Selection criteria for alternative microbial methods

ir. Vicky Jasson

Laboratory of Food Microbiology and Food Preservation Department 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 Methods

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 Pour 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!
distribution in foods) (low numbers, sub-lethally injured, heterogenious

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)

Listeria monocytogenes detection ISO 11290-1/ AM1:2004

24h at 30 C

24h at 30 C Selective enrichment Culture, 0.1 ml + Fraser broth (10 ml) 48h at 37 C

Selective plating medium Plate on Agar Listeria according to Ottaviani and Agosti (ALOA) and any other selective medium 24-48h at 37 C Pick five presumptive L. monocytogenes colonies from each agar plate and inoculate on tryptone soy agar + yeast extract (TSA-Y) 24 3h at 37 C Biochemical confirmation of Listeria spp. Morphology on TSA upon indirect illumination, Gram stain, motility, catalase activity Biochemical confirmation of L. monocytogenes -Haemolyis, CAMP test fermentation of carbohydrates (rhamnose, xylose, mannitol)
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Conventional Methods
Large volumes of materials Time consuming Laborious THE GOLDEN STANDARDS!!! Most ISO methods based on conventional methods

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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 any method or system that reduces the time taken to obtain a microbiological test result
(Feng, 1996; Fung, 1994)

Rapid =
a shorter time to detection better flow through automation

rapid methods replaced alternative methods.

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Overview: Alternative enumeration methods

pPCR Flow cytometer SimPlate Modified cultural methods Bio-chemically based enumeration methods ATP bioluminescence

Impedance DEFT

Chromogenic and fluorogenic isolation media

Compact Dry

Classical cultural methods TEMPO 3MTMPetrifilmTM Microscopic based enumeration methods Colilert

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Overview: Alternative detection methods PCR

Immuno-assays Flow cytometer ELFA Biosensors Bio-chemically based detection methods FISH

Modified cultural methods

Lateral flow devices Microarrays Chromogenic and fluorogenic isolation media ELISA Molecular based detection methods

Microscopically based detection methods

Hybridization
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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 alternative EM and DM

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Use of alternative method

Utmost importance
Reliable Agreement between parties

You need objective evidence Regulation: 2073/2005 Validation -> ISO 16140-2003: Microbiology of food and animal feeding stuffs -- Protocol for the validation of alternative methods
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Validation bodies
AOAC (Association of Official Analytical Chemist, USA) AFNOR (Association Franais de Normalisation, France) NordVal (part of the Nordic Committee on Food Analysis, Norway) MicroVal (European Validation and Certification Organisation, Europe)
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Validation bodies
Validation certificates of AFNOR, NordVal and MicroVal are based on ISO 16140 AOAC ~ ISO 16140
AOAC Performance Tested Methods 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 specificity 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 Competent authority Food business operator Industry association Purpose Verification Control activities Alerts Type of sample Food Feed Environment Parameter Utility Hygiene Pathogens

Managerial selection criteria

Total Time to result Cost

Technological selection criteria

Operational requirements Routine analysis Ad hoc analysis Performance characteristic of method Infrastructure Lab (place) Bio-Safety Level

Sustainability selection criteria

Communication Credibility Service Logistics

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Objective of MA
The type of stakeholder -> validation certificate. The purpose of the microbial analysis -> total time to result and validation certificate. The type of sample -> scope of the method. The microbial parameter -> multifunctionality.

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Objective of microbial analysis

Stakeholder Competent authority Food business operator Industry association Purpose Verification Control activities Alerts Type of sample Food Feed Environment Parameter Utility Hygiene Pathogens

Managerial selection criteria

Total Time to result Cost

Technological selection criteria

Operational requirements Routine analysis Ad hoc analysis Performance characteristic of method Infrastructure Lab (place) Bio-Safety Level

Sustainability selection criteria

Communication Credibility Service Logistics

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Managerial selection criteria

Total time to result -> as small as possible but may differ for a negative, a presumptive or confirmed positive test result. The cost -> as small as possible but depends on the amount of samples or runs. Also cost of investment/ training/maintenance.
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Objective of microbial analysis

Stakeholder Competent authority Food business operator Industry association Purpose Verification Control activities Alerts Type of sample Food Feed Environment Parameter Utility Hygiene Pathogens

Managerial selection criteria

Total Time to result Cost

Technological selection criteria

Operational requirements Routine analysis Ad hoc analysis Performance characteristic of method Infrastructure Lab (place) Bio-Safety Level

Sustainability selection criteria

Communication Credibility Service Logistics

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Technological selection criteria

The operational requirements: connected: -> high numbers of samples -> investment in equipment -> 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) and area classification.

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Objective of microbial analysis

Stakeholder Competent authority Food business operator Industry association Purpose Verification Control activities Alerts Type of sample Food Feed Environment Parameter Utility Hygiene Pathogens

Managerial selection criteria

Total Time to result Cost

Technological selection criteria

Operational requirements Routine analysis Ad hoc analysis Performance characteristic of method Infrastructure Lab (place) Bio-Safety Level

Sustainability selection criteria

Communication Credibility Service Logistics

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Sustainability selection criteria

The sustainability selection criteria -> -More than one type of method is taken 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 When different parties are involved

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 low cost. Select the methods that fits the purpose

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