SIST ISO 17995:2007

SLOVENSKI SIST ISO 17995:2007

STANDARD
februar 2007
Kakovost vode - Ugotavljanje prisotnosti in števila toplotno obstojnih vrst
Campylobacter
Water quality - Detection and enumeration of thermotolerant Campylobacter species
ICS 07.100.20 Referenčna številka
SIST ISO 17995:2007(en)
©  Standard je založil in izdal Slovenski inštitut za standardizacijo. Razmnoževanje ali kopiranje celote ali delov tega dokumenta ni dovoljeno

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INTERNATIONAL ISO
STANDARD 17995
First edition
2005-06-15

Water quality — Detection and
enumeration of thermotolerant
Campylobacter species
Qualité de l'eau — Recherche et dénombrement d'espèces
thermotolérantes du genre Campylobacter




Reference number
ISO 17995:2005(E)
©
ISO 2005

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ISO 17995:2005(E)
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ii © ISO 2005 – All rights reserved

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ISO 17995:2005(E)
Contents Page
Foreword. iv
Introduction . iv
1 Scope .1
2 Normative references .1
3 Terms and definitions .1
4 Principle.2
5 Apparatus .2
6 Culture media and reagents .3
7 Sampling, transport and storage .3
8 Procedure .3
9 Quality assurance.6
10 Expression of results .6
11 Test report .6
Annex A (informative) Flow diagram of method .7
Annex B (informative) Semiquantitative analysis .8
Annex C (normative) Culture media and reagents .9
Annex D (informative) Supplementary tests for further confirmation .14

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ISO 17995:2005(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards 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 right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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 rights.
ISO 17995 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,
Microbiological methods.
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ISO 17995:2005(E)
Introduction
Campylobacter jejuni subsp. jejuni and Campylobacter coli are common causes of intestinal infections in
humans. Campylobacter upsaliensis may be of like importance. Campylobacter lari is less frequently
associated with human infections. The vehicles for campylobacter infections are usually food, farm animals,
pets and person-to-person contact, but water is also important.

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INTERNATIONAL STANDARD ISO 17995:2005(E)

Water quality — Detection and enumeration of thermotolerant
Campylobacter species
WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. This standard does not purport to address all of the safety problems, if any, associated with
its use. It is the responsibility of the user to establish appropriate safety and health practices and to
ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this International
Standard be carried out by suitably trained staff.
1 Scope
This International Standard specifies a method for the detection and semiquantitative enumeration of
thermotolerant Campylobacter species. The method can be applied to all kinds of filterable waters.
NOTE 1 The method can also be used as a presence/absence test for Campylobacter species in a specified sample
volume.
NOTE 2 A more quantitative result can be obtained using a most probable number (MPN) set-up (see ISO 8199).
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 3696:1987, Water for analytical laboratory use — Specification and test methods
ISO 8199, Water quality — General guidance on the enumeration of micro-organisms by culture
ISO 19458, Water quality — Sampling for microbiological analysis
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
thermotolerant Campylobacter species
bacteria retained on filters during the filtration described in 8.2, multiplying during the selective enrichment
described in 8.3, forming typical colonies during incubation at elevated temperatures on the selective medium
described in 8.4, forming no visible colonies during incubation in air under the conditions specified in 8.6,
being highly motile, slender rods with spiral morphology and a motility characterized by darting or corkscrew-
like movements.
NOTE 1 Thermotolerant Campylobacter species of relevance in human infections include Campylobacter jejuni subsp.
jejuni (hereafter referred to as C. jejuni), C. coli, C. lari and possibly C. upsaliensis.
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ISO 17995:2005(E)
NOTE 2 Thermotolerant Campylobacter species are Gram-negative, oxidase-positive and catalase-positive (strains of
C. upsaliensis are reported to be catalase-negative or weakly positive) curved or spiral-shaped rods with a characteristic
darting, often rotating, motility. In older cultures, coccoid forms occur.
NOTE 3 Campylobacter species require enriched media for optimum growth. They are very sensitive to toxic oxygen
derivatives like peroxides and superoxide anions which can arise in media exposed to light and oxygen. They are
microaerophilic and prefer an atmosphere containing approximately 5 % oxygen and approximately 10 % carbon dioxide
(CO ). Some Campylobacter species may require an atmosphere containing hydrogen.
2
NOTE 4 Most C. jejuni, C. coli and C. lari grow at temperatures between 32 °C and 45 °C, but some strains will not
grow below 35 °C. Other strains may not grow above 43 °C.
NOTE 5 C. upsaliensis may not grow under the conditions described in this International Standard.
4 Principle
The sample is filtered through membrane filters with a pore size of 0,45 µm. The filters are transferred to
selective enrichment broths and incubated for (44 ± 4) h at (37 ± 1) °C in a microaerobic environment.
Following incubation, inoculums from each broth are streaked onto a solid medium, modified charcoal
cefoperazone desoxycholate agar (mCCDA), and incubated for (44 ± 4) h at (41,5 ± 1) °C in a microaerobic
environment. Colonies resembling campylobacters are tested for growth aerobically and, if negative,
examined by microscopy. If necessary, further biochemical reactions are performed. See flow diagram in
Annex A.
If typical campylobacters are found, the sample is positive for thermotolerant campylobacters. The result is
given as the semiquantitative estimate per volume of sample (see Annex B).
Non-filterable waters can be analysed by direct inoculation of sample into enrichment broths. The ratio of
sample to enrichment broth shall be 10 % or less.
NOTE When sufficient numbers of campylobacters are present, the sample can be streaked directly onto a solid
medium, mCCDA, without prior selective enrichment.
5 Apparatus
5.1 Incubators, thermostatically controlled at (37 ± 1) °C and at (41,5 ± 1) °C.
5.2 Equipment for membrane filtration, as specified in ISO 8199.
5.3 Membrane filters: Sterile membrane filters made of cellulose ester with a diameter of 45 mm to 50 mm
and a pore size of 0,45 µm.
Similar filters with a pore size of 0,22 µm are recommended for sterilization of supplements.
5.4 Equipment for microaerobic incubation: Jars able to maintain a modified atmosphere during
incubation, fitted with valves for outlet and inlet of gases; vacuum pump; monitor for gas composition; and a
suitable source of nitrogen, oxygen, carbon dioxide and preferably also hydrogen.
1)
NOTE Commercially available equipment (like ANOXOMAT ) can reproducibly deliver the modified atmosphere to
the jars.

