INTERNATIONAL ISO

STANDARD 10272-2

First edition
2017-06

Microbiology of the food chain -

Horizontal method for detection and
enumeration of Campylobacter spp. -
Part 2:
Colony-count technique
Microbiologie de la chalne alimentaire - Methode horizontale pour
la recherche et le denombrement de Campylobacter spp. -
Portie 2: Technique par comptage des colonies

Reference number
ISO 10272-2:2017(E)
ISO 10272-2:2017(E)

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 ISO 10272-2:2017(E)

Contents Page

Foreword ........................................................................................................................................................................................................................................ iv
lntroduction.................................................................................................................................................................................................................................. v
1 Scope ................................................................................................................................................................................................................................. 1
2 Normative references ...................................................................................................................................................................................... 1
3 Terms and definitions ..................................................................................................................................................................................... 1
4 Principle ........................................................................................................................................................................................................................ 2
4.1 General........................................................................................................................................................................................................... 2
4.2 Preparation of dilutions .................................................................................................................................................................. 2
4.3 En umeration ............................................................................................................................................................................................. 2
4.4 Confirmation ............................................................................................................................................................................................. 2
5 Culture media and reagents ..............................................- ..................................................................................................................... 2
6 Equipment and consumables .................................................................................................................................................................. 3
7 Sampling........................................................................................................................................................................................................................ 3
B Preparation of test sample............................................ ........................................................................................... ............................ 3
9 Procedure................................................................................................................................................................................................................. 4
9.1 Test portion, initial suspension and dilutions ........................................................................ ..................................... 4
9.2 Inoculation and incubation .......................................................................................................................................................... 4
9.3 Enumeration of characteristic colonies ............................................................................................................................. 4
9.4 Confirmation of Campylobacter................................................................................................................................................ 4
9.4.1 General...................................................................................................................................................................................... 4
9.4.2 Selection of colonies for confirmation .......................................................................................................... 5
9.4.3 Examinatio n of morphology a nd motility.................................................................................................. 5
9.4.4 Study of aerobic growth at 25 °C ....................................................................................................................... 5
9.4.5 Detection of oxidase activity .................................................................................................................................. 5
9.4.6 Interpretation ..................................................................................................................................................................... 5
9.5 Identification of Campylobacter species (optional)................................................................................................. 6
9.5.1 General...................................................................................................................................................................................... 6
9.5.2 Detection of catalase activity................................................................................................................................. 6
9.5.3 Detection ofhippurate hydrolysis ..................................................................................................................... 6
9.5.4 Detection ofin doxyl acetate hydrolysis ....................................................................................................... 6
9.5.5 Interpretation ............................................................................................................................................................... 7
10 Expression of results ........................................................................................................................................................................................ 7
11 Performance characteristics of the method .............................................................................................................- ............ 7
11.1 lnterlaboratory study........................................................................................................................................................................ 7
11.2 Repeatability limi t................................................................................................................................................................................ 7
11.3 Rep roducibility li mit .......................................................................................................................................................................... 8
12 Test report. ................................................................................................................................................................................................................. 9
Annex A (normative) Diagram ofprocedure.............................................................................................................................................10
Annex B (normative) Culture media and reagents .............................................................................................................................11
Annex C (informative) Method validation studies and performance characteristics.....................................16
Bibliography .............................................................................................................................................................................................................................19
ISO 10272-2:2017(E)

Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Sta ndards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the r·ight to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, a lso take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (I EC) on a ll matters of
electrotechnical standard izatio n.
The procedures used to develop this document and those in tended for its further mai ntenance are
described in the lSO/IEC Directives, Part 1. In particular the different approva l criteria needed for the
d ifferent types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www jso or g/djrectjyes).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent ri ghts. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the I SO list of patent declarations received (see www iso org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning ofISO specific terms and expressions related to conformity assessment,
as well as information about ISO's adherence to the World Trade Organization (WTO) principles in the
Technical Barriers to Trade (TBT) see the following URL: www jso.org/iso/foreword htm l.
This document was prepared by the European Committee for Standardization (CEN), Technical
Committee CE N/TC 275, Food Analysis - Horizontal methods, in collaboration with ISO Technical
Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology in accordance with the
agreement on technical cooperation between ISO and CEN (Vienna Agreement).
This first edition cancels and replaces 1$0/TS 10272-2:2006, which has been technically revised with
the following main changes:
samples from the primary production stage have been added to the scope;
ser.ial dilutions are plated in single instead of in duplicate, to be in line with ISO 7218;
the confirmation tests on study ofmicroaerobic growth at 25 °C and aerobic growth at 41,5 °C were
replaced by the study of aerobic growth at 25 °C;
performance testing for the quality assurance of the culture media has been added to Annex B:
perfo rmance characteristics have been added to Ann ex C.
A list of a ll parts in the ISO 10272 series can be found on the ISO website.
 ISO 10272-2:2017(E)

Introduction
The main changes, listed in the foreword, introduced in this document compared to ISO/TS 10272-
2:2006 are considered as minor (see ISO 17468).
Because of the large variety of food and feed products, this horizontal method may not be appropriate
in every detail for certain products, and for some other products, it may be necessary to use different
methods. Nevertheless, it is hoped that in all cases, every attempt will be made to apply this horizontal
method as far as possible and that deviations from this will only be made if absolutely necessary for
t echnical reasons.
When this document is next reviewed, account will be taken of all information then ava ilable regarding
the extent to which th is horizontal method has been followed and the reasons for deviations from this
in the case of particular product s. The harmonization of test methods cannot be immediate and, for
certain group of products, International Standards and/or national standards may already exist that do
not comply with this horizontal method. It is hoped that when such standards are reviewed, they will
b e changed to comply with this document, so that eventually, the only remaining departures from this
horizonta l method will be those necessary for well-established technical reasons.
INTERNATIONAL STANDARD ISO 10272-2:2017(E)

