Microbiology of the food chain - Detection and enumeration of Cryptosporidium and Giardia in fresh leafy green vegetables and berry fruits (ISO 18744:2016)

ISO 18744:2016 specifies a method that is applicable for the detection and enumeration of Cryptosporidium oocysts and Giardia cysts on or in food products that are described herein as fresh leafy green vegetables and berry fruits. With suitable controls, it may also be applicable for the examination of other fresh produce.
The microscopy descriptions are for Cryptosporidium spp. oocysts and Giardia duodenalis cysts of size ranges which include those species (Cryptosporidium) or assemblages (Giardia) known to be pathogenic to humans.
This method does not include any molecular analysis and therefore is not suitable for the determination of the species or genotypes/assemblages of Cryptosporidium oocysts and Giardia cysts. The method will detect all species and genotypes/assemblages that are known to be pathogenic for humans and also others that are not. For further identification, molecular typing assays are required. However, these cannot be reliably performed if process positive controls have been spiked into the samples, as the result of molecular typing assays will be obfuscated.
This method does not allow the determination of viability or infectivity of any Cryptosporidium oocysts and Giardia cysts which may be present.

Mikrobiologie der Lebensmittelkette - Nachweis und Zählung von Cryptosporidium und Giardia in frischem grünem Blattgemüse und Beeren (ISO 18744:2016)

Diese Internationale Norm legt ein Verfahren  zum Nachweis und zur Zählung von Cryptosporidium-Oozysten und Giardia Zysten auf oder in Lebensmitteln, die hier als frisches grünes Blattgemüse und Beeren beschrieben werden, fest. Mit geeigneten Kontrollen ist es möglicherweise auch für die Untersuchung anderer Frischwaren geeignet.
Die Beschreibungen für die Mikroskopie sind für Oozysten von Cryptosporidium spp. und Zysten von Giardia duodenalis in den Größenbereichen, die die Arten (Cryptosporidium) oder Genogruppen (Giardia) einschließen, die als pathogen für den Menschen bekannt sind.
Dieses Verfahren schließt keine molekulare Analyse ein und ist deshalb nicht zur Bestimmung der Arten oder Genotypen/Genogruppen von Cryptosporidium Oozysten und Giardia Zysten geeignet. Mit dem Verfahren werden alle Arten und Genotypen/Genogruppen nachgewiesen, die als pathogen für den Menschen bekannt sind, und auch andere, die das nicht sind. Zur weiteren Identifizierung sind molekulare Typisierungstests erforderlich. Diese können jedoch nicht zuverlässig durchgeführt werden, wenn die Proben mit positiven Prozesskontrollen beimpft (en: spiked) wurden, da das Ergebnis von molekularen Typisierungstests verschleiert wird.
Dieses Verfahren ermöglicht keine Bestimmung der Lebensfähigkeit oder Infektiosität beliebiger möglicherweise vorhandener Cryptosporidium Oozysten und Giardia Zysten.

Microbiologie de la chaîne alimentaire - Recherche et dénombrement des Cryptosporidium et Giardia dans les légumes verts frais à feuilles et les fruits à baies (ISO 18744:2016)

ISO 18744:2016 spécifie une méthode applicable à la détection et au dénombrement des oocystes de Cryptosporidium et aux kystes de Giardia sur ou dans des produits alimentaires qui sont décrits ici comme étant des légumes verts frais à feuilles et des fruits à baies. Grâce à des contrôles appropriés, elle peut être également applicable à l'examen d'autres produits frais.
Les descriptions faites au microscope concernent les oocystes de Cryptosporidium spp. et les kystes de Giardia duodenalis dont la taille inclut les espèces (Cryptosporidium) ou assemblages (Giardia) connus pour être pathogènes pour l'homme.
Cette méthode n'inclut pas l'analyse moléculaire et n'est donc pas applicable à la détermination des espèces ou des génotypes/assemblages d'oocystes de Cryptosporidium et de kystes de Giardia. La méthode permettra de détecter toutes les espèces et tous les génotypes/assemblages connus pour être pathogènes pour l'homme ainsi que d'autres qui ne le sont pas. Pour mieux les identifier, des essais de typage moléculaire sont requis. Toutefois, ceux-ci ne peuvent pas être effectués de manière fiable si des contrôles de processus positifs ont été ajoutés aux échantillons, car le résultat des essais de typage moléculaire sera erroné.
Cette méthode ne permet pas de déterminer la viabilité ou l'infectivité des oocystes de Cryptosporidium et des kystes de Giardia qui peuvent être présents.

