selection criteria for alternative microbial methods_vicky_131009
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Selection criteria foralternative microbial
Food Quality and Safety
ir. Vicky Jasson
Laboratory of Food Microbiology and Food PreservationDepartment of Food Safety and Food Quality
Part of Food2Know (www.food2know.be)Faculty of Bio-science Engineering
University of Ghent
Food Quality and Safety
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Introduction Standardized methods
e.g. ISO methods
Classical Culture Methods or Conventional
Food Quality and Safety
Developed historically
International consensus
Open access methods
The Golden standards
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Conventional Methods:Enumeration
Colony count method
Serial dilution Inoculation on growth media
Spread plates
Food Quality and Safety
Counting colonies on agar plates
Most probable number (MPN) Serial dilution
Three serial dilution in 3x3 or 3x5 tubes Number of positive tubes for each dilution
MPN table for calculation of numbers
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Conventional Methods:
Detection Pathogens! (low numbers, sub-lethally injured, heterogenious
distribution in foods)
Resuscitation phase = pre-enrichment
Enrichment phase
Food Quality and Safety
Isolation of the pathogen
Identification/confirmation
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Primary enrichment
Test portion (25g) + Demi-Fraser broth (225ml)
Selective enrichment
Selective plating medium
Plate on Agar Listeria according to Ottaviani and Agosti (ALOA) and any other selective medium
Culture, 0.1 ml + Fraser broth (10 ml)
24h at 30C48h at 37C
Listeriamonocytogenes
detection
ISO 11290-1/
AM1:2004
24h at 30C
Food Quality and Safety
Pick five presumptive L. monocytogenescolonies from eachagar plate and inoculate on tryptone soy agar + yeast
extract (TSA-Y)
Biochemical confirmation of Listeriaspp.
Morphology on TSA upon indirect illumination, Gram stain, motility, catalaseactivity
Biochemical confirmation of L. monocytogenes
-Haemolyis, CAMP testfermentation of carbohydrates (rhamnose, xylose, mannitol)
24-48h at 37C
24 3h at 37C
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Conventional Methods
Large volumes of materials
Time consuming
Food Quality and Safety
Laborious
THE GOLDEN STANDARDS!!!
Most ISO methods based on conventionalmethods
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Conventional Methods
No expensive infrastructure needs
Cheap in consumables
Food Quality and Safety
Laborious to perform
Large volumes of materials
Time consuming
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Alternative methods Based on Golden standard:
a rapid method can be defined as anymethod or system that reduces the timetaken to obtain a microbiological test result
Food Quality and Safety
eng, 1996; ung, 1994
Rapid = a shorter time to detection
better flow through
automation
rapid methods replaced alternative methods.
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Overview:
Alternative enumeration methods
Bio-chemically based
enumeration methods
SimPlate ATP bioluminescence
Flow cytometer
pPCR
Food Quality and Safety
Microscopic basedenumeration methods
Chromogenic andfluorogenic isolation
media
Classical cultural methods
TEMPO
Compact Dry
3MTMPetrifilmTM
Colilert
Impedance
DEFT
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Overview:
Alternative detection methods
Immuno-assays
Modified cultural methods
Bio-chemically baseddetection methods
Biosensors
PCR
Flow cytometerELFA
FISH
Food Quality and Safety
Microscopically based
detection methods
Chromogenic andfluorogenic isolation media Molecular based detection
methods
Microarrays
Hybridization
ELISA
Lateral flow devices
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Differences in methods techniques: PCR, ELISA, FISH
sample preparations: broth, time, temp
formats: apparatus / reagentia
Food Quality and Safety
applicability: Food matrices, MO BUT ALSO
Upgrades
Company take over, different distributors, etc.