Microbiology of the food chain - Horizontal method for

detection and enumeration of Campylobacter spp. -
Part 2:
Colony-count technique
WARNING - In order to safeguard the h ealth oflaboratory personnel, it is essential that tests for
enumeration of Campylobacter are only undertaken in properly equipped laboratories, under
the control of a skiJled microbiologist, and that great care is taken in the disposal of all incubated
materials. Persons using this document should be familiar with normal laboratory practice. This
document does not purport to address all of the safety aspects, if any, associated with its use. It
is the responsibility of the user to establish appropriate safety and health practices.

1 Scope
This document specifies a horizontal method for the enumeration of Campy!obacter spp. It is applicable to
products inte nded for human cons umption,
products in tended for animal feeding,
environme nta l samples in the area of food and feed production, handl"ing, a nd
samples from the primary production st age such as animal faeces, dust, a nd swa bs.

2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this do cument. For dated references, only the edition cited appUes. For
undated references, the latest edition of the referenced document (incl ud ing any amendmen ts) appl ies.
ISO 6887 (all parts), Microbiology of the food chain - Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination

ISO 7218, Microbiology of food and animal feeding stuffs - General requirements and guidance for
microbiological examinations
ISO 11133, Microbiology of food, animal feed and water - Preparatton, production, storage and
performance testing of culture media

3 Terms and definitions

For the purposes of this document, the following terms and definitions apply.
ISO and IEC mainta in terminological databases for use in standardization at the following addresses:
IEC Electropedia: available at http://www.electropedia.org/
ISO Online browsing platform: available at http:/jwww.iso.org/obp
ISO 10272-2:2017(E)

3.1
Campylobacter
microorganism forming characteristic colonies on solid selective media when incubated in a
microaerobic atmosphere at 41,5 °C, and which possesses the charact eristic morpho logy and motility
and biochemical and growth properties described when the tests are conducted in accordance with
this document
Note 1 to entry: This document targets the thermotolerant Campy/obacter species relevant to human health.
The most frequently encountered and relevant to human health are Campylobacterjejuni and Campylobacter coli.
Howeve1-. other species have been described (Campylobacter lari, Campylobacter upsaliensis and others).

3.2
enumeration of Campylobacter
determination of the number of colony· forming units (cfu) of Campylobacter (l l) found per gram, per
millilitre, per square centimetre or per sampling device when the test is conducted in accordance with
this document

4 Principle

4.1 General
The enumeratio n of Campylobacter requires three successive stages as specified in Annex A.

4.2 Preparation of dilutions

For the preparation of decimal di lutions from the test portion, see ISO 6887.

4.3 Enumeration
The solid selective medium modified Charcoal Cefoperozone Deoxycholate agar (mCCD agar) is
inoculated w ith a specifi ed quantity of the test portion if the product is liquid or of t he initial suspension
in the case of other products.
Other plates are prepared under the same conditions, using decimal dilutions of the test portion or of
t he init ial suspension.
The plates a re incubated at 41,5 °C in a microaerobic atmosphere and examined after 44 h to record the
number of sus pect Campy/obacter colonies.

4.4 Gonfirmation
The suspect Campy/obacter colonies are examined for morphology and mot ility using a microscope
and sub-cultured on a non-selective blood agar, and then confirmed by detection of oxidase activity
and an aerobic growth test at 25 °C. Optionally, the Campylobacter species are identified by specific
biochemical tests and/or mo lecular methods.
The number of colony-fo rming units (cfu) of Campylobacter per unit of the test portion is calculated
from t he number of confirmed typical colonies per plate.

5 Culture media and reagents

For current laboratory practice, see ISO 7218 and ISO 11133.
Composition of culture media and reagents and their preparation are described in Annex B.
For performance testing of culture media, see Annex B.
 ISO 10272-2:2017(E)

6 Equipment and consumables

Disposable eq uipment is an acceptable alternative to reusable glassware if it has suitable specifications.
Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following.

6.1 Incubators, capable of operating at 25 "C ± 1°C,37 °C ± 1 °C and 41,5 °C ± 1 °C.

6.2 Water bath, capable of operating at 37 °C ± 1 °C.

6.3 Sterile loops, of 10 µI volume and of 1 ~ti volume, and inoculation needle or wire.

A nickel/chromium loop is not s uitable for use in the oxidase test (see 2...1:..5).

6.4 Microscope, preferably with phase contrast (for observing the characteristic morphology and
motility of Campylobacter).

6.5 Appropriate apparatus for achieving a microaerobic atmosphere with oxygen content of
5 % ± 2 %. carbon dioxide 10 % ± 3 %, optional hydrogen $10 %, with the balance nitrogen.

The appropriate microaerobic atmosphere can be obtained using gas tight jars and gas-generating kits,
following precisely the manufacturer's instructions. Alternatively, the jar or incubator may be filled
with an appropriate gas mixture prio-r to incubation.

6.6 Sterile Petri dishes, with a diameter of approximately 90 mm and (optional) large size (diameter
approx imately 140 mm), preferably with vents to facili tate microaerobic incubation.