Mikrobiologija v prehranski verigi - Ugotavljanje prisotnosti in števila "Cryptosporidium" in "Giardia" v sveži zeleni listni zelenjavi in jagodičevju (ISO 18744:2016)

Ta mednarodni standard določa metodo, ki se uporablja za ugotavljanje prisotnosti in števila oocist »Cryptosporidium« in cist »Giardia« na ali v živilih, ki so v tem dokumentu opisana kot sveža zelena listna zelenjava in jagodičevje. Z ustreznim nadzorom se lahko uporablja tudi za preiskovanje drugih svežih proizvodov. Ta metoda ne omogoča ugotavljanja sposobnosti za življenje ali infektivnosti vseh oocist Cryptosporidium in cist Giardia, ki so lahko prisotne. Opisi mikroskopije se uporabljajo za oociste Cryptosporidium in ciste Giardia velikostnih obsegov, ki vključujejo vrste, za katere je znano, da so patogene za ljudi.

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Publication Date
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6060 - Definitive text made available (DAV) - Publishing
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SLOVENSKI STANDARD
SIST EN ISO 18744:2016
01-junij-2016
0LNURELRORJLMDYSUHKUDQVNLYHULJL8JRWDYOMDQMHSULVRWQRVWLLQãWHYLOD

&U\SWRVSRULGLXPLQ*LDUGLDYVYHåL]HOHQLOLVWQL]HOHQMDYLLQMDJRGLþHYMX ,62



Microbiology of the food chain - Detection and enumeration of Cryptosporidium and

Giardia in fresh leafy green vegetables and berry fruits (ISO 18744:2016)

Mikrobiologie der Lebensmittelkette - Nachweis und Zählung von Cryptosporidium und

Giardia in frischem grünem Blattgemüse und Beeren (ISO 18744:2016)
Microbiologie de la châine alimentaire - Recherche et dénombrement des

Cryptosporidium et Giardia dans les légumes verts frais à feuilles et les fruits à baies

(ISO 18744:2016)
Ta slovenski standard je istoveten z: EN ISO 18744:2016
ICS:
07.100.30 Mikrobiologija živil Food microbiology
67.080.01 Sadje, zelenjava in njuni Fruits, vegetables and
proizvodi na splošno derived products in general
SIST EN ISO 18744:2016 en

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

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SIST EN ISO 18744:2016
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SIST EN ISO 18744:2016
EN ISO 18744
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2016
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of the food chain - Detection and
enumeration of Cryptosporidium and Giardia in fresh leafy
green vegetables and berry fruits (ISO 18744:2016)

Microbiologie de la chaîne alimentaire - Recherche et Mikrobiologie der Lebensmittelkette - Nachweis und

dénombrement des Cryptosporidium et Giardia dans Zählung von Cryptosporidium und Giardia in frischem

les légumes verts frais à feuilles et les fruits à baies grünem Blattgemüse und Beeren (ISO 18744:2016)

(ISO 18744:2016)
This European Standard was approved by CEN on 11 March 2016.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels

© 2016 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 18744:2016 E

worldwide for CEN national Members.
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SIST EN ISO 18744:2016
EN ISO 18744:2016 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 18744:2016
EN ISO 18744:2016 (E)
European foreword

This document (EN ISO 18744:2016) has been prepared by Technical Committee ISO/TC 34 “Food

products” in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”

the secretariat of which is held by DIN.

