Water quality - Detection and enumeration of thermotolerant Campylobacter species

This standard specifies a method for the detection, semi-quantitative and quantitative (MPN) enumeration of thermotolerant Campylobacter species. The method can be applied to all kinds of waters including: drinking water, ground water and well water, fresh, brackish and saline surface water, swimming pools, spa and hydrotherapy pools, recreational waters, agricultural waters and runoff, untreated and treated wastewater and also sand and other sediments. This method can be used for the detection of Campylobacter species in a specified sample volume. Clean water samples with low turbidity can be membrane filtered for either a qualitative method, semiquantitative or quantitative (MPN) method. Water samples with higher turbidity, such as primary and secondary wastewater effluents and sediments, are analysed using the same qualitative, semiquantitative or quantitative MPN method by direct inoculation of material into bottles or tubes. Sediments can be suspended in a suitable diluent or inoculated directly into enrichment broths. Users wishing to employ this method are expected to verify its performance for the particular matrix under their own laboratory conditions.

Qualité de l'eau - Recherche et dénombrement d'espèces thermotolérantes du genre Campylobacter

Kakovost vode - Ugotavljanje prisotnosti in števila termotolerantnih vrst Campylobacter

Ta standard določa metodo za ugotavljanje prisotnosti ter izračuna delnega kvantitativnega in kvalitativnega (MPN) števila termotolerantnih bakterij iz roda Campylobacter. Metodo je mogoče uporabiti za vse vrste voda, vključno s: pitno vodo, podzemno vodo in vodo iz vodnjakov, sladko, slankasto in slano površinsko vodo, vodo v bazenih, vodo v zdraviliških bazenih in bazenih za hidroterapijo, rekreacijsko vodo, kmetijsko vodo in iztoki, neočiščeno in očiščeno odpadno vodo ter tudi vključno s peskom in drugimi usedlinami. Metodo je mogoče uporabiti za odkrivanje bakterij iz rodu Campylobacter v določeni količini vzorca. Vzorce čiste vode z nizko motnostjo je mogoče filtrirati skozi membrano bodisi s kvalitativno metodo, delno kvantitativno ali kvantitativno (MPN) metodo. Vzorci vode z večjo motnostjo, na primer primarne in sekundarne odpadne vode ter usedline, se analizirajo po isti kvalitativni, delni kvantitativni ali kvantitativni metodi MPN z neposredno inokulacijo materiala v steklenice ali epruvete. Usedline je mogoče odložiti v primerno razredčilo ali inokulirati neposredno v obogatitvene bujone. Od uporabnikov, ki želijo uporabiti to metodo, se pričakuje, da bodo v svojih laboratorijskih pogojih preverili učinkovitost metode za ustrezno matriko.

General Information

Status
Published
Public Enquiry End Date
03-Nov-2019
Publication Date
09-Jan-2020
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
04-Nov-2019
Due Date
09-Jan-2020
Completion Date
10-Jan-2020

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INTERNATIONAL ISO
STANDARD 17995
Second edition
2019-11
Water quality — Detection and
enumeration of thermotolerant
Campylobacter spp
Qualité de l'eau — Recherche et dénombrement d'espèces
thermotolérantes du genre Campylobacter
Reference number
ISO 17995:2019(E)
ISO 2019
---------------------- Page: 1 ----------------------
ISO 17995:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 17995:2019(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

4.1 General ........................................................................................................................................................................................................... 2

4.2 Inoculation and enrichment in selective liquid medium .................................................................................... 2

4.3 Isolation and selection for confirmation .......................................................................................................................... 2

4.4 Confirmation ............................................................................................................................................................................................. 2

5 Culture media and reagents ...................................................................................................................................................................... 3

6 Apparatus ..................................................................................................................................................................................................................... 3

7 Sampling, transport and storage ......................................................................................................................................................... 4

8 Sample processing .............................................................................................................................................................................................. 4

8.1 General ........................................................................................................................................................................................................... 4

8.2 Direct inoculation into enrichment broth ....................................................................................................................... 4

8.3 Membrane filtration ........................................................................................................................................................................... 4

9 Procedure..................................................................................................................................................................................................................... 5

9.1 Enrichment ................................................................................................................................................................................................. 5

9.2 Plating on solid, selective medium......................................................................................................................................... 6

9.3 Reading of presumptive results ................................................................................................................................................ 6

9.4 Confirmation ............................................................................................................................................................................................. 6

9.4.1 General...................................................................................................................................................................................... 6

9.4.2 Selection of colonies for confirmation .......................................................................................................... 6

9.4.3 Absence of growth on non-selective agar plates .................................................................................. 7

9.4.4 Motility and cell morphology ................................................................................................................................ 7

9.4.5 Detection of oxidase activity .................................................................................................................................. 7

