Validation of Microbiological

Methods for Use in the Food

Industry
Brazilian Association for Food Protection
6th International Symposium
Sao Paulo, Brazil
June 15th, 2007

Introduction

Hundreds of new methods developed each year

Pathogenic organisms
Non-Pathogenic organisms
Detection
Identification

How do you know if you need a new method?

How do you decide if it is the right method for
your purpose?

Introduction

Goal of methods evaluation is to find an

innovative technology that will allow for
quick and efficient detection and/or
quanitation of pathogens and spoilage
organisms

Performance Criteria

The Three Ss

Sensitivity
What is the sensitivity of current method
What degree of sensitivity is needed

Specificity
What is the false positive rate
What is the false negative rate

Speed
What is speed of current method (samples
processed/day)
How quickly are results needed

Performance Criteria

Costs

What
What
What
What

is
is
is
is

cost of current method

cost of instrumentation
cost of disposables/reagents
the cost per test

Reagents

Prep time
Stability
Availability
Consistency (Quality Control)

Performance Criteria

Versatility

Product only

Environmental samples only

Pathogens only
Microorganisms only

Variety of food matrixes

Bacteria and/or Fungi

Acceptability of method by scientific community

and/or Regulators

AOAC, AOAC-RI, USDA-FSIS, FDA, AFNOR

Performance Criteria

Vendor company reputation

Training

First product on market

Vendor provided training on site

How much, how long

Technical Service

Speed of service
Availability of service (24-7)
Service contract required

Technical Evaluation

Objective
Justification (benefit of method to company)
Acceptance Criteria
Material and Methods

Microorganisms

Test Media/Conditions

Genus, species, source

Inoculum preparation

Inoculation Procedure
Statistical Analysis
Results
Next Steps

Case Study #1
Dichloran-Rose Bengal Agar Yeast
and Mold Method Evaluation

Dichloran-Rose Bengal Agar Yeast

and Mold Method Evaluation

Objective: Determine validity of a 2 day

yeast and mold method using DRB agar
incubated at 30C or 35C
Justification: Reduced product holding
time, resulting in significant cost savings
to the plant
Acceptance Criteria: Recovery
efficiencies must be equivalent to the
current 5 day PDA method

Dichloran-Rose Bengal Agar Yeast

and Mold Method Evaluation

Microorganisms:

Mold Cultures

Yeast Cultures

A.niger, Penicillium spp., and Paecilomyces spp.

Z.ballii, S.cerevisiae, and a plant isolate

Inoculum Preparation:

Organisms were harvested from aPDA plates by

washing with sterile water
1ml from each individual mold or yeast suspension
was added to 20 mls DI water

Molds serially diluted

Yeast adjusted to a spec reading of 1.00, then serially diluted

Dichloran-Rose Bengal Agar Yeast

and Mold Method Evaluation

Material and Methods:

product was inoculated with 100 cfu/g of

target organisms
0.1ml of inoculated product surface plated
onto each media (aPDA, DRBA)
aPDA incubated at 25C

Counted at 3 and 5 days

DRBA incubated at 30C and 35C

Counted at 2, 3, 4, and 5 days

Dichloran-Rose Bengal Agar Yeast

and Mold Method Evaluation

Statistical Analysis:
An analysis of variance (AOV) was done to
test if the total counts for DRB at 2 and 5
days was significantly different from aPDA
at 5 days

Dichloran-Rose Bengal Agar Yeast

and Mold Method Evaluation

Results:

DRB at 2 days-30C was statistically equivalent to

aPDA at 5 days for mold recovery

Molds were pale in color; Penicillium spp. was white

on DRB (green on aPDA). The other 2 test molds
were pale yellow

Yeast counts on DRB at 30C were significantly

lower than counts on aPDA at 2 and 5 days
Mold and Yeast counts were significantly lower
on DRB at 35C vs. aPDA

Dichloran-Rose Bengal Agar Yeast

and Mold Method Evaluation

Conclusion:

Due to overall decreased recovery of yeast

and mold, and the mold visual observations;
the Dichloran-Rose Bengal Agar Yeast and
Mold recovery medium is not recommended.