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Validation of alternativeEM and DM
Food Quality and Safety
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Use of alternative method Utmost importance
Reliable Agreement between parties
Food Quality and Safety
Regulation: 2073/2005
Validation
-> ISO 16140-2003: Microbiology of foodand animal feeding stuffs -- Protocol forthe validation of alternative methods
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Validation bodies AOAC (Association of Official Analytical
Chemist, USA) AFNOR(Association Franais de
Food Quality and Safety
, NordVal (part of the Nordic Committee
on Food Analysis, Norway)
MicroVal (European Validation andCertification Organisation, Europe)
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Validation bodies Validation certificates of AFNOR, NordVal
and MicroVal are based on ISO 16140 AOAC ~ ISO 16140
Food Quality and Safety
er ormance es e e o s AOAC Official Methods
In own lab
Secondary validation
Verification
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ISO 16140:2003 For all foods -> select 5 relevant food categories
Two stages 1ste stage: Method comparison study
Relative accuracy
Food Quality and Safety
Relative sensitivity (60 test portions)
Relative detection limit (5x3x6 test)
Inclusivity (50 target strains)
Exclusivity (30 not target strains)
2de stage: Inter-laboratory study Variability of results
10 collaborative laboratories
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Selection criteria
Food Quality and Safety
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Objective of microbial analysis
Stakeholder Purpose Type of sample Parameter
Competent authority
Food business operator
Industry association
Managerial selection criteria
Total Time to result Cost
Verification
Control activities
Alerts
Food
Feed
Environment
Utility
Hygiene
Pathogens
Food Quality and Safety
Performance characteristic of method
Technological selection criteria
Operational requirements Infrastructure
Lab (place)
Bio-Safety Level
Sustainability selection criteria
Communication Credibility Service Logistics
Routine analysis
Ad hoc analysis
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Objective of MA The type of stakeholder -> validation certificate.
The purpose of the microbial analysis -> total
Food Quality and Safety
The type of sample -> scope of the method.
The microbial parameter -> multifunctionality.
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Objective of microbial analysis
Stakeholder Purpose Type of sample Parameter
Competent authority
Food business operator
Industry association
Managerial selection criteria
Total Time to result Cost
Verification
Control activities
Alerts
Food
Feed
Environment
Utility
Hygiene
Pathogens
Food Quality and Safety
Performance characteristic of method
Technological selection criteria
Operational requirements Infrastructure
Lab (place)
Bio-Safety Level
Sustainability selection criteria
Communication Credibility Service Logistics
Routine analysis
Ad hoc analysis
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Managerial selection criteria Total time to result -> as small as possible
but may differ for a negative, apresumptive or confirmed positive testresult.
Food Quality and Safety
The cost -> as small as possible butdepends on the amount of samples or
runs. Also cost of investment/training/maintenance.
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Objective of microbial analysis
Stakeholder Purpose Type of sample Parameter
Competent authority
Food business operator
Industry association
Managerial selection criteria
Total Time to result Cost
Verification
Control activities
Alerts
Food
Feed
Environment
Utility
Hygiene
Pathogens
Food Quality and Safety
Performance characteristic of method
Technological selection criteria
Operational requirements Infrastructure
Lab (place)
Bio-Safety Level
Sustainability selection criteria
Communication Credibility Service Logistics
Routine analysis
Ad hoc analysis
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Technological selection criteria The operational requirements: connected: ->
high numbers of samples -> investment inequipment -> automated -> multifunctional ->appropriate training!
Performance characteristic
Food Quality and Safety
quantitative methods -> inclusivity, exclusivity,relative accuracy, detection limit, quantification limit,reproducibility, repeatability and linearity.
qualitative methods -> specificity, sensitivity, relative
accuracy and detection limit. The infrastructure -> Bio-Safety Level (BSL) andarea classification.
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Objective of microbial analysis
Stakeholder Purpose Type of sample Parameter
Competent authority
Food business operator
Industry association
Managerial selection criteria
Total Time to result Cost
Verification
Control activities
Alerts
Food
Feed
Environment
Utility
Hygiene
Pathogens
Food Quality and Safety
Performance characteristic of method
Technological selection criteria
Operational requirements Infrastructure
Lab (place)
Bio-Safety Level
Sustainability selection criteria
Communication Credibility Service Logistics
Routine analysis
Ad hoc analysis
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Sustainability selection criteriaThe sustainability selection criteria ->
-More than one type of method istaken under consideration.
Food Quality and Safety
-In case of a defined type of method,but available in various formats from
different supplies.
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In conclusion ISO methods -> conventional methods Rapid alternative methods
Need of validation certificate for Verification/validation FSMS Governmental monitoring systems Accreditation
Food Quality and Safety
en eren par es are nvo ve
BUT more criteria are important The selection criteria can help to select the methods
that fits best the purpose in a particular context. No method = 100% sensitive, 100% specific, can
provide results without hands-on time and at lowcost.
Select the methods that fits the purpose