1Rapid Methods for Microbiological Analyses of FoodsByAlejandro CastilloFood Science and TechnologyTexas A&M UniversityTesting is a part of the sampling plan in a microbiological criterionComponents of a microbiological criterion9Identity of the food or ingredient 9Agent of concern9Analytical method9Sampling plan9Microbiological limitsThis approach is good for quality control, but not for safety2Establishing critical limitsHazard analysis YesIdentification of CCP NoYes/NoMonitoring Yes/NoCorrective action Yes*Record keeping NoVerification YesPrincipleRapid methodsapplicableApplicability of rapid testing methods in a HACCP programFung, 1995Rapid methods can help speed the decision-making processRelative interest of microbiologists on rapid methods 02468101965 1975 1985 1995 2000YearRelative interestMedical MicrobiologistsFood MicrobiologistsFung, 19953Advantages of rapid testing methods9Same (or greater) sensitivity, accuracy, and specificity as conventional methods9Savings of space and materials9Reduction in human errors and labor cost (when automated methods are used)The microbiologist can judge data, organize activities, and repeat tests if necessaryRapid MethodsMicrobiological methods that produce results in 24 h or less9In the practice, results are not always obtained within 24 h9Ex. ELISA for Salmonella ID (6 h)ÆPreenrichment: 18h, M broth: 18 h, ELISA: 6 h.Æ18+18+6 = 42 h—Time for conventional method: 4-5 daysSpecific characters can be used to develop rapid testsExamples997% E. coli produce $-D-glucuronidase9Methyl umbelliferyl glucuronide (MUG) test, E. coli PetrifilmTM9>90% S. aureus produce TNase9Lachica TNase test4Application of rapid tests 9Microbial enumeration9Quantification of microbial toxins9Identification of microorganisms and their toxins 9Verification of sanitizing procedures in food plants Microbial EnumerationVarious systems for estimation of microbial numbers ImpedanceDEFTBactometerAutomated Microbiological SystemMalthus-ATThermal Activity MonitorConductivityMicrocalorimetryRespirometerChanges in CO2concentration5Other systems for microbial enumeration 9Redigel9Isogrid9Petrifilm9Spiral platerSperman correlation coefficient of different methods for simplified bacterial countChain and Fung, 199100.20.40.60.81RAPC Redigel Petrifilm Spiral IsogridAPCRedigelPetrifilmSpiralIsogridWhat is Petrifilm?6Petrifilm9Simple method 9Little space used 9Minimizes time for analysis 9Material preparation 9Inoculation time 9Colony counting time PetrifilmAerobic plate count Coliforms and E. coli Yeasts and molds TTC helps visualizing colonies $-glucuronidase reagent differentiates between total coliforms and E. coliNo need for acidificationCounts of different heat-stressed indicators in cow manure by PetrifilmTMand conventional plate methods01234567Log10 CFU g-1APC Enterob. Coliforms FecalcoliformsE. coliConventionalPetrifilm7Pathogen DetectionMost common methods9Immunoassays9Immunofluorescence9Coagglutination (latex agglutination technique)9Immunomobilization9Enzyme immunoassays (ELISA)9DNA hybridization9Filter hybridization9Liquid hibridization9Polymerase chain reaction (PCR)Immunoassays Sandwich enzyme-linked immunosorbent assay (ELISA)8Immunoassays Sandwich ELISA Immunoassay 12345 6Salmonella 1-2BioControl Systems, Inc.ImmunoimmobilizationFor flagellated bacteria onlyDNA hybridization9Polymerase Chain Reaction (PCR)Increases numbers of a specific DNA sequence in a sample9Multiple chains of the same DNA sequence are more easily detected by hybridization 9High specificity and sensitivity ÆDetects as little as ~50 ng DNACombined methodsImmunomagnetic separationDynabeads10E. coli VIPTMListeriaClearviewTMSalmonellaMicro-ScreenCombined methodsBacterial Identification9Automated systems9MinikitsAutomated systemsVITEK: Automated colorimeter11Most common minikits9Miniaturized multiple tests9Enterotube IIÆRoche Diagnostics—for Enterobacteriaceae9APIÆbioMPrieux—Kits for specific microorganisms 9Micro-IDÆOrganon Teknika—Kits for specific microorganisms Methods to assess plant sanitationATP bioluminiscence9ATP = Adenosin triphosphate9energy-rich compound9Most important energy carrier in metabolic processes 9Present in all living cells9Animals9Plants9Microorganisms (bacteria, yeasts, molds)12Total ATP measured9Microorganisms9Food residues9“Free”ATP bioluminiscencetechniqueATP bioluminiscence techniqueATP + luciferin + luciferaseMg++Oxyluciferin + CO2+ LIGHTATP Bioluminiscence9 Rapid results9 Sensitive9 Non-microbial specific9 Allows for immediate corrective action13Conclusions 9Different products, more choices9Rapid testing will not modify usefulness of microbiological testing 9Positive results by rapid methods have to be confirmed by conventional