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Applicable analytical methods according to the Commission Regulation

(EC) No 2073/2005 on microbiological criteria for foodstuffs

According to the regulation (EC 2073/2005 Article 5), food business operators may use other
methods than listed in Annex 1 if it can be demonstrated to the satisfaction of the competent
authority that these procedures provide at least equivalent guarantees.

The use of alternative analytical methods is acceptable when the methods are validated against
the reference method in Annex I, and if a proprietary method, certified by a third party in
accordance with ISO 16140 or another international recognised protocol (like NordVal protocol).

By “..procedures provide at least equivalent guarantees” we understand methods that give

equivalent results, i.e. methods that provide results that do not differ significant statistically. This
means that alternative methods to those referred to in Annex 1 do not necessarily have to apply
the same principle, however, need to provide equivalent results.

The table below lists the microorganisms and the referred reference methods given in the EC
2073/2005 Annex 1 along with alternative NMKL and NordVal methods that provide at least
equivalent guarantees / results.

Microorganism Reference Alternative Alternative

method NMKL method NordVal methods
Expected to give (Validated acc. to
equivalent results NordVal/ISO 16140)

Listeria EN/ISO 11290-1 NMKL 136 NordVal 022 RAPID’ L.mono

Monocytogenes Horizontal method for Listeria monocytogenes. Detection + enumeration
the detection and Detection and enumeration in (Ref. method: ISO 11290-1)
- detection enumeration of foods and feeding stuffs
thereof Listeria NordVal 025 LightCycler
monocytogenes Validation status: L.mono Detection Kit
Part 1: Detection Collaboratively validated (Ref. method: ISO 11290-1)
method
Comment: NordVal 037 IQ-Check
Validation status: The method applies similar Listeria monocytogenes II
Collaboratively principle as EN/ISO 11290- (Ref. method: ISO 11290-1)
validated 1.
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Microorganism Reference Alternative Alternative

method NMKL method NordVal methods
Expected to give (Validated acc. to
equivalent results NordVal/ISO 16140)

Listeria EN/ISO 11290-2 NMKL 136 NordVal 022 RAPID’ L.mono

Monocytogenes Horizontal method for Listeria monocytogenes. Detection + enumeration
the detection and Detection and enumeration in (Ref. method: ISO 11290-2)
- enumeration enumeration of foods and feeding stuffs
thereof Listeria
monocytogenes Validation status:
Part 2: Enumeration Collaboratively validated
method
Comment:
Validation status: The method applies similar
Not collaboratively principle as EN/ISO 11290-
validated 2.

Salmonella EN/ISO 6579 NMKL 71 NordVal 001

Horizontal method for Salmonella. Detection in Transia Plate Salmonella Gold
the detection of foods. (Ref. method: ISO 6579)
Salmonella spp.
NordVal 010
Validation status: Validation status: Bioline Salmonella ELISA
Partly validated Collaboratively validated Test Optima / RayAl
Salmonella Optima
(Ref. method: ISO 6579)
Comments:
ISO 6579 includes two NordVal 023
selective enrichment LightCycler Salmonella
procedures (RVS and Detection Kit
MKTTn), NMKL 71 includes (Ref. method: ISO 6579)
the RVS procedure. The
principles of the methods NordVal 030
are similar for the RVS part. BAX Salmonella PCR (BAX
The methods have also Classic and BAX Q7) BAX
been compared in studies. System with Automated
Detection PCR Assay for
Screening Salmonella
(Ref. method: ISO 6579)

NordVal 031
Salmonella detection method
by real-time PCR
(Ref. method: NMKL 71,
NMKL 187)

NordVal 032
RAPID' Salmonella - 24 hours
test method
(Ref. method: ISO 6579)

NordVal 038
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Microorganism Reference Alternative Alternative

method NMKL method NordVal methods
Expected to give (Validated acc. to
equivalent results NordVal/ISO 16140)
iQ-Check TM Salmonella II kit
(Ref. method: ISO 6579)

NordVal 041
Salmonella detection method:
DNA extraction by boiling
followed by real-time PCR
(Ref. method: NMKL 187)

E.coli ISO 16649 NMKL 125 NordVal 014

Horizontal method for Thermotolerant coliform 3M Petrifilm E.coli/Coliform
the enumeration of bacteria and Count Plate
beta-glucuroni-dase- Escherichia coli. (Ref. method: ISO 16649-2,
positive Escherichia Enumeration in food and ISO 4832, AOAC 991.14)
coli feed.
Part 1: Colony-count NordVal 020
technique at 44 Validation status: RAPID’ E.coli 2 AGAR
degrees C using Collaboratively validated (Ref. method: ISO 16649-2,
membranes and 5- ISO 4832)
bromo-4-chloro-3-
indolyl beta-D NordVal 036
NMKL 96
Part 2: Colony-count Compact Dry EC Method for
Bacterial Examinations the Enumeration of E.coli and
technique at 44 in Fresh and
degrees C using 5- Coliforms
Frozen seafood. (Ref. method: ISO 16649-2,
bromo-4-chloro-3-
indolyl beta-D- ISO 4832)
Validation status:
glucuronide
Collaboratively validated NordVal 027
Part 3: Most probable
number technique 3M Petrifilm Select E.coli
using 5-bromo-4- Count Plate
chloro-3-indolyl-beta- Comments: (Ref. method: ISO 16649-2)
D-glucuronide The method applies a
different principle than ISO
16649. However, the two
Validation status: Not methods are considered to
collaboratively provide equivalent results.
validated None of the methods will
cover E. coli that does not
grow at lower temperatures
(such as E. coli O157), but
both methods will work fine
for the enumeration of E.
coli in hygiene purposes.