6.7 Refrigerators, capable of operating at 3 °C ± 2 °C and at 5 °C ± 3 °C.

7 Sampling
Sampling is not part of the method specified in this document. See the specific International Standard
dealing with the product concerned. If there is no specific International Standard dealing with the
sampling of the product concerned, it is recommended that the parties concerned come to an agreement
on this subject.
A recommended sampUng method is given in ISO/TS 17728 for food and anima l feed, in ISO 13307 for
sampling at the primary production stage, in ISO 17604 for sampling of carcasses, and in ISO 18593 for
sampling of s urfaces.
It is important that the laboratory receives a sample that is representative and the sample should not
have been damaged or changed during trans port or storage.
Since Campylobacter is very sensitive to freezing but survives best at low temperatures, samples to be
t ested should not be frozen, but stored at 3 °C (.6..2.) and subjected to analysis as rapidly as possible.
Also, take care to prevent the samples from drying.

8 Preparation of test sample

Prepare the test sample from the laboratory sample in accordance with the specific International
Standard dealing with the product concerned; see ISO 6887 (all parts). If there is no specific International
Standard, it is recommended that the parties concerned come to an agreement on this subject.
ISO 10272-2:2017(E)

9 Procedure

9 .1 Test portion, initial suspension and dilutions

See ISO 6887 and the specific International Standard dealing with the product concerned.
Prepare a s ingle decimal dilut ion ser ies from t he test portion if the product is liquid or from t he initial
suspension in the case of other products.

9.2 Inoculation and incubation

9 .2.1 Using a sterile pipette, transfer 0,1 ml of the initial suspension (or sample if liquid) (.2..1) to the
mCCD agar plate (.11..1). Repeat the procedure using further decima l dilutions if necessary. If only the
initial suspension is used, prepare duplicate p lates using an additional agar plate.

When, for certain products, it is necessary to estimate low numbers of Campylobacter. the limit of
enumeration may be lowered by a factor of 10 by examining 1,0 ml of t he initial suspension. Distribute
the 1,0 m l of inoculum either on the surface of t he agar medi um in a large Petri d.ish (140 mm) or three
regular plates (90 mm). In bot h cases, prepare duplica tes by using two large plates or six r eg ular plates.

9 .2.2 Evenly spread the inoculum, as quickly as possible, over the surface of the agar plate, using a
sterile spreader. Avoid touching the s ides of the Petri dish with the spreader.

NOTE Drying of the plates is critical to produce countable plates. Each labo ratory has to use its own
standardised way to dry the plates in a proper way.

9 .2.3 Incubate th e plates (9.2 2) at 41,5 °C (t i) in a microaerobic atmosphere(~).

9.3 Enumeration of characteristic colonies

9 .3.1 Aher 44 h ± 4 h of incubation, examine the plates (2.U) for typical and/ or suspect colonies of
Campylobacter.

Typical colonies are greyish on mCCD agar, often wit h a metallic sheen, and are flat a nd moist, with a
tendency to spread. Colonies tend to spread less on drier agar surfaces. Other forms of colonies may occur.
NOTE The recognition of colonies of Campy/obacter is to a large extent a matter of experience and their
appea r a nee can va ry somewhat, not only from stra in to strain, but also from batch to batch of the selective
culture medium used.

9 .3.2 Select the plates (2...3...l) conta ining less than 150 typical or suspect colonies; coun t these colonies
and reco rd their number as presumptive colonies per dish. Then choose at random five such colonies for
subculturing for the confirmation tests (t i) .

9.4 Confirmation of Campy/obacter

9 .4.1 Gen er al

As Campylobacter rapidly loses culturability in air, follow the procedure described in !M2 to ~
without de lay.
For a clear disti nction bet ween posit ive and negative confirmation reactions, it is helpful to verify this
with well-characterized positive and negative co ntrol strains. Examples of suitable control strains are
Campylobacter jejuni WDCM 00005 (positive control)[lQJ and Escherichia coli WDCM 00013 (negative
control).
 ISO 10272-2:2017(E)

As an alternative, or in addition, to the confirmation and identification tests described in this document,
other tests (PCR tests, serological methods, matrix-assisted laser desorption/ionization time-of-
flight mass spect rometer (MALDl-TOF-MS) analysis, etc.) can be used, providing the suitab ility of t he
alternative procedure is verified (see ISO 7218).

9.4.2 Selection of colonies for confirmation

9.4.2.1 For confirmation, take five presumptive colonies from each dish retained for enumeration
(ll..2.).

9.4.2.2 Streak each of the selected colonies onto a non-selective blood agar plate, e.g. Columbia
blood agar (.BA) in order to allow the development of well-isolated colonies. Incubate the plates in a
microaerobic atmosphere (.6.5.) at 41,S °C (i l) for 24 h to 48 h. Use well-isolated freshly grown colonies
for examination of morphology and motility (il.:l), absence of aerobic growth at 25 °C (.2..i.i) and the
presence of oxidase activity (ti.5.).
NOTE The suspect colony could be previewed for characteri stic morphology and motility before streaking
on blood agar.

9.4.3 Examination of morphology and motility

9.4.3.1 Examine a freshly grown colony from each individual plate (.9.Al.Z) for morphology and
motility using a microscope (M ).

9.4.3.2 Retain for further examination all cultures (9.4.2.2) in which curved bacilli with a spiralling
"corkscrew" motility are found (.9A:.ll).