9.4.6 Interpretation ..................................................................................................................................................................... 7

9.4.7 Further verification ....................................................................................................................................................... 8

9.5 Identification of Campylobacter species (optional)................................................................................................. 8

9.5.1 General...................................................................................................................................................................................... 8

9.5.2 Detection of catalase ........................................................................................................................................... .......... 8

9.5.3 Detection of hippurate hydrolysis ..................................................................................................................... 8

9.5.4 Detection of indoxyl acetate hydrolysis ....................................................................................................... 9

9.5.5 Interpretation ..................................................................................................................................................................... 9

10 Quality assurance ................................................................................................................................................................................................ 9

11 Expression of results ........................................................................................................................................................................................ 9

12 Test report ................................................................................................................................................................................................................10

Annex A (normative) Flow diagram of the method ............................................................................................................................11

Annex B (normative) Semi‑quantitative analysis .................................................................................................................................12

Annex C (normative) Composition and preparation of culture media and reagents ......................................13

Annex D (normative) Performance testing for the quality assurance of the culture media ...................22

Annex E (informative) Performance characteristics .........................................................................................................................23

Annex F (informative) Additional information about campylobacters .........................................................................24

Bibliography .............................................................................................................................................................................................................................25

© ISO 2019 – All rights reserved iii
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ISO 17995:2019(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.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different 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 .iso .org/ directives).

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. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO 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 of the voluntary nature of standards, the meaning of ISO 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 www .iso .org/

iso/ foreword .html.

This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,

Microbiological methods.

This second edition cancels and replaces the first edition (ISO 17995:2005), which has been technically

revised. The main changes compared to the previous edition are as follows:

— the inclusion of direct inoculation of enrichment broths in addition to membrane filtration with

additional information about sample processing.
— methods for the speciation of Campylobacter.

— performance testing for the quality assurance of culture media has been added to Annex D.

— performance characteristics of the method have been added as an Annex E.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2019 – All rights reserved
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ISO 17995:2019(E)
Introduction

The thermotolerant Campylobacter species are not thought to propagate as free living but are zoonotic

pathogenic bacteria of mammals and birds and which cause disease in humans. Campylobacter

jejuni subsp. jejuni and Campylobacter coli are common causes of intestinal infections in humans.

Campylobacter upsaliensis is found mainly in cats and dogs and is of minor importance for human

infections. Campylobacter lari is less frequently associated with human infections. Campylobacter

infections give rise to a flu-like illness with malaise, fever and myalgia followed by diarrhoea. The

vehicles for Campylobacter infections are usually food, farm animals, pets and person-to-person

contact; water is also important. They can be isolated from waters contaminated with human or animal

faeces such as wastewater and surface waters. The bacteria have been demonstrated to survive within

amoebae. Outbreaks of campylobacteriosis have been reported in relation to the use of contaminated

drinking water and sporadic cases from recreational water use.
© ISO 2019 – All rights reserved v
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INTERNATIONAL STANDARD ISO 17995:2019(E)
Water quality — Detection and enumeration of
thermotolerant Campylobacter spp

WARNING — Persons using this document should be familiar with normal laboratory practice.

This document 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.

IMPORTANT — It is absolutely essential that tests conducted in accordance with this document

be carried out by suitably trained staff.
1 Scope

This document specifies a method for the detection, semi-quantitative and quantitative (MPN)

enumeration of thermotolerant Campylobacter species.

The method can be applied to all kinds of waters including: drinking water, ground water and well

water, fresh, brackish and saline surface water, swimming pools, spa and hydrotherapy pools,

recreational waters, agricultural waters and runoff, untreated and treated wastewater and also sand

and other sediments.

This method can be used for the detection of Campylobacter species in a specified sample volume. Clean

water samples with low turbidity can be membrane filtered for either a qualitative method, semi-

quantitative or quantitative (MPN) method. Water samples with higher turbidity, such as primary

and secondary wastewater effluents and sediments, are analysed using the same qualitative, semi-

quantitative or quantitative MPN method by direct inoculation of material into bottles or tubes.

Sediments can be suspended in a suitable diluent or inoculated directly into enrichment broths.