1) ANOXOMAT is a trade name. This information is given for the convenience of users of this International Standard and
does not constitute an endorsement by ISO of this product.
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ISO 17995:2005(E)
Alternatively, gas-generating pouches can be used if they are able to maintain an atmosphere with
approximately 5 % oxygen, approximately 10 % carbon dioxide and preferably also approximately 10 %
hydrogen.
5.5 Microscope, preferably with phase contrast.
5.6 Bottles, 150 ml to 250 ml, with airtight screw caps for the selective enrichments.
5.7 Vented Petri dishes, sterile, 9 cm.
5.8 Usual laboratory equipment.
6 Culture media and reagents
All ingredients and chemicals shall be of recognized quality, “for microbiology” or better. Water used shall be
distilled or of like quality, as specified for ISO 3696:1987, grade 3. Follow the instructions given in Annex C.
Use of commercially available dehydrated substrates is encouraged, provided they comply with the
descriptions in Annex C. They shall be prepared in accordance with the manufacturer's instructions.
Other grades of chemicals may be used provided they can be shown to lead to the same results.
7 Sampling, transport and storage
In addition to the instructions given in ISO 19458, be aware that campylobacters are very sensitive to adverse
conditions. Keep samples cool (3 ± 2) °C and in the dark until the filtrations have been done. Avoid
unnecessary mixing with air. Filter the samples as soon as possible after collection. Store for a maximum of
30 h prior to analysis.
NOTE Campylobacters survive well in clean water at (3 ± 2) °C. At higher temperatures or in other media, they may
quickly deteriorate.
8 Procedure
8.1 General
Parallel to samples, run a positive control spiked in sample material or in sterile water through all the steps of
the procedure to demonstrate the proper functioning of the apparatus, culture media and procedure, and to
facilitate recognition of campylobacters (8.5 to 8.6).
Parts of each sample shall be enriched in the highly selective Preston broth (C.1.1) and parts in the less
selective Bolton broth (C.1.2).
NOTE Preston broth may be too selective to allow the recovery of some strains of C. coli. Bolton broth may not be
selective enough to counteract the growth of non-campylobacters in some samples. If the available sample size is limited,
the most appropriate enrichment broth should be used. For waters with an expected high background count, it is more
appropriate to use Preston broth, and for clean waters or where the background count is likely to be low Bolton broth is
more appropriate.
NOTE 2 The amount of sample to be analysed varies with the sample material and the scope of the investigation. In
Annex B, sample volumes for the analyses of drinking water are proposed.
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ISO 17995:2005(E)
8.2 Membrane filtration
Filter the appropriate volumes of sample through sterile membrane filters (5.3). Avoid unnecessary exposure
to air.
With turbid samples, it may be necessary to use two or more filters for the largest sample volumes to
counteract clogging. All these filters are transferred to the same portion of enrichment broth. The use of a
“filter aid” will facilitate the filtration of turbid samples. For a short description of filter aids, see ISO 8199.
8.3 Enrichment
Preheat enrichment broths (C.1.1, C.1.2) to 20 °C to 30 °C before inoculation.
Transfer the filters (see 8.2) to bottles with 100 ml of enrichment broth immediately after filtration. Put the
inoculated broths in jars (see 5.4). Leave the caps off or loosely placed on the inoculated broths during
incubation to allow the modified atmosphere to reach the broths. Apply the modified atmosphere (see 5.4) and
incubate at (37 ± 1) °C for (44 ± 4) h.
NOTE 1 100 ml volumes of enrichment broth are used to make sure that the contaminating bacteria present on the
filters are diluted sufficiently to avoid their inhibition of the growth of campylobacters during the enrichment.
NOTE 2 Some laboratories report successful isolation of campylobacters without the use of a modified atmosphere,
closed bottles or tubes with only a small headspace of air being used instead. This procedure may need careful
standardization to avoid false negative results due to sub-optimum conditions during enrichment.
NOTE 3 The Preston campylobacter-selective supplement (C.1.1.2) contains antibiotics (polymyxin B and rifam
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