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by october 2016, and conflicting national standards shall

be withdrawn at the latest by october 2016.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent

rights.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,

France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,

Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and the United Kingdom.
Endorsement notice

The text of ISO 18744:2016 has been approved by CEN as EN ISO 18744:2016 without any modification.

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SIST EN ISO 18744:2016
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SIST EN ISO 18744:2016
INTERNATIONAL ISO
STANDARD 18744
First edition
2016-04-01
Microbiology of the food chain —
Detection and enumeration of
Cryptosporidium and Giardia in fresh
leafy green vegetables and berry fruits
Microbiologie de la chaîne alimentaire — Recherche et
dénombrement de Cryptosporidium et Giardia dans les légumes verts
frais à feuilles et les fruits à baies
Reference number
ISO 18744:2016(E)
ISO 2016
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SIST EN ISO 18744:2016
ISO 18744:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland

All rights reserved. Unless otherwise specified, 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
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved
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SIST EN ISO 18744:2016
ISO 18744:2016(E)
Contents Page

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

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

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

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

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

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

5 Reagents ........................................................................................................................................................................................................................ 2

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

7 Sampling and transport ................................................................................................................................................................................ 4

7.1 Sampling ....................................................................................................................................................................................................... 4

7.2 Transport ..................................................................................................................................................................................................... 4

7.3 Receipt of samples ............................................................................................................................................................................... 4

7.4 Storage ........................................................................................................................................................................................................... 4

7.5 Preparation of test sample ............................................................................................................................................................ 4

8 Procedure..................................................................................................................................................................................................................... 5

8.1 Removal of parasites from leafy green vegetables ................................................................................................... 5

8.2 Removal of parasites from berry fruits .............................................................................................................................. 6

8.3 Immunomagnetic separation (IMS) ...................................................................................................................................... 7

8.4 Sample staining ...................................................................................................................................................................................... 7

8.5 Microscopy ................................................................................................................................................................................................. 8

8.5.1 General comments .......................................................................................................................................................... 8

8.5.2 Examination of sample preparations using epifluorescence microscopy ...................... 9

8.5.3 Examination of sample preparations using DIC microscopy ..................................................11

9 Quality control procedures.....................................................................................................................................................................12

9.1 General ........................................................................................................................................................................................................12

9.2 Inclusion and interpretation of controls ........................................................................................................................12

9.3 Equipment cleaning .........................................................................................................................................................................13

10 Reporting of results ........................................................................................................................................................................................13

10.1 Expression of results .......................................................................................................................................................................13

10.2 Test report ................................................................................................................................................................................................13

Annex A (normative) Preparation of reagents ........................................................................................................................................15

Annex B (normative) Preparation of positive process control suspensions ............................................................17

Annex C (informative) Percentage recoveries achieved by the methods .....................................................................19

Bibliography .............................................................................................................................................................................................................................20

© ISO 2016 – All rights reserved iii
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SIST EN ISO 18744:2016
ISO 18744:2016(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 on the meaning of ISO specific terms and expressions related to conformity

assessment, as well as information about ISO’s adherence to the WTO principles in the Technical

Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information

The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 9,

Microbiology.
iv © ISO 2016 – All rights reserved
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SIST EN ISO 18744:2016
ISO 18744:2016(E)
Introduction

Cryptosporidium spp. and Giardia duodenalis (syn. G. lamblia, G. intestinalis) are protozoan parasites

that can cause enteric illness in humans. Both organisms are characterized by a robust transmission

stage, the Cryptosporidium oocyst and the Giardia cyst, which can survive in moist environments

for prolonged periods. These transmission stages are hereafter referred to collectively as (oo)cysts.