Users wishing to employ this method are expected to verify its performance for the particular matrix

under their own laboratory conditions.
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 document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 8199, Water quality — General requirements and guidance for microbiological examinations by culture

ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and

performance testing of culture media
ISO 19458, Water quality — Sampling for microbiological analysis
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org ./ obp
— IEC Electropedia: available at http:// www .electropedia .org/
© ISO 2019 – All rights reserved 1
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ISO 17995:2019(E)
3.1
Campylobacter

microorganisms forming characteristic colonies on solid selective media when incubated in a

microaerobic atmosphere at 41,5 °C and which possess the characteristic morphology, motility,

biochemical and growth properties described when tests are conducted in accordance with ISO 17995

Note 1 to entry: Thermotolerant Campylobacter species of relevance in human infections include Campylobacter

jejuni subsp. jejuni (hereafter referred to as Campylobacter jejuni), Campylobacter coli, Campylobacter lari and

possibly Campylobacter upsaliensis. Campylobacter upsaliensis is found mainly in cats and dogs and is of minor

importance for human infection.
3.2
detection of Campylobacter

determination of the presence or absence, semi-quantitative enumeration or quantitative enumeration

using a most probable number (MPN) system of Campylobacter in water and solid materials when the

test is conducted in accordance with ISO 17995
4 Principle
4.1 General

In general, the detection of Campylobacter requires enrichment followed by isolation of colonies and

their confirmation. The flow diagram of the procedure in Annex A shall be applied.

4.2 Inoculation and enrichment in selective liquid medium

Samples are inoculated either directly or after concentration using membrane filtration into one of two

selective enrichment broths depending on the expected level of background microorganisms: Bolton

broth for clean water and Preston broth for more heavily contaminated water. A single sample volume

is processed for Campylobacter detection and, where necessary, at least three 10-fold volumes (for

example 10 ml, 100 ml and 1 000 ml) are used for a semi-quantitative determination (Annex B shall

be applied). For a quantitative (MPN) determination, volumes of 500 ml, 5 × 100 ml, 5 × 10 ml and,

where counts may be high, smaller volumes are used or the initial sample is diluted. The broths are

then incubated microaerobically at (37 ± 1) °C for (44 ± 4) h.

The ratio of sample to enrichment broth shall be 10 % or less if single strength broth is used.

4.3 Isolation and selection for confirmation

From the enrichment broth cultures in 4.2, liquid selective media are inoculated onto modified charcoal

cefoperazone deoxycholate agar (mCCDA).

The mCCDA plates are then incubated at (41,5 ± 1) °C for (44 ± 4) h in a microaerobic atmosphere

and examined for characteristic colonies after incubation. Colonies with typical characteristics are

presumed to be Campylobacter (see 9.3). It is necessary to confirm at least one colony from each plate to

demonstrate that the corresponding enrichment culture is positive for the presence of Campylobacter

species.
4.4 Confirmation

Confirmation of suspect colonies of Campylobacter species involves biochemical, morphological and

physiological tests. See flow diagram in Annex A.

The colonies presumed to be Campylobacter are inoculated on the non-selective Columbia agar or other

appropriate solid non-selective media, and are then confirmed by means of microscopic examination,

failure to grow aerobically at 25 °C and appropriate biochemical tests. Optionally, Campylobacter

species are further identified by specific biochemical tests.
2 © ISO 2019 – All rights reserved
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ISO 17995:2019(E)

For species identification, it is recommended that more than one isolate per enrichment culture is tested.

If typical Campylobacter species are confirmed or identified, the result is given as the qualitative,

semi-quantitative or quantitative (MPN) estimate per volume of sample.
5 Culture media and reagents
For current laboratory practice see ISO 8199 and ISO 11133.

The composition and preparation of the culture media and reagents given in Annex C shall be used. The

performance testing requirements given in Annex D shall be applied.
6 Apparatus

Usual microbiological laboratory equipment (as specified in ISO 8199) and, in particular, the following:

6.1 Water baths or incubators, capable of operating at (37 ± 1) °C and (41,5 ± 1) °C.

6.2 Water bath, capable of operating between 47 °C and 50 °C.
6.3 Membrane filtration equipment, as specified in ISO 8199.

6.4 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. Larger diameter membranes may be used with suitable filter holders

provided they have a pore size of 0,45 µm.
6.5 pH‑meter, accurate to within 0,2 pH units at 25 °C.

6.6 Apparatus suitable for achieving a microaerobic atmosphere, with oxygen content of (5 ± 2) %,

carbon dioxide (10 ± 3) %, optional hydrogen at ≤10 %, with the balance being nitrogen.

Appropriate gastight containers of, for example, 3,5 l capacity are used to hold Petri dishes and/

or flasks or bottles for the enrichment broth, such as bacteriological anaerobic jars. The appropriate

microaerobic atmosphere can be obtained using commercially available gas-generating kits, following

precisely the manufacturer's instructions, particularly those relating to the volume of the jar and the

capacity of the gas-generating kit. The jar may be flushed and filled with an appropriate gas mixture

prior to incubation. Alternatively, an incubator with appropriately modified atmosphere (approximately

5 % oxygen and 10 % carbon dioxide) can also be used.

Gas-generating pouches can be used if they are able to maintain an atmosphere with approximately 5 %

oxygen and approximately 10 % carbon dioxide.