Aerobic microbes ISO 4833 Horizontal On surfaces: On surfaces:

– surface method for the
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Microorganism Reference Alternative Alternative

method NMKL method NordVal methods
Expected to give (Validated acc. to
equivalent results NordVal/ISO 16140)
enumeration of NMKL 5 NordVal 018
microorganisms. Aerobic microorganisms Hygicult TPC
Colony-count and presumptive (Ref. method: NMKL 5)
technique at 30°C . Enterobacteriaceae.
Enumeration on surfaces a
Validation status: nd utensils.
Collaboratively
validated on milk Validation status:
Collaboratively validated
The ISO method is a
colony count method
tested on milk, and
does not describe
enumeration on
surfaces, which is
the scope in the
regulation.
Recommended
method should be
methods similar to
NMKL 5

Aerobic microbes ISO 4833 NMKL 86 NordVal 012

- in foods Horizontal method for Aerobic microorganisms. 3M Petrifilm, AerobicCount
the enumeration of Determination in foods at Plate
microorganisms. 30ºC, 20ºC or 6,5ºC. (Ref. method: ISO 4833)
Colonycount
technique at 30 Validation status:
degrees C. Not collaboratively validated NordVal 033
Compact Dry TC Method for
Validation status: Based on: the Enumeration of Total
Collaboratively The method applies similar Viable Organisms
validated on milk principle as ISO 4833. (Ref. method: ISO 4833)

Entero- ISO 21528-2

bacteriaceae Horizontal methods for
– surface the detection and
enumeration of
Enterobacteriaceae -
Part 2: Colony-count
method

Validation status:
Not validated
collaboratively

This ISO method is a

colony count method
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Microorganism Reference Alternative Alternative

method NMKL method NordVal methods
Expected to give (Validated acc. to
equivalent results NordVal/ISO 16140)
and does not
describe
enumeration on
surface, which is the
scope in the
regulation.
Recommended
method should be
methods similar to
NMKL 5, NordVal 018

Entero- ISO 21528-2 NMKL 144 NordVal 029

bacteriacea Horizontal methods for Enterobacteriaceae. 3M Petrifilm
- in foods the detection and Determination in foods and Enterobacteriaceae Count
enumeration of feeds. Plate
Enterobacteriaceae - (Ref. method: ISO 21528-2)
Part 2: Colony-count Validation status:
method Collaboratively validated NordVal 034
Compact Dry ETB Method for
Validation status: Comments: the Enumeration of
Not validated The method applies similar Enterobacteriaceae
collaboratively principle as ISO 21528-2, (Ref. method: ISO 21528-2)
except for two minor
differences: 1) The ISO
method prescribes two
plates of VRBG-agar per
dilution and NMKL one
plate. 2) The ISO method
prescribes confirmation by
tests for fermentation of
glucose and presence of
oxidase while NMKL is
using oxidase test only.

Stafylococcus ISO 6888-1 + 2 NMKL 66 NordVal 019

aureus Horizontal method for Coagulase positive staphylo 3M Petrifilm Express Count
the enumeration of cocci. Enumeration in foods. System
coagulase-positive (Ref. method: ISO 6888-1)
staphylococci Validation status:
(Staphylococcus Collaboratively validated
aureus and other
species) Part 1: Comments:
Technique using The method applies similar
Baird-Parker agar principle as ISO 6888.
medium Part 2: The NMKL method includes
Technique using the possibility of including
rabbit plasma Blood agar, but does not
fibrinogen agar recommend using this as a
medium single medium, but more
like an extra plate. The ISO
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Microorganism Reference Alternative Alternative

method NMKL method NordVal methods
Expected to give (Validated acc. to
equivalent results NordVal/ISO 16140)
Validation status: method states that the pour-
Validated plate method is used with
collaboratively BP-RPF agar thereby
lowering the detection limit.

E.coli O 157 EN/ISO 16654 NMKL 164 NordVal 026

Horizontal method for Escherichia coli O157. LightCycler food proof E. coli
the detection of Detection in food and O 157 Detection Kit in
Escherichia coli O157 feeding stuffs. combination with ShortPrep
foodproof II Kit
Validation status: Validation status: (Ref. method: ISO 16654)
Not validated Collaboratively validated
collaboratively
Comments:
The method applies similar
principle as ISO 16654.
There are only minor non-
technical differences.