9.4.4 Study of aerobic growth at 25 °C

Using the colonies isolated in l l l l inoculate with the aid of a loop (23) the surface of a non-selective
blood agar plate, e.g. Columbia blood agar (M ).
Incubate the plate at 25 °C (hl) aerobically for 44 h ± 4 h.
Examine the plate for absence of growth of colonies.

9.4.5 Detection of oxidase activity

Using a loop (23), take a portion of a well-isolated colony from each individual plate (2A.2..Z) and streak
it onto a filter paper moistened with the oxidase reagent (.B...!i); the appearance of a mauve, violet or
deep blue colour within 10 s indicates a positive reaction. If a commercially available oxidase test kit is
used, follow the manufacturer's instructions.

9.4.6 Interpr etation

Campylobocter gives results in accordlance with :J::ahk.1.
ISO 10272-2:2017(E)

Table 1 - Characteristics of Campy lobacter

Morphology (2.i.l) Small curved bacilli•

Motility (li.l) Characteristic corkscrew darting•
Aerobic growth at 25 °C (2,i.4) -
Ox idase activity (.2.1..5.) +
+ Positive.
- Negative.
• Older cultures may rapidly lose their characteristic shape and motility and turn into less motile
coccoid- forms.

9.5 Identification of Campylobacter species (optional)

9 .5.1 Genera l

Among the Campylobacter spp. grow ing at 41,5 °C, the most freq uently encountered species
are Campylobacter jejuni and Campylobacter coli. Other species have, however, been described
(Campylobacter lari, Campylobacter upsaliensis and others); the characteristics given in Table 2 permit
their differentiation.

9.5.2 Detection of catalase activity

F'o r each colony selected in 9.4.2.2 depos it a loop of cult ure in to a drop of hydrogen peroxide solu t ion
(B.6) on a clean mi croscope slide.
The test is posit ive if bubbles appear within 30 s.
Confirm the results us ing positive and negative controls. Examples of suitable control strains are
Campylobacter jejuni WDCM 00005 (positive control) and Enterococcus faecalis WDCM 00087 (negative
co nt rol).

9 .5.3 Detection ofhippurate hydrolys is

For each colony selected i n ~ use a 10 µ I loop (.12..3) with a heavy inoc ul um to prepare a suspension
in a tube of appropriate size containing 0,4 ml of a sodium hippurate solution (R.'.ZJ.), taking care not to
incorporate any agar.

Shake in order to mix t horoughly and incubat e for 2 h ± 5 min in a water ba th at 37 °C (2,2) or 4 h ± 5 min
in an incubator at 37 °C (hl).
Carefully add 0,2 ml of a ninhyd rin solut ion (.!iU) on top of the sodi um hippurate solution. Do no t shake.
Interpret after incubation of 5 min to 10 min at 37 °C (.6..l or .6..1.) -
A dark v iolet colour indicates a positive reaction.
A pale violet colour or no colour cha nge indicates a negative reac tion.
Confirm the results us ing positive and negative controls. Examples of suitable control strains are
Campylobacter jejuni WDC M 00005 (positive control) and Campylobacter coli WDC M 00004 (negative
co nt rol).

9 .5.4 Detection ofindoxyl acetate hydrolysis

Place a 1 µI loopful of colony material (l l U) on an indoxyl acetate disc (JUl) and add a drop of sterile
distilled water.
 ISO 10272-2:2017(E)

If the indoxyl acetate is hydrolysed, a colour change to dark blue occurs within 5 min to 10 min. No
colour change indicates hydrolysis has not taken place.
Confirm the results using positive and negative controls. Examples of suitable control strains are
Campylobacter jejuni WDCM 00005 (positive control) and Campylobacter /ari WDCM 00204 (negative
control).
If commercially available indoxyl acetate discs are used, follow the manufacturer's instructions.

9.5.5 Interpretation

Campylobacter species grow ing at 41, 5 °C may be identified at species level according to Ia.b.le..2..

Table 2 - Characteristics of Campylobacter species

Characteristic C.jej1111i C. coli C. l ari C. upsaliensis
Catalase act ivity (2.5.2) + + + - or weak
Hippurate hydrolysis (.2.5...1) +a - - -
Jndoxyl acetate hydrolysis(~) + + - +
+ Positive.
- Negative.
a Some hippurate·negative C.jeju11i strain.shave been reported.

10 Expression of results
See ISO 7218. Calculate and report the result as the number of Campy/obacter in cfu per gram, per
millilitre, per sq uare centimeter or per sampling device.

11 Performance characteristics of the method

11.1 Interlaboratory study

Results of the in terlaboratory study to determine the precision of the method are summarized in
Annex C. Repeatability and reproducibility limit s were determined using 5 sample types (broiler caecal
material, frozen spinach, frozen minced meat, raw milk, chicken skin) contaminated at various levels.
The values derived from the interlaboratory study may not be applicab le to concentration ranges and
sample types other than those given in Annex C.

11.2 Repeatability Umit

The absolute difference between two independent single (log1 o-transformed) test results (number
of cfu per gram or per millilitre) or the ratio of the higher to the lower of the two test results on the
n ormal scale, obtained using the same method on identical test material in the same laboratory by t he
same operator usi ng the same appa ratus within the shortest feasible time inter val, will, in not more
t han 5 % of cases, exceed the repeatability limit, r.
As a general indication of repeatabi lit y limit (r), the following overall va lues (derived from the mean of
the variance estimates for a ll levels per matrix tested in t he ILS, see data in Annex C) may be used when
t esting broiler caecal material samples:
r = 0,38 (expressed as a difference between log10-transformed test resu lts), or

r = 2,38 (expressed as a ratio between test results).