6.7 Microscope, preferably with phase contrast or differential interference contrast. Dark ground

illumination can also be used.
6.8 Bottles, 100 ml to 250 ml, with screw caps for the selective enrichments.
6.9 Vented Petri dishes, sterile, 90 mm.
6.10 Disinfected forceps, for handling membrane filters.
6.11 Refrigerator, capable of operating at (5 ± 3) °C.
© ISO 2019 – All rights reserved 3
---------------------- Page: 8 ----------------------
ISO 17995:2019(E)
7 Sampling, transport and storage

It is important that the laboratory receive a sample which is truly representative and has not been

damaged or changed during transport or storage. Be aware that campylobacters are very sensitive

to adverse conditions. Keep samples cool (5 ± 3) °C and in the dark until they are processed. The

temperature should not be above that at which the sample was taken, nor should the sample be allowed

to freeze. Avoid unnecessary mixing with air. Process the samples as soon as possible after collection,

but within a maximum of 30 h. In all other aspects of sampling, transport and storage, follow the

instructions given in ISO 19458.

NOTE 1 Campylobacters survive well in clean water at (3 ± 2) °C. At higher temperatures or in other media,

they can quickly deteriorate.

NOTE 2 It is important that sample bottles are filled to the rim. This avoids air spaces in the container and

helps to ensure Campylobacter survival.
8 Sample processing
8.1 General

Depending on the sample properties and expected level of faecal contamination, use direct

inoculation (see 8.2) or membrane filtration (see 8.3) for sample processing. Direct inoculation is

suitable for samples with a high contamination level such as wastewater, whereas membrane filtration

is suitable for surface water, groundwater and drinking water samples. Direct inoculation is only

suitable for relatively small volumes, for example 100 ml or less and membrane filtration is suitable for

larger volumes.

The ratio of a maximum of 10% of sample to enrichment broth is used (for example 100 ml of sample

to 1 L of broth) to avoid significant change to the composition of the medium. In addition, background

microorganisms present in the sample are diluted sufficiently to avoid their inhibition of the growth of

campylobacters during enrichment.

Throughout the processing steps, care should be taken to minimise the risk of cross-contamination

of enrichment broths. The use of pipette tips with sterile filters is essential if automated pipettes are

used. In addition any positive controls should be processed separately.
8.2 Direct inoculation into enrichment broth

Samples with expected high contamination levels are inoculated directly into Preston broth (see C.2).

Where the expected level of background microorganisms is low and samples cannot be processed by

membrane filtration, Bolton broth (see C.3) may be used. If no information about the contamination

level is available, both broths should be used.

Double strength broth should be used for large volumes. Single strength broth should be used for

relatively small volumes of water where the ratio of sample to enrichment broth shall be 10 % or less.

Generally, volumes of 100 ml, 50 ml and 10 ml are inoculated into an equal volume of double strength

broth. Volumes of 1 ml and 0,1 ml if required are inoculated into 10 ml of single strength broth.

Solid material can be weighed directly into single strength enrichment broths. Aliquots of 10 g can

be weighed into 100 ml of enrichment broth, 1 g and smaller aliquots should be weighed into 10 ml of

enrichment broth.

NOTE When high numbers of campylobacters are expected, the sample can also additionally be streaked

directly onto mCCDA (see C.4) without prior selective enrichment.
8.3 Membrane filtration

Samples with expected low levels of contamination are filtered using membrane filters with a pore size of

0,45 µm (see 6.4). Select one of the two enrichment broths depending on the expected level of background

4 © ISO 2019 – All rights reserved
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ISO 17995:2019(E)

microorganisms and use 100 ml of single strength broth for the enrichment of each sample volume.

Bolton broth (see C.3) is used for clean samples and Preston broth (see C.2) for heavily contaminated

water. If no information about the contamination level is available, both broths should be used.

For detection of campylobacters in drinking water, other treated waters and clean surface waters, a

1 000 ml sample is filtered. If required, larger volumes may be processed using more filters or, where

necessary, larger diameter filters may be used. Alternative concentration methods may be used, for

example ultra-filtration or centrifugation providing that they are verified for recovery by the user. For

more contaminated water, smaller volumes should be used and, for samples containing more particulate

material, several membrane filters may be used to filter the required volume. All membrane filters from

one sample volume shall be placed in the same container of enrichment broth.

For a semi-quantitative determination, volumes of 10 ml, 100 ml and 1 000 ml are filtered (see Annex B).

The smaller volumes of 10 ml can also be pipetted directly into 100 ml of single strength medium or

10 ml of double strength medium.

