EN ISO 6222:1999

Water quality - Enumeration of culturable micro-organisms - Colony count by inoculation in a nutrient agar culture medium (ISO 6222:1999)

This European Standard specifies a method for the enumeration of culturable micro-organisms in water by counting the colonies formed in a nutrient agar culture medium after aerobic incubation at 36 °C and 22 °C. The method is intended to measure the operational efficiency of the treatment process of public drinking water supplies and for general application to all types of water. It is particulary applicable to the examination of water intended for  human consumption, including water in closed containers and to natural mineral waters.

Wasserbeschaffenheit - Quantitative Bestimmung der kultivierbaren Mikroorganismen - Bestimmung der Koloniezahl duch Einimpfen in ein Nähragarmedium (ISO 6222:1999)

Diese Europäische Norm beschreibt ein Verfahren für den quantitativen Nachweis von kultivierbaren Mikroorganismen im Wasser durch Zählung der Kolonien, die sich auf Nähragar nach aerober Inkubation bei 36 °C und 22 °C bilden. Das Verfahren ist anwendbar zur Beurteilung der Leistungsfähigkeit des Aufbereitungsprozesses der öffentlichen Trinkwasserversorgungen und fu(r allgemeine Anwendungen für alle Arten von Wasser. Es ist besonders geeignet für die Untersuchung von Wasser für den menschlichen Gebrauch, einschließlich Wasser in geschlossenen Behältnissen und natu(rliche Mineralwa(sser.

Qualité de l'eau - Dénombrement des micro-organismes revivifiables - Comptage des colonies par ensemencement dans un milieu de culture nutritif gélosé (ISO 6222:1999)

La présente norme internationale spécifie une méthode de dénombrement des micro-organismes revivifiables présents dans l'eau par comptage des colonies se formant dans un milieu de culture nutritif gélosé après incubation en aérobiose à 36 °C et 22 °C. La méthode vise à mesurer l'efficacité de fonctionnement du procédé de traitement des alimentations publiques en eau potable et, plus généralement de tous les types d'eau. Elle est plus particulièrement applicable à l'analyse des eaux destinées à la consommation humaine, y compris des eaux en récipients fermés et des eaux minérales naturelles.

Kakovost vode – Ugotavljanje števila mikroorganizmov, sposobnih tvorbe kolonij - Štetje kolonij z nasajanjem v hranljivi agar (ISO 6222:1999)

General Information

• Status: Published
• Publication Date: 14-May-1999
• ICS: 13.060.30 - Sewage water
• Technical Committee: CEN/TC 230 - Water analysis
• Drafting Committee: CEN/TC 230/WG 3 - Water analysis - Microbiological methods
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Start Date: 21-May-2008
• Completion Date: 21-May-2008
• Ref Project: SIST EN ISO 6222:1999 - Water quality - Enumeration of culturable micro-organisms - Colony count by inoculation in a nutrient agar culture medium (ISO 6222:1999)

Overview

EN ISO 6222:1999 specifies a standardized culture-based method for the enumeration of culturable micro-organisms in all types of water. The procedure uses a nutrient agar medium and the pour-plate technique with aerobic incubation at 36 °C and 22 °C. Results are reported as colony-forming units per millilitre (cfu/ml) and are intended to support operational monitoring of drinking-water treatment, distribution integrity and general water-quality surveillance.

Key Topics

• Scope and purpose: Method for counting colonies formed on nutrient agar after aerobic incubation at two temperatures to assess treatment efficiency and water suitability for human consumption (including bottled and natural mineral waters).
• Principle: Inoculate measured sample volumes or dilutions into molten yeast-extract nutrient agar, allow it to set and incubate at specified temperatures; count colonies and calculate cfu/ml.
• Procedure highlights:
  • Pour-plate inoculation (sample volume not exceeding 2 ml) mixed with 15–20 ml molten medium.
  • Incubation: one set at 36 ± 2 °C for 44 ± 4 h; another set at 22 ± 2 °C for 68 ± 4 h.
  • Examination immediately after incubation (or store at 5 ± 3 °C and examine within 48 h if necessary).
• Media and preparation: Yeast-extract agar (composition given in the standard) prepared using high-purity water (EN ISO 3696 grade), sterilized and held molten at 45 ± 1 °C for use.
• Result expression: Report counts as cfu/ml for each incubation temperature; if no colonies are found in plates inoculated with undiluted sample, express as "not detected in 1 ml"; counts above 300 colonies are reported as > 300 or approximate.

