selection criteria for alternative microbial methods_vicky_131009

8/2/2019 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



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Introduction Standardized methods

e.g. ISO methods

Classical Culture Methods or Conventional


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


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


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


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


Laborious

THE GOLDEN STANDARDS!!!

Most ISO methods based on conventionalmethods


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Conventional Methods

No expensive infrastructure needs

Cheap in consumables


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


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


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


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


applicability: Food matrices, MO BUT ALSO

Upgrades

Company take over, different distributors, etc.


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Validation of alternativeEM and DM



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Use of alternative method Utmost importance

Reliable Agreement between parties


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


, 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


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


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



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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


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


The type of sample -> scope of the method.

The microbial parameter -> multifunctionality.


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Competent authority





Verification

Control activities

Alerts

Food

Feed

Environment

Utility

Hygiene

Pathogens





Lab (place)

Bio-Safety Level



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.


The cost -> as small as possible butdepends on the amount of samples or

runs. Also cost of investment/training/maintenance.


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Competent authority





Verification

Control activities

Alerts

Food

Feed

Environment

Utility

Hygiene

Pathogens





Lab (place)

Bio-Safety Level



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


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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Competent authority





Verification

Control activities

Alerts

Food

Feed

Environment

Utility

Hygiene

Pathogens





Lab (place)

Bio-Safety Level



Routine analysis

Ad hoc analysis


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Sustainability selection criteriaThe sustainability selection criteria ->

-More than one type of method istaken under consideration.


-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


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

selection criteria for alternative microbial methods_vicky_131009

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