. 2009. Biosens.Bioelectron. 24:1685mg/ml1050EPSProteinsµg/ml3002001000Pentosesand uronicacidsHexoseshttp://bs.kaist.ac.kr/~mbtlab/Gram_Neg.jpgGram-negative cell wallhttp://www.mbl.edu/marine_org/images/animals/Limulus/blood/lpstyl01.gifLipopolysaccharide of Gram-negative bacteriaBARDOT images of E. coli colonies on BHIE. coli strain Time (h) Images on BHIO157:H7 DEL933O157:H7 SEA13A531212O127:H6 EPECO142:H6 EPECO25:K98 NM ETEC121212BARDOT images of E. coli colonies on BHIE. coli strain Time (h) Images on BHIO5 NMO46:H381212O111:H11 12O91:H21 12O78:H11 ETEC12BARDOT images of E. coli colonies on SMACE. coli strain Time (h) Color Images on SMACBARDOT images of E. coli colonies on SMACO157:H7 EDL93312greyE. coli strain Time (h) Color Images on SMACO157:H7 SEA13A5313greyO5 NM13greyO127:H6 EPEC12greyO46:H3813pinkO142:H6 EPEC13greyO111:H1113greyO25:K98 NM ETEC12pinkO91:H2113pinkO78:H11 ETEC13pink2

BARDOT images of E. coli colonieson SMAC and CT-SMACE. coli strain Time (h) Media BARDOT ImagesBARDOT analysis of Salmonella serovarswith selective and non-selective media(A. Bettasso et .al.)O157:H7 EDL933O127:H6 EPEC12141218SMACCT-SMACSMACCT-SMAC• Salmonella serovars cultured overnight inRappaport Vassiliadis R10broth• Spread plated on BHI (general) and XLD(selective) and Brilliant Green agars• Incubated at 37°C for 12h• Screened with BARDOT14Salmonella serovar scatter patternson BHIS. Typhimuriumvar copenhagenS. LitchfieldSalmonella serovar scatter patterns on BHIS. AgonaS. CholerasuisS. IndianaS. HeidelbergSalmonellaTyphimurium on BHIS. PoonaS. TyphiBHI, 12 h,37°CS. EnteriditisPT14bBHI/12 h/ 37°CS. SchottmuelleriS. SeftenbergSalmonella on selective media (XLD)Salmonella on selective media (XLD)S. TyphimuriumvarcopenhagenS. LitchfieldS. AgonaS. CholerasuisSalmonellaTyphimurium on XLDS. IndianaS. HeidelbergS. TyphiS. PoonaXLD/12h/37°CS. EnteriditisPT14b12h /XLD/37°CS.SchottmuelleriS. Seftenberg3

Micro-scale : colony dipole model constructorModeling Results from DDSCATModel Parameters:Bacteria No. = 200Bacteria Size = 0.855 µm x 0.315µm x0.315µmColony Size = 13.5 µm x 1.35 µmDipole Spacing = 0.045 µmWavelength = 0.6328 µm (p-polarized planewave with normal incidence)Bacteria Refractive Index = 1.38Extracellular Refractive Index = 1.40FS is favored against BS with1000 times stronger signalWavefront and Scattering Pattern Generatedby Ray Optics Model (I)Wavefront and Scattering Pattern Generatedby Ray Optics Model (II)-2-1.5-1-0.500.511.52Incoming wave(laser)Bacterial colony as aamp/phase modulatorScattered lightAt image planeWavefront emerging from the colonyimprinted with its 3D phase structureinformation-2 -1.5 -1 -0.5 0 0.5 1 1.5 2Intensity field distribution of thepropagating wave calculated byFresnel DiffractionConclusionsBARDOT show differential scatter signatures fordifferent E. coli serovars primarily onnonselective mediaBARDOT show differential scatter signatures fordifferent Salmonella serovars primarily onselective mediaSalmonella was successfully detected from spikedpeanut butter samples with or withoutenrichment from samples stored for 0, 7, 14, 21and 28 days at RT.Vibrio cholerae, V. parahemolyticus, V. vulnificuscan be detected from a mixture of other Vibrio35The new version of the BARDOT system• Developed at Purdue University and fundedby USDA• Commercialized by Advanced BioimagingSystems• Fully customizable for anyworkflow/requirements• Deployed at USDA July, 2009White lightimagerRed-laserlight sourceSample interogation areaForwardscatterdetector6