SIST EN ISO 19458:2007

SLOVENSKI STANDARD
SIST EN ISO 19458:2007
01-februar-2007
.DNRYRVWYRGH9]RUþHQMH]DPLNURELRORãNHDQDOL]H,62
Water quality - Sampling for microbiological analysis (ISO 19458:2006)
Wasserbeschaffenheit - Probenahme für mikrobiologische Untersuchungen (ISO
19458:2006)
Qualité de l'eau - Échantillonnage pour analyse microbiologique (ISO 19458:2006)
Ta slovenski standard je istoveten z: EN ISO 19458:2006
ICS:
13.060.45 Preiskava vode na splošno Examination of water in
general
SIST EN ISO 19458:2007 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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EUROPEAN STANDARD
EN ISO 19458
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2006
ICS 13.060.45

English Version
Water quality - Sampling for microbiological analysis (ISO
19458:2006)
Qualité de l'eau - Échantillonnage pour analyse Wasserbeschaffenheit - Probenahme für mikrobiologische
microbiologique (ISO 19458:2006) Untersuchungen (ISO 19458:2006)
This European Standard was approved by CEN on 1 July 2006.
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, Romania,
Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2006 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 19458:2006: E
worldwide for CEN national Members.

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EN ISO 19458:2006 (E)





Foreword


This document (EN ISO 19458:2006) has been prepared by Technical Committee ISO/TC 147
"Water quality" in collaboration with 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 February 2007, and conflicting national
standards shall be withdrawn at the latest by February 2007.

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, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.


Endorsement notice

The text of ISO 19458:2006 has been approved by CEN as EN ISO 19458:2006 without any
modifications.

2

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INTERNATIONAL ISO
STANDARD 19458
First edition
2006-08-01


Water quality — Sampling
for microbiological analysis
Qualité de l'eau — Échantillonnage pour analyse microbiologique





Reference number
ISO 19458:2006(E)
©
ISO 2006

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ISO 19458:2006(E)
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ISO 19458:2006(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Sampling point . 1
4 Sampling technique. 2
5 Transport and storage. 10
Annex A (informative) A priori determination of the number of samples to analyse to determine
the mean concentration of microbes in water with a given confidence, for quantitative
determination derived by cultivation of microorganisms . 13
Annex B (informative) Recommended (R) and acceptable (A) values for maximum sample storage
times including transport time and temperatures unless otherwise specified in specific
standards. 16
Bibliography . 17

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ISO 19458:2006(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 19458 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,
Microbiological methods.
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ISO 19458:2006(E)
Introduction
Appropriate sampling is essential to provide representative samples to the laboratory in charge of testing.
Sampling features depend on the objective of sampling, but also on the nature of the sample. Microorganisms
are living organisms. In addition, when they are introduced into water, they do not form a perfect solution, but
a suspension with an inherent degree of variability.
Sampling objectives may serve different purposes, which are described in the ISO 5667 series of standards
(ISO 5667-1, ISO 5667-2 and ISO 5667-3):
a) determination of the compliance of a water with a regulatory quality specification;
b) characterization of any contamination, its level (mean) and its variations:
1) what is its random variation?
2) is there a trend?
3) are there cycles?
c) identification of the sources of pollution.
Regarding the number or frequency of samples, it will vary according to the aim of the sampling.
The minimum number of samples will be low if the mean concentration differs greatly from the specification
(much lower or much higher), and the minimum number of samples will be higher if the mean concentration
and the specification are close to one another. Similarly, in case b), when looking for a trend: the less obvious
the trend, the higher the frequency of sampling (see also Annex A).