For quantitative estimates, an MPN series can be used. In an MPN series, 100 ml and larger volumes

can be filtered and membrane filters from different volumes are placed into 100 ml of separate single

strength broth. Smaller volumes of 10 ml can be pipetted into 100 ml of single strength medium or

10 ml of double strength medium, and 1 ml can be pipetted into 10 ml of single strength medium. For

example, volumes of 5 × 100 ml and 500 ml can be filtered and membranes placed into 100 ml of single

strength broth. Volumes of 10 ml can be directly inoculated into 5 × 100 ml of single strength medium

or 5 × 10 ml of double strength medium. Volumes of 5 × 1 ml can be inoculated directly into 10 ml of

single strength broth.

After filtration, the membranes are transferred using disinfected forceps (see 6.10) into enrichment

broths.
9 Procedure
9.1 Enrichment

For samples known to have low concentrations of background microorganisms, use the less selective

Bolton broth for enrichment and for samples expected to have a high concentration of background

microorganisms use the highly selective Preston broth. For samples where the background microbial

concentration is unknown, enrich parts of each sample in both Preston and Bolton broths. 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, chose one of the enrichment broths which is considered to be the most appropriate.

NOTE 1 The amount of sample (volume or weight) to be analysed can vary with the sample material and the

scope of the investigation.
Bring enrichment broths to room temperature prior to inoculation.

Transfer the samples (see 8.2 and 8.3) to bottles with the appropriate volumes of enrichment broth(s).

Put the inoculated broths in jars (6.6) or in an incubator with modified atmosphere. Leave the caps

loosely placed on the inoculated broths during incubation to allow the modified atmosphere to reach

the broths. Apply the modified atmosphere to the jars (6.6) and incubate at (37 ± 1) °C for (44 ± 4) h.

NOTE 2 The Preston campylobacter-selective supplement (see C.2.2) contains antibiotics (polymyxin B and

rifampicin) known to be rather toxic towards C. coli and towards sub-lethally injured C. jejuni. Accordingly,

pre-enrichment for 4 h in Preston broth without the selective supple
...

SLOVENSKI STANDARD
SIST ISO 17995:2020
01-februar-2020
Nadomešča:
SIST ISO 17995:2007
Kakovost vode - Ugotavljanje prisotnosti in števila termotolerantnih vrst
Campylobacter

Water quality - Detection and enumeration of thermotolerant Campylobacter species

Qualité de l'eau - Recherche et dénombrement d'espèces thermotolérantes du genre
Campylobacter
Ta slovenski standard je istoveten z: ISO 17995:2019
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST ISO 17995:2020 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST ISO 17995:2020
---------------------- Page: 2 ----------------------
SIST ISO 17995:2020
INTERNATIONAL ISO
STANDARD 17995
Second edition
2019-11
Water quality — Detection and
enumeration of thermotolerant
Campylobacter spp
Qualité de l'eau — Recherche et dénombrement d'espèces
thermotolérantes du genre Campylobacter
Reference number
ISO 17995:2019(E)
ISO 2019
---------------------- Page: 3 ----------------------
SIST ISO 17995:2020
ISO 17995:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
---------------------- Page: 4 ----------------------
SIST ISO 17995:2020
ISO 17995:2019(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

4.1 General ........................................................................................................................................................................................................... 2

4.2 Inoculation and enrichment in selective liquid medium .................................................................................... 2

4.3 Isolation and selection for confirmation .......................................................................................................................... 2

4.4 Confirmation ............................................................................................................................................................................................. 2

5 Culture media and reagents ...................................................................................................................................................................... 3

6 Apparatus ..................................................................................................................................................................................................................... 3

7 Sampling, transport and storage ......................................................................................................................................................... 4

8 Sample processing .............................................................................................................................................................................................. 4

8.1 General ........................................................................................................................................................................................................... 4

8.2 Direct inoculation into enrichment broth ....................................................................................................................... 4

8.3 Membrane filtration ........................................................................................................................................................................... 4

9 Procedure..................................................................................................................................................................................................................... 5

9.1 Enrichment ................................................................................................................................................................................................. 5

9.2 Plating on solid, selective medium......................................................................................................................................... 6

9.3 Reading of presumptive results ................................................................................................................................................ 6

9.4 Confirmation ............................................................................................................................................................................................. 6

9.4.1 General...................................................................................................................................................................................... 6

9.4.2 Selection of colonies for confirmation .......................................................................................................... 6

9.4.3 Absence of growth on non-selective agar plates .................................................................................. 7

9.4.4 Motility and cell morphology ................................................................................................................................ 7

9.4.5 Detection of oxidase activity .................................................................................................................................. 7

9.4.6 Interpretation ..................................................................................................................................................................... 7

9.4.7 Further verification ....................................................................................................................................................... 8

9.5 Identification of Campylobacter species (optional)................................................................................................. 8

9.5.1 General...................................................................................................................................................................................... 8

9.5.2 Detection of catalase ........................................................................................................................................... .......... 8

9.5.3 Detection of hippurate hydrolysis ..................................................................................................................... 8