Case Study #2
Rapid Check Salmonella
Test Kit Evaluation

Rapid Check Salmonella

Test Kit Evaluation

Objective: Determine validity of the

Strategic Diagnostics Inc. Rapid Check
antibody lateral flow method for the
detection of Salmonella in comparison to
the BAX PCR test method
Justification: Reduce testing cost, false
positives rate and technician time

Rapid Check Salmonella

Test Kit Evaluation

Acceptance Criteria:

Cost

Speed; shorter time to results vs. PCR?

Sensitivity; greater or equivalent to PCR?
Specificity; greater or equivalent to PCR
Less than or equal to BAX PCR system
Cost per test

Versatility; food products only, environmental

samples only, or both?

Rapid Check Salmonella

Test Kit Evaluation

Organisms and Inoculum Preparation:

A cocktail of 5 Salmonella spp.

A cocktail of 7 non-Salmonella spp.

E.coli (2), Citrobacter, Bacillus, Klebsiella,

Enterobacter (2)

Individual cultures grown overnight in BHI at

35C
Salmonella strains pooled, diluted to 100cfu/ml
Non-Salmonella strains pooled, diluted to 1,000
cfu/ml

Rapid Check Salmonella

Test Kit Evaluation

Methods:

Inoculation of samples

Pre-enrichment of samples

With Salmonella
With non-Salmonella strains
With both
Traditional medium; Lactose for 24 hours
SDI medium for 5 hours

Secondary enrichment

Tetrathionate for 24 hours

Rapid Check Salmonella

Test Kit Evaluation

Methods (cont):

BAX PCR analysis

3 hour re-growth
Cell lysis
4-8 hour PCR cycle

SDI lateral flow assay

Load 150ul onto SDI cartridge
Develop for 10 minutes

Rapid Check Salmonella

Test Kit Evaluation

Results:

Sensitivity

Results were more consistent with SDI when recovering at

the threshold level (1000 cfu/ml in the TT broth)
Equivalent results with both methods above the threshold
level
SDI 5 hour pre-incubation media did not consistently support
growth above the threshold level (acceptance criteria)

Specificity

No cross reactivity with non-Salmonella organisms with

either method

Rapid Check Salmonella

Test
Kit
Evaluation
Results: Speed
BAX-PCR

SDI w/ 5 hour
medium

SDI

Enrichment

24 hours

5 hours

24 hours

Secondary

18 hours

18 hours

18 hours

Re-growth

3 hours

0 hours

0 hours

Cell lysis

0.5 hours

0 hours

0 hours

Analysis
time

8 hours

10 minutes

10 minutes

Total

53.5 hours

23 hours, 10 minutes

42 hours, 10 minutes

Rapid Check Salmonella

Test Kit Evaluation

Conclusions:

SDI shown to be as sensitive as BAX-PCR

5 hour medium not recommended

No cross reactivity observed with SDI

SDI gave results sooner than PCR
PCR has more steps, more prone to technician error
Some degree of subjectivity with SDI
SDI easier to use; 1 step inoculation of 1 single
cartridge

Rapid Check Salmonella

Test Kit Evaluation

Conclusions:

SDI can be successfully used for food and

environmental samples
No additional equipment needed (heat blocks,
thermal cycler)
Cost per test of SDI less than BAX-PCR
SDI approved for use in place of PCR
Appropriate for use by labs analyzing a
smaller number of samples

Value of Method Validation

Need to validate method on your intended product; rule

out matrix interference
Determine minimum regulatory requirements (AOAC,
AFNOR, etc)
Determine what is the right method for your lab based
on volume of testing and number of technicians
Base selection of methodology on need

Sensitivity
Specificity
Speed
Cost
Lab space