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INTERNATIONAL STANDARD ISO 19458:2006(E)

Water quality — Sampling for microbiological analysis
WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. This standard does not purport to address all of the safety problems, if any, associated with
its use. It is the responsibility of the user to establish appropriate safety and health practices and to
ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted according to this standard be carried
out by suitably trained staff.
1 Scope
This International Standard provides guidance on planning water sampling regimes, on sampling procedures
for microbiological analysis and on transport, handling and storage of samples until analysis begins. It focuses
on sampling for microbiological investigations.
General information in respect to the sampling from distinct water bodies is given in the respective parts of
ISO 5667.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 5667-1, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes and
sampling techniques
ISO 5667-2, Water quality — Sampling — Part 2: Guidance on sampling techniques
ISO 5667-3, Water quality — Sampling — Part 3: Guidance on the preservation and handling of water
samples
3 Sampling point
The sampling site shall provide representative characteristics and account for any vertical, horizontal and
temporal variations and shall be identified precisely following the general recommendations of ISO 5667-1 and
ISO 5667-2, taking into account additional aspects specific to microbiology.
Sampling points where conditions are unstable should be avoided, and the heterogeneity of the hydraulic
system shall be taken into consideration. In studies on the efficacy of disinfection, the sampling point shall be
chosen to ensure that the reaction is complete.
EXAMPLE Examples of how the heterogeneity of the system may influence the results are given below.
⎯ It is not equivalent to take a subsurface or a surface sample, or a subsurface sample “contaminated” during recovery
through the surface film. In some instances (e.g. lakes, swimming pools), the concentration in the surface film can be
1 000 times higher than in the subsurface.
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ISO 19458:2006(E)
⎯ All the points of a network are not equivalent, as there may be dead ends and sections where the flow is reduced,
particularly if the network is fed from two sources.
⎯ The quality at the outlet of a well-mixed tank is generally the same as in the body of water, but can be quite different
from the inlet.
4 Sampling technique
4.1 Personnel
Formal training, training records and determination of competence shall be described for all those who sample,
and this information shall be properly documented.
4.2 Sample containers
4.2.1 General
For routine samples (for example, sampling at taps, recreational waters, swimming pool waters), use clean,
sterile bottles. The volume of the bottles should be adequate for analysis of all requested parameters.
For sampling by immersion in clean waters, use bottles that are sterile both inside and out and protected, for
example, by kraft paper (to keep dry after autoclaving), aluminium foil or by plastic outer bags.
If not autoclavable, sterilization with gamma rays or by ethylene oxide may be used. The bag can then be
opened just before sampling and can also serve as a glove to hold the bottle to provide maximum asepsis
before being placed on a pole or other sterilizable sampling apparatus.
Alternatively, the outside of sample bottles may be disinfected immediately prior to immersion by a suitable
disinfectant such as isopropanol (4.3.1.1) and allowed to dry before use. This is not suitable for analysis of
spore-forming bacteria.
In most cases, 500 ml bottles are sufficient, as less than five categories of microorganisms are measured,
each involving inoculation of a maximum of 100 ml.
In some cases, larger volumes are necessary, e.g.:
⎯ for bottled water analysis (250 ml per parameter);
⎯ for Legionella spp. or Salmonella spp. (up to 1 l);
⎯ for viruses, Giardia cysts, Cryptosporidium oocysts, amoebae in clean waters, from 10 to several hundred
litres or more are examined. Usually, a concentration step is made on site using a cartridge filter which is
then transported to the laboratory.
Bottles can be made of glass or various plastics (polypropylene, polystyrene, polyethylene, polycarbonate).
Usually glass is preferred for re-use, and polyethylene is used as disposable.
Adhesion to surfaces can lower the detection of microorganisms, and the critical tangential surface tension γ
[13]
has to be considered if a non-standard material is used .
Closures can be a ground glass or plastic stopper for glass bottles, a plastic press-on lid for plastic bottles or
jars, or a plastic or metal screw cap for either. Bottle openings closed with plastic or glass stoppers should be
further protected from contamination by, e.g. aluminium foil.
When larger volumes are necessary for the assay of, for example, viruses, Salmonella spp., amoebae,
Cryptosporidium oocysts, Giardia cysts, it is sometimes necessary to analyse tens of litres or hundreds of
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ISO 19458:2006(E)