9.5.4 Detection of indoxyl acetate hydrolysis ....................................................................................................... 9

9.5.5 Interpretation ..................................................................................................................................................................... 9

10 Quality assurance ................................................................................................................................................................................................ 9

11 Expression of results ........................................................................................................................................................................................ 9

12 Test report ................................................................................................................................................................................................................10

Annex A (normative) Flow diagram of the method ............................................................................................................................11

Annex B (normative) Semi‑quantitative analysis .................................................................................................................................12

Annex C (normative) Composition and preparation of culture media and reagents ......................................13

Annex D (normative) Performance testing for the quality assurance of the culture media ...................22

Annex E (informative) Performance characteristics .........................................................................................................................23

Annex F (informative) Additional information about campylobacters .........................................................................24

Bibliography .............................................................................................................................................................................................................................25

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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.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different 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 .iso .org/ directives).

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. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO 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 of the voluntary nature of standards, the meaning of ISO 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 www .iso .org/

iso/ foreword .html.

This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,

Microbiological methods.

This second edition cancels and replaces the first edition (ISO 17995:2005), which has been technically

revised. The main changes compared to the previous edition are as follows:

— the inclusion of direct inoculation of enrichment broths in addition to membrane filtration with

additional information about sample processing.
— methods for the speciation of Campylobacter.

— performance testing for the quality assurance of culture media has been added to Annex D.

— performance characteristics of the method have been added as an Annex E.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
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Introduction

The thermotolerant Campylobacter species are not thought to propagate as free living but are zoonotic

pathogenic bacteria of mammals and birds and which cause disease in humans. Campylobacter

jejuni subsp. jejuni and Campylobacter coli are common causes of intestinal infections in humans.

Campylobacter upsaliensis is found mainly in cats and dogs and is of minor importance for human

infections. Campylobacter lari is less frequently associated with human infections. Campylobacter

infections give rise to a flu-like illness with malaise, fever and myalgia followed by diarrhoea. The

vehicles for Campylobacter infections are usually food, farm animals, pets and person-to-person

contact; water is also important. They can be isolated from waters contaminated with human or animal

faeces such as wastewater and surface waters. The bacteria have been demonstrated to survive within

amoebae. Outbreaks of campylobacteriosis have been reported in relation to the use of contaminated

drinking water and sporadic cases from recreational water use.
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INTERNATIONAL STANDARD ISO 17995:2019(E)
Water quality — Detection and enumeration of
thermotolerant Campylobacter spp

WARNING — Persons using this document should be familiar with normal laboratory practice.

This document 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.

IMPORTANT — It is absolutely essential that tests conducted in accordance with this document

be carried out by suitably trained staff.
1 Scope

This document specifies a method for the detection, semi-quantitative and quantitative (MPN)

enumeration of thermotolerant Campylobacter species.

The method can be applied to all kinds of waters including: drinking water, ground water and well

water, fresh, brackish and saline surface water, swimming pools, spa and hydrotherapy pools,

recreational waters, agricultural waters and runoff, untreated and treated wastewater and also sand

and other sediments.

This method can be used for the detection of Campylobacter species in a specified sample volume. Clean

water samples with low turbidity can be membrane filtered for either a qualitative method, semi-

quantitative or quantitative (MPN) method. Water samples with higher turbidity, such as primary

and secondary wastewater effluents and sediments, are analysed using the same qualitative, semi-

quantitative or quantitative MPN method by direct inoculation of material into bottles or tubes.

Sediments can be suspended in a suitable diluent or inoculated directly into enrichment broths.

Users wishing to employ this method are expected to verify its performance for the particular matrix

under their own laboratory conditions.
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 document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 8199, Water quality — General requirements and guidance for microbiological examinations by culture

ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and

performance testing of culture media
ISO 19458, Water quality — Sampling for microbiological analysis
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org ./ obp
— IEC Electropedia: available at http:// www .electropedia .org/
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3.1
Campylobacter

microorganisms forming characteristic colonies on solid selective media when incubated in a

microaerobic atmosphere at 41,5 °C and which possess the characteristic morphology, motility,

biochemical and growth properties described when tests are conducted in accordance with ISO 17995

Note 1 to entry: Thermotolerant Campylobacter species of relevance in human infections include Campylobacter

jejuni subsp. jejuni (hereafter referred to as Campylobacter jejuni), Campylobacter coli, Campylobacter lari and

possibly Campylobacter upsaliensis. Campylobacter upsaliensis is found mainly in cats and dogs and is of minor

importance for human infection.
3.2
detection of Campylobacter

determination of the presence or absence, semi-quantitative enumeration or quantitative enumeration

using a most probable number (MPN) system of Campylobacter in water and solid materials when the

test is conducted in accordance with ISO 17995
4 Principle
4.1 General

In general, the detection of Campylobacter requires enrichment followed by isolation of colonies and

their confirmation. The flow diagram of the procedure in Annex A shall be applied.