Cryptosporidium oocysts in particular are highly resistant to chlorine at the concentrations used in

the treatment of drinking water, and chemical disinfection of leafy green vegetables and berry fruits,

where performed during processing, may also be ineffective. Consequently, the absence of vegetative

bacteria on fresh produce as indicators of faecal contamination does not necessarily indicate the

absence of (oo)cysts. No practical method exists to culture Cryptosporidium spp. and Giardia duodenalis

for the purpose of detection, and therefore, in order to detect contamination with these parasites,

direct removal of the (oo)cysts from the food sample must be performed, followed by visualization of

the (oo)cysts by microscopy. The methods described in this International Standard are for determining

whether Cryptosporidium and/or Giardia (oo)cysts are present on the surfaces of fresh produce and for

their enumeration. This International Standard is based on published methods that have been tested in

a multicentre collaborative trial. Alternative methods can be used following a demonstration of their

[1]

equivalence with this International Standard following the protocol described in ISO 16140.

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SIST EN ISO 18744:2016
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SIST EN ISO 18744:2016
INTERNATIONAL STANDARD ISO 18744:2016(E)
Microbiology of the food chain — Detection and
enumeration of Cryptosporidium and Giardia in fresh leafy
green vegetables and berry fruits

WARNING — Persons using this International Standard should be familiar with normal

laboratory practice. This International 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.
1 Scope

This International Standard specifies a method that is applicable for the detection and enumeration of

Cryptosporidium oocysts and Giardia cysts on or in food products that are described herein as fresh leafy

green vegetables and berry fruits. With suitable controls, it may also be applicable for the examination

of other fresh produce.

The microscopy descriptions are for Cryptosporidium spp. oocysts and Giardia duodenalis cysts of size

ranges which include those species (Cryptosporidium) or assemblages (Giardia) known to be pathogenic

to humans.

This method does not include any molecular analysis and therefore is not suitable for the determination

of the species or genotypes/assemblages of Cryptosporidium oocysts and Giardia cysts. The method will

detect all species and genotypes/assemblages that are known to be pathogenic for humans and also

others that are not. For further identification, molecular typing assays are required. However, these

cannot be reliably performed if process positive controls have been spiked into the samples, as the

result of molecular typing assays will be obfuscated.

This method does not allow the determination of viability or infectivity of any Cryptosporidium oocysts

and Giardia cysts which may be present.
2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are

indispensable for its application. For dated references, only the edition cited applies. For undated

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

ISO 7218:2007, Microbiology of food and animal feeding stuffs — General requirements and guidance for

microbiological examinations
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
Cryptosporidium oocyst
transmission stage of Cryptosporidium spp.

Note 1 to entry: Its detection is based on reaction with specific anti-Cryptosporidium antibodies and morphological

characteristics as described in Clause 8.
© ISO 2016 – All rights reserved 1
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SIST EN ISO 18744:2016
ISO 18744:2016(E)
3.2
Giardia cysts
transmission stage of Giardia spp.

Note 1 to entry: Its detection is based on reaction with specific anti-Giardia antibodies and typical morphological

characteristics as described in Clause 8.
3.3
fresh leafy green vegetable

plant leaves eaten as a vegetable, which have not been subjected to any process, except perhaps cutting

and washing
3.4
fresh berry fruit

small, round or oblong, fleshy and juicy fruit, which has not been subjected to any process except

perhaps cutting and washing
3.5
internal extraction control

(oo)cysts labelled with specific fluorogenic reporters that may be added in defined numbers to the

sample prior to processing to assure that the method is operating properly
3.6
positive control

sample to which (oo)cysts have been added in defined numbers prior to extraction to verify that the

method after the elution step is operating efficiently
3.7
negative control

sample having an equivalent quantity of material to the tested sample, which is considered to be free of

(oo)cysts and processed in the same manner as the tested sample
4 Principle

The principle of the method is based on removal of the (oo)cysts from the sample by elution procedures,

followed by concentration in the eluate by centrifugation and isolation by immunomagnetic separation

(IMS). Detection of the (oo)cysts is performed by microscopy after labelling with specific monoclonal

antibodies (mAbs) conjugated to a fluorochrome.
5 Reagents

5.1 Reagents required for eluting (oo)cysts from leafy green vegetables and berry fruits

5.1.1 Glycine buffer, pH 5,5 for leafy green vegetables (A.2.1).
5.1.2 Glycine buffer, pH 3,5 for berry fruits (A.2.2).

5.2 Reagents required for concentrating, fixing, staining, detection, and quality control

5.2.1 Methanol, analytical grade.

5.2.2 Paramagnetic beads, coupled with antibodies specific to the walls of Cryptosporidium oocysts

and/or Giardia cysts.
5.2.3 Hydrochloric acid (HCl), 0,1 mol/l (A.3.1).
2 © ISO 2016 – All rights reserved
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SIST EN ISO 18744:2016
ISO 18744:2016(E)
5.2.4 Sodium hydroxide (NaOH), 1 mol/l (A.3.2).

