EN 14486:2005

SLOVENSKI STANDARD
01-december-2005
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Water quality - Detection of human enteroviruses by monolayer plaque assay
Wasserbeschaffenheit - Nachweis humaner Enteroviren mit dem Monolayer-Plaque-
Verfahren
Qualité de l'eau - Détection des entérovirus humains par culture cellulaire par la méthode
des plages
Ta slovenski standard je istoveten z: EN 14486:2005
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN 14486
NORME EUROPÉENNE
EUROPÄISCHE NORM
May 2005
ICS 07.100.20
English version
Water quality - Detection of human enteroviruses by monolayer
plaque assay
Qualité de l'eau - Détection des entérovirus humains par Wasserbeschaffenheit - Nachweis humaner Enteroviren mit
culture cellulaire par la méthode des plages dem Monolayer-Plaque-Verfahren
This European Standard was approved by CEN on 8 April 2005.
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 Central Secretariat 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 Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,
Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
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EUROPÄISCHES KOMITEE FÜR NORMUNG
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© 2005 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 14486:2005: E
worldwide for CEN national Members.

Contents           Page
Foreword .3
Introduction.4
1 Scope .5
2 Normative references .5
3 Terms and definitions.5
4 Principle.5
5 Apparatus .6
6 Media.6
7 Cell culture .6
8 Assay procedure.7
9 Confirmation of enterovirus.8
10 Expression of results.8
11 Analytical Quality Control .9
12 Test report .9
Annex A (informative) Media: Suggested media for cell culture components.10
Annex B (informative) BGM cell culture.12
Annex C (informative) Alternative examination procedures .13
Bibliography.14

Foreword
This document (EN 14486:2005) has been prepared by Technical Committee CEN/TC 230 “Water analysis”, 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 November 2005, and conflicting national standards shall be withdrawn at the latest
by November 2005.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark,
Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
Introduction
Enterovirus infection of man occurs all year round with a seasonal peak in the summer and autumn. These viruses
are species specific and replicate primarily in the gastro-intestinal tract. Most infections are asymptomatic but some
may result in a flu-like illness that in a minority of cases leads to meningitis or paralysis. Gastro-enteritis may occur
as part of wider systemic disease. The viruses infect all ages but are particularly common in children. Many
serotypes exist and the most common serotypes change from year to year.
Like all other viruses, enteroviruses are obligate intracellular parasites and will not replicate in the environment.
Enteroviruses have been used as a marker for the presence of human faecal pollution for many years because
they were the first isolated and still remain the most readily detectable human viruses in environmental samples.
BGM (Buffalo Green Monkey) cells have been shown to be sensitive to infection by enteroviruses including
poliovirus and Coxsackievirus B (Dahling and Wright 1986, Morris 1985) and are in widespread use throughout the
world. The BGM monolayer plaque assay is approximately half as sensitive as the suspended cell plaque assay
(Dahling and Wright 1988) but utilises significantly fewer cells and is more practical for most laboratories. The
Council Directive of 8 December 1975 concerning the quality of bathing water (76:160/EEC) specifies that the
enumeration of enterovirus should be reported as confirmed 'Plaque forming units' per 10 l of original sample. The
method described below for infectious enteroviruses, therefore meets the requirements of the EU Bathing Water
Directive, is the most practical for most virus laboratories, is well established throughout the world and will detect a
group of viruses known to be common in environmental samples.
WARNING — Enteroviruses are pathogens capable of causing illness. All samples and controls shall be
handled by trained staff in a laboratory with appropriate equipment. Staff shall be fully vaccinated against
poliovirus. Persons using this standard shall be familiar with normal virology laboratory practice. This
standard does not purport to address fully all of the safety issues 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 European Standard describes a method for the detection of those serotypes of enterovirus that replicate and
form plaques in BGM cells under agar. These will largely be poliovirus and Coxsackievirus B serotypes. Most
serotypes of echovirus, Coxsackievirus A and animal enteroviruses are unlikely to produce plaques under these
conditions. Reoviruses are unlikely to be detectable within the specified seven day incubation period. It is
applicable to any type of water and processed water sample although toxic elements may interfere with cell culture.
The methods to confirm that the plaques seen contain enterovirus are given. Methods to identify which serotype of
enterovirus is found in the plaque are not included as this is not required by the EU Bathing Water Directive.
Basic cell culture procedures including the preparation, maintenance and enumeration of BGM cell culture are not
described but suggestions are made for media that may be used (see Annex A).
2 Normative references
The following referenced documents are indispensable for the application of this European Standard. 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 guide to the enumeration of micro-organisms by culture
3 Terms and definitions
For the purposes of this European Standard, the following term and definition applies.
3.1
enterovirus
virus that replicates and forms plaques in BGM cells under agar when incubated at (36 ± 2) °C for up to seven days
and is confirmed by the production of a characteristic enterovirus cytopathic effect (CPE) when sub-cultured into
fresh cells
4 Principle
4.1 Detection
Confluent monolayers of BGM cells in flasks or cell culture grade dishes are inoculated with sample and incubated
for 1 h at (36 ± 2) °C or for 2 h at room temperature. Excess sample is removed from each flask or dish if
necessary, and an overlay mixture containing agar is poured over the cell sheet and allowed to set. The cultures
are incubated at (36 ± 2) °C for up to 7 d.
4.2 Enumeration
The pale areas of cell death (plaques) are counted daily. It is assumed that a plaque is the progeny of a single
infectious unit of virus and the plaques counted and reported as 'plaque forming units (pfu)'.
The whole of the sample concentrate is assayed and the number of pfu in the original sample calculated.
4.3 Confirmation
The cells from a plaque are sub-cultured into fresh cells. An enterovirus in a plaque will be confirmed if it produces
a characteristic cytopathic effect (CPE) when sub-cultured into fresh cells under liquid maintenance medium.
The identification of the serotype of the enterovirus may be done by a serum neutralisation test, by
immunofluorescence using specific monoclonal antibodies, or by enterovirus specific polymerase chain reaction
(PCR). The details of these methods are beyond the scope of this European Standard.
5 Apparatus
5.1 General
All plastics ware and glassware shall be sterile.
Except for disposable glassware which is delivered sterile, glassware shall be sterilised in accordance with
instructions given in ISO 8199.
5.2 Incubator, capable of being maintained at (36 ± 2) °C, with 5 % (volume fraction) CO supply if Petri dishes
are used.
5.3 Water bath, capable of maintaining (45 ± 2) °C.
5.4 Water bath, boiling.
5.5 Refrigerator, capable of maintaining (5 ± 3) °C.
5.6 Deep freeze, capable of maintaining (-20 ± 5) °C.
5.7 Petri dishes, sterile, tissue culture grade, with a nominal diameter of 90 mm or 60 mm.
3 3 3
5.8 Tissue culture flasks, sterile, volume 25 cm , 75 cm , 80 cm
5.9 Pipettes, sterile, graduated, volumes 1 ml and 10 ml.
5.10 Micropipette, with sterile pipette tips, 1 ml to 5 ml.
5.11 Measuring cylinder, sterile, volume 50 ml.
5.12 Vertical laminar air-flow cabinet, optional for cell culture procedures.
5.13 Liquid nitrogen, optional for cell culture storage facility.
6 Media
Media shall be suitable for the culture and maintenance of BGM cells. Examples are given in Annex A.
7 Cell culture
7.1 Stock culture
Grow stock BGM cells on a seven-day cycle using local cell culture protocols.
Growth medium (A.1) may be replaced with maintenance medium (A.2) on day four or five.
Use the harvest of cells to produce confluent mono
...