4.2 Inoculation and enrichment in selective liquid medium

Samples are inoculated either directly or after concentration using membrane filtration into one of two

selective enrichment broths depending on the expected level of background microorganisms: Bolton

broth for clean water and Preston broth for more heavily contaminated water. A single sample volume

is processed for Campylobacter detection and, where necessary, at least three 10-fold volumes (for

example 10 ml, 100 ml and 1 000 ml) are used for a semi-quantitative determination (Annex B shall

be applied). For a quantitative (MPN) determination, volumes of 500 ml, 5 × 100 ml, 5 × 10 ml and,

where counts may be high, smaller volumes are used or the initial sample is diluted. The broths are

then incubated microaerobically at (37 ± 1) °C for (44 ± 4) h.

The ratio of sample to enrichment broth shall be 10 % or less if single strength broth is used.

4.3 Isolation and selection for confirmation

From the enrichment broth cultures in 4.2, liquid selective media are inoculated onto modified charcoal

cefoperazone deoxycholate agar (mCCDA).

The mCCDA plates are then incubated at (41,5 ± 1) °C for (44 ± 4) h in a microaerobic atmosphere

and examined for characteristic colonies after incubation. Colonies with typical characteristics are

presumed to be Campylobacter (see 9.3). It is necessary to confirm at least one colony from each plate to

demonstrate that the corresponding enrichment culture is positive for the presence of Campylobacter

species.
4.4 Confirmation

Confirmation of suspect colonies of Campylobacter species involves biochemical, morphological and

physiological tests. See flow diagram in Annex A.

The colonies presumed to be Campylobacter are inoculated on the non-selective Columbia agar or other

appropriate solid non-selective media, and are then confirmed by means of microscopic examination,

failure to grow aerobically at 25 °C and appropriate biochemical tests. Optionally, Campylobacter

species are further identified by specific biochemical tests.
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For species identification, it is recommended that more than one isolate per enrichment culture is tested.

If typical Campylobacter species are confirmed or identified, the result is given as the qualitative,

semi-quantitative or quantitative (MPN) estimate per volume of sample.
5 Culture media and reagents
For current laboratory practice see ISO 8199 and ISO 11133.

The composition and preparation of the culture media and reagents given in Annex C shall be used. The

performance testing requirements given in Annex D shall be applied.
6 Apparatus

Usual microbiological laboratory equipment (as specified in ISO 8199) and, in particular, the following:

6.1 Water baths or incubators, capable of operating at (37 ± 1) °C and (41,5 ± 1) °C.

6.2 Water bath, capable of operating between 47 °C and 50 °C.
6.3 Membrane filtration equipment, as specified in ISO 8199.

6.4 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. Larger diameter membranes may be used with suitable filter holders

provided they have a pore size of 0,45 µm.
6.5 pH‑meter, accurate to within 0,2 pH units at 25 °C.

6.6 Apparatus suitable for achieving a microaerobic atmosphere, with oxygen content of (5 ± 2) %,

carbon dioxide (10 ± 3) %, optional hydrogen at ≤10 %, with the balance being nitrogen.

Appropriate gastight containers of, for example, 3,5 l capacity are used to hold Petri dishes and/

or flasks or bottles for the enrichment broth, such as bacteriological anaerobic jars. The appropriate

microaerobic atmosphere can be obtained using commercially available gas-generating kits, following

precisely the manufacturer's instructions, particularly those relating to the volume of the jar and the

capacity of the gas-generating kit. The jar may be flushed and filled with an appropriate gas mixture

prior to incubation. Alternatively, an incubator with appropriately modified atmosphere (approximately

5 % oxygen and 10 % carbon dioxide) can also be used.

Gas-generating pouches can be used if they are able to maintain an atmosphere with approximately 5 %

oxygen and approximately 10 % carbon dioxide.

6.7 Microscope, preferably with phase contrast or differential interference contrast. Dark ground

illumination can also be used.
6.8 Bottles, 100 ml to 250 ml, with screw caps for the selective enrichments.
6.9 Vented Petri dishes, sterile, 90 mm.
6.10 Disinfected forceps, for handling membrane filters.
6.11 Refrigerator, capable of operating at (5 ± 3) °C.
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7 Sampling, transport and storage

It is important that the laboratory receive a sample which is truly representative and has not been

damaged or changed during transport or storage. Be aware that campylobacters are very sensitive

to adverse conditions. Keep samples cool (5 ± 3) °C and in the dark until they are processed. The

temperature should not be above that at which the sample was taken, nor should the sample be allowed

to freeze. Avoid unnecessary mixing with air. Process the samples as soon as possible after collection,

but within a maximum of 30 h. In all other aspects of sampling, transport and storage, follow the

instructions given in ISO 19458.