ISO 10272-2:2017(E)

The following overall values may be used w hen testing chicken skin samples:

r= 0,98 (ex pressed as a difference between log10·transformed test results), or

r: 9,52 (expressed as a r at io between test results).

EXAMPLE A test result of l 000 000 or 1,0 x 106 or log10 6,0 cfu per gram of broi ler caecal material was
observed in a given laboratory. Under repeatability conditions, the difference between log10-transformed resu lts
should not be greater than ±0,38 log10 units. So the result from a second test of the same sample should be
between 5,62 (6,0 - 0,38) and 6,38 (6,0 + 0,38) log10 units.
For non-log-transformed resu lts, the ratio between the fi rst test resu lt and the second test resu lt from the same
sample should not be greater than 2,38. So the second test result should be between 420 000 (= 1 000 000/2,38)
and 2 400 000 (1 000 000 x 2,38) cfu per gram.

11.3 Reproducibility limit

The absol ute difference between two si ngle ( log10-tra nsfo rmed) test r esults (numbe r of cfu per gram or
per millili tre) or t he rat io of t he higher t o t he lower of t he two test res ul ts on t he normal scale, ob tained
using th e same method on identical test material in different laboratories w ith different operators
using different equipment, will, in not more than S % of cases, exceed the reproducibility limit. R.

As a general indication of reprod ucibility limit (R), the following overall val ues (derived from the mean
of the variance estimates for all levels per matrix tested in the ILS, see data in Annex C) may be used
when testing broiler caecal material samples in genera l:

R = 0,91 (expressed as a d ifference between log10-transfonn ed test result s), or

R = 8,14 (expressed as a ratio between test results).

T he following overall va lues may be used when testing chicken skin samples:

R = 1,31 (expressed as a difference between log10-transformed test resu lts), or

R = 20,43 (expressed as a ratio between test r esults).

EXAMPLE 1 A test result of 1 000 000 or 1,0 x 106 or log10 6,0 cfu per gram of broi ler caecal material was
observed in a first laboratory. Under reproducibility conditions, the difference between log10-transformed
results should not be greater than ±0.91 logio units. So the result from a second laboratory should be between
5.09 (6,0 - 0,91) and 6.91 (6,0 + 0,91) log10 units.
For non -log-transformed results, the ratio between the test result from this first laboratory and a second
laboratory should not be greater than 8.14. So the result from the second laboratory should be between 120 000
(= 1 000 000/8,14) and 8 100 000 (1 000 000 x 8,14) cfu per gram.

EXA MPLE 2 A laboratory wants to know the maximum value it may find for a poultry skin sample, which is
still in compliance with a pre-set limit (e.g. a limit of 1 000 or log10 3). For th is, the R value (on the log scale) has to
be muJtipl ied by a factor of0,59.
The factor 0,59 reflects the fact that a test with a one-sided 95 % interval is used to test whether the limit is
64
exceeded; it is obtained from the following formula: 0,59 = l, fi.
1,96x 2
The max imum va lue is 0,77 (1,31 x 0,59) as a difference between log1o·transformed test resu lts or 5,93 (100.77)
as a ratio between test results. So results up to log10 3,77 (log10 3 + log10 0,77) or 5 900 (1 000 x 5,93) do not
indicate non-compliance with the limit.
 ISO 10272-2:2017(E)

12 Test report
The test report shall specify t he following:
the test method used, with a reference to this document, i.e. ISO 10272-2;
the sampling method used, if known;
the nature of the objects examined;
all operating details not specified in document, or regarded as optiona I, together with details of any
incidents which may have infl uenced the test r esult(s);
any deviation in the media or the incubation conditions used;
all informat ion necessary for the complete ident ificat io n of the sample;
the test result(s) obtained.
ISO 10272-2:2017(E)

Annex A
(normative)

Diagram of procedure

Test portion (x g or x ml)

..
Dllue11t for the prepa ration of the initial
suspension (generally 9 x g or 9 x m l)

l
Furthe.r decimal dilution series

1
Surface inocula tion of mCCD agar (B.3)

1
Incubation In a microaeroblc atmosphere
a t 41,5 •c for H b :t 4 h

l
Enume ration of characteristic colonies (9.3)

l
Confirmation (9.4.2 to 9.4.6)

l
Identification (optional) (9.5)

l
Expression of result< {l 0)
and test report (12)

Figure A.1 - Diagram of the procedure for enumeration of Campylobacter in the food chain
 ISO 10272-2:2017(E)

AnnexB
(normative)

Culture media and reagents

B.1 General
The general specificat ions of ISO 11133 are applicable to the preparation and perfor mance testing of
th e culture media described in this annex. If culture media or reagent s are prepared from dehydrated
complete media/reagents or if ready-to-use media/reagents are used, follow the manufacturer's
instructions regarding preparation, storage conditions, expiry date and use.

The shelf lives of t he media indicated in this annex have been determined in some studies. The user
should verify these under t heir own storage conditions (as specified in ISO 11133).

Performance testi ng of culture media is described in .11.ft.

B.2 Diluent
See ISO 6788.