litres. To avoid the difficulties of handling, refrigerating and agitating such volumes, a concentration step in situ
(by flocculation, centrifugation or filtration) is recommended. Peristaltic pumps can be used with sterile tubing.
NOTE 1 Metal caps, especially aluminium, can produce toxicity when autoclaved. This can be prevented by
incorporating a heat-resistant leak-proof liner.
NOTE 2 Certain materials can also give toxic by-products when heat sterilized, even in a dry oven, or induce pH
changes.
NOTE 3 Some brands of cotton wool used to make plugs for glassware may become toxic if they are heated for too
long at too high temperatures.
NOTE 4 Press-on plastic lids attached to the bottle or jar have several advantages in that they are as leak-proof as
screw-caps, and the lids can stand open, which facilitates filling and pipetting. When open, the lid remains linked to the
bottle, so bottles and closures are kept together, and the lid is also protected from contamination.
4.2.2 Sterilization of bottles
If re-used, clean glass bottles and their closures with a non-toxic, phosphorus-free detergent, followed by a
thorough rinse with deionized or distilled water.
Autoclave bottles at (121 ± 3) °C for at least 15 min. Keep the closure of the bottles loose, to allow the steam
to replace all the air during the temperature rise, and to prevent plastic bottles from collapsing when cooling.
Tighten screw caps after sterilization. Autoclave glass stoppers separately from the bottle, or use a paper or
aluminium separator to prevent the stopper sticking on cooling.
If necessary, sterilize bottles in a dry oven for at least 1 h at (170 ± 10) °C. Separate ground glass stoppers
from the neck by a paper strip or a piece of string to avoid jamming during cooling. The bottles should be
traceable to the sterilization date.
Control the effectiveness of the sterilization process by chemical or biological indicators.
When sterilization is not possible with any other means, disinfect by immersing open bottles in boiling water
for at least 30 min. Immediately after boiling, empty the bottles and close them with boiled caps and wrapped
in clean paper.
NOTE 1 Polyethylene bottles can be sterilized by exposure to ethylene oxide gas, but, because of its toxicity, the
procedure is carried out in specialized facilities and time allowed for desorption of the ethylene oxide. It is therefore not
used as a routine laboratory procedure.
60 137
NOTE 2 Exposure to gamma rays produced by a Co or Cs source or to accelerated electrons of sufficient energy
4 4
(1 × 10 Gy to 2 × 10 Gy) is a very efficient sterilization technique, available in specialized installations. There is no
residual antibacterial activity, but some materials may be altered by polymerization after repeated irradiation.
4.2.3 Inactivation of disinfectants
To assess the microbiological quality of water disinfected by an oxidant (e.g. chlorine, chloramine, bromine or
ozone), stop the action of the oxidant as soon as the sample is taken. Add a reducing agent such as sodium
thiosulfate to the sample bottles.
The theoretical mass of sodium thiosulfate (pentahydrate) necessary to inactivate 1 mg of chlorine is 7,1 mg.
Thus, 0,1 ml of sodium thiosulfate pentahydrate solution (4.3.1.2) is added for each 100 ml of bottle capacity.
This will inactivate at least 2 mg/l and up to 5 mg/l of free chlorine residual, depending on inactivation
dynamics, which is sufficient for the majority of samples.
In certain circumstances, such as foot baths in swimming pools, disinfection measures (e.g. Legionella
eradication in drinking water distribution systems), higher chlorine concentrations can be found and a
proportionately higher dosage of sodium thiosulfate will be necessary.
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ISO 19458:2006(E)
Sodium thiosulfate is not destroyed by autoclaving or dry heat. Ensure that the pH of the sodium thiosulfate
solution is around neutral (low pH can cause decomposition).
Sodium thiosulfate has no effect on the sample and can be used for non-chlorinated waters too.
NOTE It has been claimed that Legionella are sensitive to sodium and that potassium thiosulfate is preferable, but no
adverse effect of sodium has been detected at the concentration used to inactivate usual chlorine concentrations.
For other disinfectants, corresponding inactivation measures need to be taken. If inactivation is not possible or
feasible, it has to be reported.
Chelating agents have been recommended to protect bacteria from the toxic action of heavy metals such as
copper or zinc. Ethylene dinitrilotetraacetic acid (EDTA) or sodium nitrilotriacetate (NTA) (Na C H NO ) can
3 6 6 6
be used as a filter-sterilized solution at a final concentration of about 50 mg per litre but should only be added
when necessary (e.g. water treated with silver or copper). Silver can also be inactivated by sodium sulfide.
Add 1 ml of a sodium sulfide solution (4.3.1.3) to 1 l of sample.
4.2.4 Quality control of sample bottles
4.2.4.1 Testing of sterility
The laboratory shall ensure the sterility of the sample bottles, whether they are prepared in-house or