NOTE 1 Campylobacters survive well in clean water at (3 ± 2) °C. At higher temperatures or in other media,

they can quickly deteriorate.

NOTE 2 It is important that sample bottles are filled to the rim. This avoids air spaces in the container and

helps to ensure Campylobacter survival.
8 Sample processing
8.1 General

Depending on the sample properties and expected level of faecal contamination, use direct

inoculation (see 8.2) or membrane filtration (see 8.3) for sample processing. Direct inoculation is

suitable for samples with a high contamination level such as wastewater, whereas membrane filtration

is suitable for surface water, groundwater and drinking water samples. Direct inoculation is only

suitable for relatively small volumes, for example 100 ml or less and membrane filtration is suitable for

larger volumes.

The ratio of a maximum of 10% of sample to enrichment broth is used (for example 100 ml of sample

to 1 L of broth) to avoid significant change to the composition of the medium. In addition, background

microorganisms present in the sample are diluted sufficiently to avoid their inhibition of the growth of

campylobacters during enrichment.

Throughout the processing steps, care should be taken to minimise the risk of cross-contamination

of enrichment broths. The use of pipette tips with sterile filters is essential if automated pipettes are

used. In addition any positive controls should be processed separately.
8.2 Direct inoculation into enrichment broth

Samples with expected high contamination levels are inoculated directly into Preston broth (see C.2).

Where the expected level of background microorganisms is low and samples cannot be processed by

membrane filtration, Bolton broth (see C.3) may be used. If no information about the contamination

level is available, both broths should be used.

Double strength broth should be used for large volumes. Single strength broth should be used for

relatively small volumes of water where the ratio of sample to enrichment broth shall be 10 % or less.

Generally, volumes of 100 ml, 50 ml and 10 ml are inoculated into an equal volume of double strength

broth. Volumes of 1 ml and 0,1 ml if required are inoculated into 10 ml of single strength broth.

Solid material can be weighed directly into single strength enrichment broths. Aliquots of 10 g can

be weighed into 100 ml of enrichment broth, 1 g and smaller aliquots should be weighed into 10 ml of

enrichment broth.

NOTE When high numbers of campylobacters are expected, the sample can also additionally be streaked

directly onto mCCDA (see C.4) without prior selective enrichment.
8.3 Membrane filtration

Samples with expected low levels of contamination are filtered using membrane filters with a pore size of

0,45 µm (see 6.4). Select one of the two enrichment broths depending on the expected level of background

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microorganisms and use 100 ml of single strength broth for the enrichment of each sample volume.

Bolton broth (see C.3) is used for clean samples and Preston broth (see C.2) for heavily contaminated

water. If no information about the contamination level is available, both broths should be used.

For detection of campylobacters in drinking water, other treated waters and clean surface waters, a

1 000 ml sample is filtered. If required, larger volumes may be processed using more filters or, where

necessary, larger diameter filters may be used. Alternative concentration methods may be used, for

example ultra-filtration or centrifugation providing that they are verified for recovery by the user. For

more contaminated water, smaller volumes should be used and, for samples containing more particulate

material, several membrane filters may be used to filter the required volume. All membrane filters from

one sample volume shall be placed in the same container of enrichment broth.

For a semi-quantitative determination, volumes of 10 ml, 100 ml and 1 000 ml are filtered (see Annex B).

The smaller volumes of 10 ml can also be pipetted directly into 100 ml of single strength medium or

10 ml of double strength medium.

For quantitative estimates, an MPN series can be used. In an MPN series, 100 ml and larger volumes

can be filtered and membrane filters from different volumes are placed into 100 ml of separate single

strength broth. Smaller volumes of 10 ml can be pipetted into 100 ml of single strength medium or

10 ml of double strength medium, and 1 ml can be pipetted into 10 ml of single strength medium. For

example, volumes of 5 × 100 ml and 500 ml can be filtered and membranes placed into 100 ml of single

strength broth. Volumes of 10 ml can be directly inoculated into 5 × 100 ml of single strength medium

or 5 × 10 ml of double strength medium. Volumes of 5 × 1 ml can be inoculated directly into 10 ml of

single strength broth.

After filtration, the membranes are transferred using disinfected forceps (see 6.10) into enrichment

broths.
9 Procedure
9.1 Enrichment

For samples known to have low concentrations of background microorganisms, use the less selective

Bolton broth for enrichment and for samples expected to have a high concentration of background

microorganisms use the highly selective Preston broth. For samples where the background microbial

concentration is unknown, enrich parts of each sample in both Preston and Bolton broths. Preston broth

may be too selective to allow the recovery of some strains of C. coli. Bolton broth may not

...

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