B.3 Modified charcoal cefoperazone deoxycholate agar (mCCD agar)

B.3.1 Basic medium

B.3.1.1 Composition

Meat extract 10,0g

Enzymatic digest of animal tissues 10,0 g

Sodium chloride (CAS No. 7647-14-5) 5,0 g

Activated charcoal (CAS No. 7440-44-0) 4,0 g

Enzymatic digest of casein 3,0 g

Sodium deoxycholate (CAS No. 302-95-4) 1,0 g

lron(ll) sulfate hydrate (CAS No. 13463-43-9) 0,25 g

Sodium pyruvate (CAS No. 113-24-6) 0,25 g

Agar 8,0 g to 18,0 ga

Water 1000 ml

a Depending on the gel strength of the agar.

ISO 10272-2:2017(E)

B.3.1.2 Preparation

Dissolve the basic components or the dehydrated complete basic medium in the water, by bringing to
the boil. Adjust the pH, if necessary, so that after steriUzation it is 7,4 ± 0,2 at 25 °C. Dispense the basic
medium into flasks of suitable capacity. Sterilize in the autoclave set at 121 °C for 15 min.

B.3.2 Antibiotic solution

B.3.2.1 Composition

Cefoperazone sodium salt (CAS No. 62893-20-3) 0,032 g

Amphotericin B (CAS No. 1397-89-3) 0,01 g
Water 5 ml

B.3.2.2 Preparation
Dissolve the components in t he water. Sterili ze by filtration.

B.3.3 Complete medium

B.3.3.1 Composition

Basic medium (B.ll) 1000 ml

Antibiotic sol ution (B...32) S ml

B.3.3.2 Preparation
Add the antibiotic solution to the basic medium, cooled down to 44 •c to 47 •c, then mix carefully. Pour
18 ml t-0 20 ml of the complete medium into sterile Petri dishes (M ). Allow to solidify. Immediately
before use, carefully dry the agar plates, preferably with the lids off and the agar surface downwards,
in a drying cabinet for 30 min or until the agar surface is free of visible moisture. If they have been
prepared in adva nce, store the undried agar plates in t he dark at S °C (.6...Z.) for up to 1 month.

B.4 Columbia blood agar

B.4.1 Bask medium

B.4.1.1 Composition

Enzymatic digest of animal tissues 23,0g

Starch soluble (CAS No. 9005-84-9) 1,0 g
Sodium chloride (CAS No. 7647-14-5) 5,0 g

Agar 8,0 g to 18,0 ga

Water 1 000 ml
a Depending on t he gel strength of the agar.
 ISO 10272-2:2017(E)

B.4 .1.2 Preparation

Dissolve the basic components or the dehydrated complete medium in the water, by heating. Adjust t he
pH, if necessary, so that after sterilization it is 7,4 ± 0,2 at 25 °C. Dispense the basic medium into flasks
of suitable capacity. Sterilize in the autoclave set at 121 °C for 15 min.

B.4.2 Sterile sheep or horse blood

B.4.3 Complete medium

B.4.3.1 Composition

Basic medium (.IM:.1) 1000 ml

Sterile blood (HA.2) 50ml

B.4.3.2 Preparation

Add the blood aseptically to t he basic medi um, cooled down to 44 °C to 47 °C, then mix. Pour 18 ml to
20 ml of t he complete medium into st erile Petri d ishes(§&). Allow to solidify. Immediately before use,
carefully dry the aga r plates, prefera.bly with the lids off and the agar surface downwards, in a drying
cabinet for 30 min or until the agar surface is free of visible moisture. If they have been prepared in
advance, store the undried agar plates in the dark at 5 °C (hl) for up to 1 month.

B.5 Reagent for the detection of oxidase activity

B.5.1 Composition

N,N,N:N'-Tetramethyl-1,4-p henylenediamine dihydrochloride (CAS No. 637-01-4) 1,0 g

Water 100ml

B.5.2 Preparation
Dissolve the component in the water immediately prior to use.

B.6 Reagent for the detection of catalase activity

B.6.1 Composition

Hydrogen peroxide solution, vo lume fraction of 30 % in water (CAS No. 7722-84-1) 1 ml

Water 9ml

B.6.2 Preparation
Dissolve the component in the water immediately prior to use.
ISO 10272-2:2017(E)

B.7 Reagents for the detection of hydrolysis ofhippurate

B.7.1 Sodium hippurate solution

B.7.1.1 Composition

Sodium hippurate hydrate (CAS No. 532-94-5) 10 g

Phosphate-buffered saline (PBS) consisting of:

Sodium chloride (CAS No. 7647-14-5) 8,5 g

Disodium hydrogen phosphate dihydrate (CAS No. 10028-24-7) 8,98 g

(Na2HP04·2H20)

Sodium dihydrogen phosphate monohydrate (CAS No. 10049-21-5) 2,71 g

(NaH2P04·H20)

Water, to a final volume of 1000 ml

B.7.1.2 Preparation

Dissolve the sodium hippurate in the PBS solution. Sterilize by filtration. Dispense the reagent
aseptically in quant ities of 0.4 ml into small tubes of suitab le capacity. Store at about -20 °C.

B.7.2 Ninhydrin solution, mass fraction of3,5 %

B.7.2.1 Composition

Ninhydrin (CAS No. 485-47-2) 1.75 g

Acetone (CAS No. 67-64-1) 25ml

2-Butanol (CAS No. 78-92-2) 25 ml

B.7.2.2 Preparation

Dissolve the ninhydrin in the acetone/butanol mixture. Store the solution in the dark.
The solution shall not be kept for more than 4 h at ambient temperature, or more than 7 days at
5 °C (Q.7).