commercially, whether they are made of glass or of plastic. Commercially prepared bottles should be
delivered with a certificate of sterilization as a condition for acceptance, and sterility tests are also advisable
on the batch in use. This relates to the batch of bottles after labelling, addition of inactivation agents where
relevant, and storage.
The sterility of bottles can usually be guaranteed by control of the sterilization process. If not, the sterility of
the containers should be tested.
EXAMPLE The following are examples of testing procedures (usually performed a rate of 1 per 100 bottles):
a) “Roll bottle” method
This consists of introducing 20 ml or 50 ml of melted nutrient agar (plate count agar) into the test bottle and lining the
walls with agar by rotating the bottle while cooling (under a trickle of water if necessary). Incubation at (22 ± 2) °C for
five days should give no visible growth.
b) Liquid broth method
This consists of placing 20 ml to 50 ml of thioglycollate or other nutrient broth inside the bottle, rolling the bottle to wet
the walls and incubating at (22 ± 2) °C for five days. No turbidity should appear if sterile.
4.2.4.2 Testing for the presence of inactivating agents
The presence of thiosulfate may be checked by an iodometric method:
2− − 2−
I + 2 S O 2 I + S O
2 2 3 4 6
Add 10 ml distilled water to the bottle and titrate with iodine solution (4.3.1.4), using starch or thiophene as an
end point titration agent.
4.2.4.3 Testing for residual toxicity in sample bottles
Residual toxicity in sample containers may result from the washing procedure of glassware, from the release
of components or additives from plastic bottles and also from the sterilization process. Routine use of glass or
polyethylene bottles does not require a regular check for toxicity, but if in any doubt, test according to
[8]
Geldreich, 1975 (for example).
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ISO 19458:2006(E)
4.3 Reagents, apparatus and materials
4.3.1 Reagents
4.3.1.1 Ethanol, volume fraction ϕ (C H OH) = 70 %, isopropanol, volume fraction
2 5
–
ϕ [(CH ) CHOH] = 70 %, or hypochlorite solution, ρ (CLO ) ≈ 1 g/l.
3 2
4.3.1.2 Sodium thiosulfate pentahydrate solution, ρ (Na S O ·5H O) = 18 mg/ml.
2 2 3 2
4.3.1.3 Sodium sulfide solution, ρ (Na S) = 0,1 mg/ml.
2
4.3.1.4 Iodine solution, c(I ) = 0,05 mol/l.
2
4.3.2 Apparatus and materials
In addition to sample containers, the following items may be necessary.
4.3.2.1 Soap and towels.
4.3.2.2 Gas blow lamp and refill.
4.3.2.3 Jars or beakers, disinfecting wipes.
4.3.2.4 Lighter, matches.
4.3.2.5 Markers, pencils, labels.
4.3.2.6 Spanners, pliers, screwdrivers, knife.
4.3.2.7 Icebox and ice or ice packs, portable refrigerators or refrigerated compartments in vehicles.
4.3.2.8 Thermometer or temperature recorder.
4.3.2.9 Ballasted bottle-carrier or equivalent, with rope or chain (preferably stainless steel, at least the
bottom part).
4.3.2.10 Pole or long forceps or samplers adapted to various depths.
4.3.2.11 Maps, list of sampling points, sampling forms.
4.3.2.12 Vehicle and papers, identity or authorization card.
4.3.2.13 Waterproof (safety) boots.
4.3.2.14 Apparatus to measure pH, chlorine, dissolved oxygen, conductivity.
4.3.2.15 Sterile gloves.
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ISO 19458:2006(E)
4.4 Filling procedure
4.4.1 Potable water from a tap
4.4.1.1 General
Sampling at a tap can have different purposes:
a) to determine the quality of the water in the distribution main (which is the responsibility of the distributor);
b) to know the quality of the water as it flows from the tap to be consumed — as it is delivered to the tap —
(which can be altered by the service network inside the building);
c) to know the quality of the water as it is consumed, i.e. as it flows out of the (possibly contaminated) tap.
Samples to assess the quality in the main [case a)] are best taken at special taps (also in the distribution
system) that are close to the main distribution, clean, without attachments and disinfectable by flaming or
suitable equivalent.
Normal taps may be used to assess the quality in the mains [still case a)], if they are disinfectable by flaming
but in case of unclear results, consider the service network as potential source of contamination.
The situation described in case b) is the method of choice to assess the quality of drinking water including the
influence of the service network inside the building. In this case, taps disinfectable by flaming are not always
available and other disinfection methods [application of hypochlorite solution, ethanol or isopropanol (4.3.1.1)]
need to be considered.
The situation described in case c) is the method to assess the quality of drinking water in special situations,
e.g. outbreaks.
Depending on the purpose, it is either necessary or incorrect to:
⎯ remove attached devices and inserts;
⎯ disinfect the tap;
⎯ flush (see Table 1).
Table 1 — Sampling at a tap for different purposes
Purpose
Water type Remove attached devices and inserts Disinfect Flush
(see above)
a) In the distribution main Yes Yes Yes
a
b) As it is delivered to the tap Yes Yes No (minimal)
c) As it is consumed No No No
a
Flush briefly only to overcome influence of disinfection of the tap.