5.2.5 Fluorescently-labelled monoclonal antibodies (mAbs), against Cryptosporidium oocysts

and/or Giardia cysts.
5.2.6 Immunofluorescence mounting medium (A.3.3).

5.2.7 4’,6’-diamidino-2-phenylindole dihydrochloride dihydrate (DAPI), freeze-dried reagent.

5.2.8 DAPI stock solution (A.3.4).
5.2.9 DAPI working solution (A.3.5).
5.2.10 Phosphate buffered saline (PBS), pH 7,3 (A.1.1).
5.2.11 Non-fluorescing immersion oil.
5.2.12 Stock suspensions of Cryptosporidium oocysts and Giardia cysts (Annex B).

5.2.13 Suspensions of pre-labelled and enumerated non-viable Cryptosporidium oocysts and

Giardia cysts.

The fluorochrome label shall be a different colour than that which is used for the detection of the target

organism.
5.2.14 Parasite storage medium (A.3.6; A.3.7).
5.2.15 Demineralized and filtered water (ultrapure water type 1).
5.2.16 Fingernail varnish (for sealing coverslips onto slides as necessary).
6 Apparatus

This method requires common microbiological laboratory equipment (refer to ISO 7218) and, in

particular, the following.
6.1 Tweezers, for handling fresh produce as necessary.
6.2 A paddle (peristaltic) blender and compatible filtered bags.

6.3 Swing out centrifuge, to accommodate at least 4 × 50 ml conical centrifuge tubes per run.

6.4 Glass Leighton tubes.
6.5 Rotating mixer, compatible with the Leighton tubes.
6.6 Magnetic clip stand, compatible with the Leighton tubes.
6.7 Magnetic clip stand, for microcentrifuge tubes.

6.8 Welled slides, with hydrophobic coating, wells capable of accommodating the 50 µl volume of

processed sample after IMS, and coverslips of appropriate size.
© ISO 2016 – All rights reserved 3
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SIST EN ISO 18744:2016
ISO 18744:2016(E)

6.9 Slide warmer tray, 37 °C to 42 °C incubator, or equivalent slide-drying apparatus.

6.10 Humidity chamber, lidded box containing damp absorbent material, e.g. a paper towel that can be

held at the temperature and humidity appropriate for the immunofluorescent reagent.

6.11 Aspiration device, vacuum source equipped with liquid trap and pipette, or equivalent

6.12 Epifluorescence microscope with 20×, 40× objectives and 100× objective immersion lens

and a calibrated eyepiece graticule (reticule), shall include fluorescein isothyocyanate (FITC) filter set

(480 nm excitation, 520 nm emission filter) and DAPI filter set (375 nm excitation, >420 nm emission).

If internal extraction controls are used, an additional filter set suitable for the fluorochrome will be

required. Differential interference contrast (DIC) optics are advantageous. A photographic recording

system attached to the microscope may be pertinent for recording positive or presumptive events.