B.8 lndoxyl acetate discs

B.8.1 Composition

lndoxyl acetate (CAS No. 608-08-2) 0,1 g

Acetone (CAS No. 67-64-1) lml

B.8.2 Preparation
Dissolve the indoxyl acetate in the acetone. Add 25 ~ti to 50 µl of tbis solution to blank paper discs
(diameter 0,6 cm to 1,2 cm). After drying at room temperat ure, store the discs at 5 °C (.6..Z.) in a brown
tube or bottle in the presence of silica gel.
 ISO 10 272-2:2017(E)

B.9 Performan ce testing for the quality assurance of the culture med ia
Performance testing of t he culture media shall be carried out according to ISO 11133, which includes
definition s for productivity and selectivity. Table B 1 gives details of cont rol strains to be used for
p erformance testing of culture media specified in t his document Where more than one strain is listed
for each aspect of performance testing (productivity, selectivity), one of t he strains which species is
indicated by the letter d has to be used as a minimum. Commercial or non-commercial suppliers are
expected to use additional strains, e.g. those shown in Table B l to further ensure the quality of t he
cult ure media t hey supply.

Table B.1 - Per for mance testing of cultu re media for Campylobacter
Cbarac·
terlstlc
Control WDCM Reference Method of Criteria< reactions
Mediu m Function Incubation strains numbers• media control of target
microor-
ganism
mCCD Productivity Campylobac· 00156 or Greyish.
aga r ter jejunid 00005 fla t and
Quantitative moist,
Campylobac· 00004 Blood aga r Pn:?: 0,5
ter coli
someti mes
with me·
(44± 4) h/ tall ic sheen
(41,5 :I: 1) °C
Selectivity Escherichia 00012 or Total or
microaerobic coJid pa rt ial No char·
00013
a tmosphere - Qualitat ive
inhibition
acteristic
colonies
(0·1)
Scaphylococ· 00034 Total
cusaureus - Quali tat ive in hibition -
(0)
Columbia Product iv ity 24 h to 48 h/ Campylohac- 00156 or Media batch
blood aga r (41,5 :!: 1) °C ter jejunid 00005 blood agar Good
a lready Qualitative -
microaerobic Campylobac· 00004 growth
a tmosphere ter coJid validated
3 WDCM: \Vorld Data Centre for Microorganisms. Refer to t he reference strain catalogue available at wwwwfrc jnfn for in formation on
culture s l rain numbers and contact detailsllQJ.
b Slra ln to be used as a minimum.
c Growth is categorized as 0: no growth: 1: weak growth: 2: good growth, PR= productivity ratio (see 150 11133).
d Strain free of choice, one of the s trains has to be used as a minimum.
ISO 10272-2:2017(E)

Annexe
(informative)

Method validation studies and performance characteristics

An interlaboratory study involving 15 laboratories in 1.2 countries was carried out. The following
samples types were involved in the study: broiler caecal material, frozen spi nach, frozen minced meat
(pork/beef), raw milk and chicken skin. The samples were each tested at three different levels of
contamination, plus a negative control. The study was organized in 2013 by the National Institute for
Public Health and the Environment (RIVM), Bilthoven, The Netherlands, as part of t he CEN Mandate
M381 from the European Commission.
The broiler caecal material samples were contaminated artific ially before sending to the participants.
The samples of the other matrices were contaminated artificially by each lab according to the detailed
standard operation procedure (SOP).

The performance characteristics shown in Tah les C.1 to id were calculated in accordance with
ISO 5725-2:1994. Data obtained by some collaborators have been excluded from t he calculations only
on the basis of clearly identified techn ical reasons (deviations to the protocol, or a ll counts below 10 cfu
per plate per sa mple).

Table C.1- Results of data analysis obtained with broiler caeca l material
Parameter Sample type: Broiler caecal material
Low level• Medium level• High level•
Number of participating collaborators 15 15 15
Number of collaborators reta ined after evaluation of the data 13 8 11
Nu111be1· of samples tested 30 30 30
Number of sample resu lts retained after evaluation of the data 26 16 22
Mean value l:a ( log10 cfu/g) 5,1 5,4 6,7
Repeatability standard deviation Sr (log10 cfu/g) 0,13 0,15 0,13
Repeatability li mit r:
as difference on log10 scale (log10 cfu/g) 0,36 0,42 0,35
a s ratio on normal scale (cfu/g) 2,3 2,6 2,2
Reproducibility standard deviation -~R (log10 cfu/g) 0,38 0,31 0,28
Reproducibility limit R:
as diffel'ence on log10 scale (log10 cfu/g) 1,07 0,86 0,78
as ratio on normal scale (cfu/g) 11,7 7,2 6,0
• Inoculation stra in: C.jejuni {DSM 24306/CNET 076}.
 ISO 10272-2:2017(E)

Table C.2 - Results of da ta analys is obtained with fn>zen s pina ch

Pa ra meter Sample type: Frozen s pinach
Low level• Medium level• High level•
Number of partici pating collaborators 15 15 15
Number of collaborators retained after evaluation of the data 11 10 14
Number of samples tested 30 30 30
Number of sample resu lts retained a fte1- eva luation of the data 22 20 28
Mean value !a (log10 cfu/g) 3,7 4,6 5,3
Repeatabil ity standard dev iation Sr (log10 cfu/g) 0,17 0,10 0,17
Repeatabil ity limit r:
as difference on log10 sca le ( log10 cfu/g) 0,47 0,28 0,48
as ratio on normal scale (cfu/g) 3,0 1,9 3,0
Reproducibility standard deviation su (log10 cfu/g) 0,32 0,40 0,50
Reproducibility limit R:
as difference on log10 sca le ( log1.0 cfu/g) 0,89 1,13 1,40
as ratio on normal scale (cfu/g) 7,7 13,4 25,0
n Inoculation st rain: C. jejuni (WOCMOOOQS).