Sampling from drinking water storage tanks is usually made from a tap on the outlet. Subsurface samples are
sometimes taken from the tank itself, in which case, bottles that are sterile both inside and outside (see 4.2.1)
are required.
Ensure samples are taken aseptically using clean hands or sterile gloves with protection of the sample from
air drifts and splashing.
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ISO 19458:2006(E)
During filling, the inside of the bottle closure shall not come in contact with anything (fingers, ground, pocket,
teeth).
Leave some air space in the bottle to allow for adequate shaking before analysis.
Close the bottle immediately. Do not use this water sample for the measurement of temperature or any other
on-site tested parameter.
For details, see as well ISO 5667-5.
4.4.1.2 Water in treatment works and storage tanks
In water treatment works and storage tanks, dedicated sample taps should be provided on each outlet main
and other sampling points. These should be capable of being sterilized by flaming, maintained in a clean state,
labelled clearly and used exclusively for sampling. For details, see ISO 5667-5 and ISO 5667-13.
Flaming a metal tap intensely with a blowlamp ensures disinfection of the mouth if the temperature there
reaches 80 °C or more. This is not the case if water remains in the heated portion.
NOTE Flaming with a lighter is only superficial (not sufficient).
4.4.1.3 Water in the distribution main
To determine the quality in the distribution main, sample in the distribution main or close to it (usually just after
the water meter). Ensure that no contamination from the outer surface of the tap reaches the sample. Do not
sample taps with leaking spindles and avoid mixer taps, if possible. Take out any faucet nozzle or other
attachment or insert (spanners and pliers shall be available). Scrape off any dirty (scale, slime, grease or
other extraneous matter) and fully open and close the tap repeatedly to rinse out the dirt from the tap.
Disinfect the tap preferentially by flaming (after flaming and opening the tap, a sizzling noise should occur).
Subsequently, open the tap to half-flow and flush until constant water temperature is reached (to overcome
the water inside the building). Then place the open bottle in the water flow and fill it under aseptic conditions.
Only if flaming is not possible, disinfect the tap by other adequate methods. To disinfect the mouth of a plastic
tap, after thorough cleaning, dip it for 2 min to 3 min in a beaker with hypochlorite solution, ethanol or
isopropanol (4.3.1.1). Alternatively, a swab or a wash bottle or similar device may be used to disinfect the
outside and as much of the inside as possible.
Subsequently, allow the water to flow long enough to minimize the influence of the network inside the building.
It is necessary to know the detailed layout of the network (volume of tanks or softeners and retention time) to
determine the flush time before sampling. Water temperature stabilization may be monit
...