6.13 FITC control slide, for evaluation of fluorescence intensity and verification of proper performance

of the optical system of the fluorescence microscope.
7 Sampling and transport
7.1 Sampling

A procedure for sampling is not specified in this International Standard. See the specific International

Standard dealing with the product concerned. If there is no specific International Standard, it is

recommended that the relevant stakeholders (e.g. competent authorities, regulators, customers) come

to an agreement on this subject.
7.2 Transport

Soft fruit samples and other delicate samples shall be handled carefully during transport to preserve

their physical integrity (refer to ISO 7218:2007, 8.2).
7.3 Receipt of samples

Samples should be assessed for acceptance criteria using appropriate guidance as required within the

purpose of testing (refer to ISO 7218:2007, 8.3). Fresh leafy green vegetables and berry fruits shall be

regarded as perishable and analysis shall commence as soon as possible after acceptance. Criteria for

rejection can include the presence of mould, decomposition of sample, or loss of sample integrity in the

case of berry fruit.
7.4 Storage

Fresh leafy green vegetables and berry fruits should be stored refrigerated (between 4 °C and 8 °C), to

reduce sample deterioration (refer to ISO 7218:2007, 8.4).
7.5 Preparation of test sample

The condition of the fresh produce shall be noted before the analytical procedure is commenced.

Test samples shall be at least 25 g.

It is recommended that no more than 100 g of any individual sample is analysed in a single procedure.

When analysing whole leafy green vegetables, such as lettuce heads, it is recommended that a random

selection of intact clean leaves (exclude stems) from different parts of the plant should be examined.

4 © ISO 2016 – All rights reserved
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SIST EN ISO 18744:2016
ISO 18744:2016(E)

For samples that are in small units, for example, raspberries, the sample shall consist of a random

sample of these units.
8 Procedure
8.1 Removal of parasites from leafy green vegetables

a) Place sample in filtered bag. Avoid excessive handling. Use tweezers if necessary.

It is recommended that each sample is spiked with an internal extraction control suspension

containing a defined number of pre-labelled and enumerated Cryptosporidium oocysts and/or

Giardia cysts following the manufacturer’s instructions. The control suspension should be pipetted

on to the surface of the leaves; this should be done after the sample has been placed in the

processing container. The fluorescent label used on the internal extraction control should differ

from that used to detect the target (oo)cysts in the test samples. If internal extraction controls are

not used, a positive process control sample is recommended (see 9.2).
b) Add 200 ml glycine buffer pH 5,5.
c) Process sample in paddle blender for 30 s at 200 rpm to 300 rpm.

d) Collect eluate into 50 ml conical-bottomed centrifuge tubes, dividing the eluate equally between

them, ensuring that all the vegetable matter is retained in the filter. Squeeze the bag and the

filter tightly in order to ensure that all the eluate is obtained from the sample. The eluate may be

collected into a vessel before transferring to the centrifuge tubes.

Alternatively, the eluate can be transferred to a single 250 ml conical-bottomed centrifuge bottle, if

an appropriate centrifuge/rotor is available for the subsequent step.

Rinsing the sample following washing could increase recovery efficiencies. After transferring

the eluate from the filter bag, and while the tubes are being centrifuged [see point e)], add

10 ml 1 mol/l glycine buffer pH 5,5 to the sample and rinse by manipulating the sample from outside

the bag. After removal of the supernatant from the centrifuged tubes, add the rinsate from the

sample to the tubes. Add a further 10 ml 1 mol/l glycine buffer pH 5,5 to the bag, again manipulate

produce and water from outside the bag, and add rinsate to tubes. Centrifuge as in point e).

e) Centrifuge the eluate at 2 500g maximum for 10 min with no braking.

NOTE 1 Centrifugation at 2 500g maximum can result in a very compact pellet. A lower speed of

1 100g maximum for 10 min with no braking has been reported to give at least equivalent recoveries of

Cryptosporidium oocysts and Giardia cysts.

f) Remove the supernatant, by using a pipette and vacuum source, ensuring the pellet is not disturbed.

If no pellet is visible, extra care shall be taken to ensure that parasites are not lost during aspiration.

This can be done by leaving a small volume of liquid in the bottom of the tube.

g) Re-suspend the pellet in the residual liquid left in the bottom of each tube and combine the pellets

into a single centrifuge tube.

h) Rinse the empty 50 ml conical centrifuge tubes with sterile distilled water and transfer the rinsate

into the tube containing the combined pellets. Repeat this process until the volume of

...

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