Table C.3 - Results of data analysis obtained with frozen minced meat (pork/beef)
Parameter Sample type: Frozen mince d meat (pork/
be ef)
Low level• Medium level• High level•
Number of participating collaborators 15 15 15
Number of coll aborato rs retained after e valuation of the data 8 9 12
Number of samples tested 30 30 30
Number of sample results retained a fter eva luation of the data 16 18 24
Mean value Ea (log10 cfu/g) 3,6 4,7 5,1
Repeatability standard deviation Sr (log10 cfu/g) 0,17 0,11 0,43
Repeatability limit r:
as difference on log10 sca le ( log10 cfu/g) 0,49 0,32 1,20
as ratio 011 nor mal scale (cfu/g) 3,1 2,1 15,7
Reproducibility standard dev iation SR (log10 cfu/g) 0,24 0,41 0,52
Reproducibility limit R:
as difference on log10 scale ( log10 cfu/g) 0,68 1.16 1,44
as ratio on normal scale (cfu/g) 4,8 14.4 27,8
a Inocu lation strai n: C. coli (WDCM 00072).
ISO 10272-2:2017(E)

Table C.4 - Results of data ana lysis obtaine d w it h raw milk

Parame ter Sample type: Raw milk
Low level• Medium level• High level•
Number of participating coll aborators 15 15 15
Number of collaborators retained after evaluation of the data 11 10 13
Number of samples tested 30 30 30
Number of sample resu lts retained after evaluation of the data 22 20 26
Mean val ue Ea ( log10 cfu/g) 3,7 4,8 5,9
Repeatability standard deviation sr (log10 cfu/g) 0,19 0,22 0,12
Repeatability li mit r:
as diffe1·ence on log10 scale (log10 cfu/g) 0.53 0,62 0,34
as ratio on normal scale (cfu/g) 3,4 4,2 2,2
Reproducibility standard deviation SR (log10 cfu/g) 0,33 0,47 0,37
Reproducibility limit R:
as difference on log10 scale (log10 cfu/g) 0,92 1,32 1,03
as ratio on normal scale (cfu/g) 8,4 21,Q 10,6
• Inoculation strain: C. jejuni (WDCM 00156) .
Table C.5 - Results of data analys is obtained with chicken skin
Pa rameter Sample type: Chicke n s kin
Low level• Medium level• High level•
Numbe1· of participating collaborators 15 15 15
Number of collaborators retained after evaluation of the data 9 11 13
Number of samples tested 30 30 30
Nurnbe1· of sample resu lts retained after evaluation of the data 18 22 26
Mean value Ea ( log10 cfu/g) 2,8 3,7 4.9
Repeatability standard deviation Sr ( log10 cfu/g) 0,17 0,38 0,44
Repeatability lim it r :
as difference on log10 scale (log10 cfu/g) 0,48 1,06 1,23
as ratio on normal scale (cfu/g) 3,0 11,5 17,1
Reproducibility standard deviation SR (log10 cfu/g) 0,45 0,41 0,54
Reproducibil ity limit R:
as difference on log10 scale (log10 cfu/g) 1,26 l,15 1,50
as ratio on normal scale (cfu/g) 18,0 14,Q 31.9
• Inoculation straJn: C. coli (W DCM 00004) .
 ISO 10272-2:2017(E)

Bibliography

[1] ISO 5725-2:1994, Accuracy (trueness and precision) of measurement methods and results -
Part 2: Basic method for the determination of repeatabilio/ and reproducibilio/ of a standard
measurement method
[2) ISO 13307, Microbiology of food and animal feed - Primary production stage - Sampling
techniques
[3) ISO/TS 17728, Microbiology of the food chain - Sampling techniques for microbiological analysis
offood and feed samples
[4) ISO 17 604, Microbiology of the food chain - Carcass sampling for microbiological analysis

[SJ ISO 18593, Microbiology of food and animal feeding stuffs - Horizontal methods for sampling
techniques from surfaces using contact plates ond swabs
(6) BOLTON F.j., HUTCH INSON D.N., COATES D. Blood-free selective medium for isolation of
Campylobacter jejuni from faeces.]. Clin. Microbial. 1984, 19 pp. 169- 171

(7) CORRY j.E.L., ATABAY H.J. Culture media for the isolation of campylobacters, helicobacters and
arcobacters. In: Handbook ofCulture media for Food and Water Microbiology, (CORRY j.E.L., CURTIS
G.D.W., BAIRD R.M., eds.). The Royal Society of Chemistry, Cambridge, UK, Third Edition, 2012

[8) HUTCHI NSON D.N., & BOLTON F.J. Improved blood-free selective medium for the isolation of
Campylobacter jejuni from faecal specimen.]. Clin. Pathol. 1984, 37 pp. 956-957
[9] RODGERS J.D., CLll>TON- HADLEY F.A., MARI N c., VIDAL A.B. An evaluation of survival and
detection of Campylobacter jejuni and C. coli in broiler caecal contents using culture-based
methods.]. Appl. Microbial. 2010, 109 pp.1244-1252

[10] World Data Centre for Microorganisms available at: h ttp://www.wfcc.i nfo
[11] ISO 17468, Microbiology of the food chain - Technical requirements and guidance on establishment
or revision of a standardized reference method
ISO 10272-2:2017(E)

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