Applications

This method provides practical value in routine laboratory monitoring and operational control:

• Verification of water-treatment stages (coagulation, filtration, disinfection) by tracking changes in culturable counts.
• Surveillance of drinking-water supplies and bottled waters to detect contamination events or distribution-system breaches.
• Quality control for food- and beverage- related water supplies where low microbial load is required.
• Long-term trend monitoring: sudden increases in colony counts serve as early warnings prompting investigation.

Practical notes: follow sampling and preservation guidance (EN ISO 5667-3, EN 25667-2), use appropriate diluents (ISO 8199, ISO 6887) and ensure plate integrity (reject confluent growth).

Related Standards

• EN ISO 3696 - Water for analytical laboratory use
• EN ISO 5667-3 - Sampling: preservation and handling
• EN 25667-2 - Sampling techniques
• ISO 6887 - Preparation of dilutions for microbiological examinations
• ISO 8199 - General guide to enumeration of micro-organisms by culture

Using EN ISO 6222:1999 ensures consistent, comparable colony-count data for water-quality assessment, regulatory compliance and operational decision-making.

Standards Content (Sample)

SLOVENSKI STANDARD
01-november-1999
1DGRPHãþD
SIST ISO 6222:1997
Kakovost vode – Ugotavljanje števila mikroorganizmov, sposobnih tvorbe kolonij -
Štetje kolonij z nasajanjem v hranljivi agar (ISO 6222:1999)
Water quality - Enumeration of culturable micro-organisms - Colony count by inoculation
in a nutrient agar culture medium (ISO 6222:1999)
Wasserbeschaffenheit - Quantitative Bestimmung der kultivierbaren Mikroorganismen -
Bestimmung der Koloniezahl duch Einimpfen in ein Nähragarmedium (ISO 6222:1999)
Qualité de l'eau - Dénombrement des micro-organismes revivifiables - Comptage des
colonies par ensemencement dans un milieu de culture nutritif gélosé (ISO 6222:1999)
Ta slovenski standard je istoveten z: EN ISO 6222:1999
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

INTERNATIONAL ISO
STANDARD 6222
Second edition
1999-05-15
Water quality — Enumeration of culturable
micro-organisms — Colony count by
inoculation in a nutrient agar culture
medium
Qualité de l'eau — Dénombrement des micro-organismes revivifiables —
Comptage des colonies par ensemencement dans un milieu de culture nutritif
gélosé
A
Reference number
ISO 6222:1999(E)
ISO 6222:1999(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 3.
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
International Standard may be the subject of patent rights. ISO shall not be
held responsible for identifying any or all such patent rights.
International Standard ISO 6222 was prepared by the European
Committee for Standardization (CEN) in collaboration with ISO Technical
Committee TC 147, Water quality, Subcommittee SC 4, Microbiological
methods, in accordance with the Agreement on technical cooperation
between ISO and CEN (Vienna Agreement).
Throughout the text of this document, read “.this European Standard.” to
mean “.this International Standard.”.
This second edition cancels and replaces the first edition (ISO 6222:1988),
which has been technically revised.
©  ISO 1999
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced
or utilized in any form or by any means, electronic or mechanical, including photocopying and
microfilm, without permission in writing from the publisher.
International Organization for Standardization
Case postale 56 • CH-1211 Genève 20 • Switzerland
Internet [e-mail]
Printed in Switzerland
ii
©
ISO
ISO 6222:1999(E)
Foreword
The text of EN ISO 6222:1999 has been prepared by Technical Committee CEN/TC 230 "Water analysis", the secretariat
of which is held by DIN, in collaboration with Technical Committee ISO/TC 147 "Water quality".
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 1999, and conflicting national standards shall be withdrawn at the latest by
November 1999.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries
are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France,
Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and
the United Kingdom.
Introduction
Waters of all kinds invariably contain a variety of micro-organisms derived from various sources, such as soil and
vegetation, and estimation of the overall numbers provide useful information for the assessment and surveillance of water
quality. Separate counts are usually made of the micro-organisms which are able to grow and form colonies on nutrient
agar media at 36 °C and 22 °C.
Colony counts are useful for assessing the integrity of ground water sources and the efficiency of water treatment
processes such as coagulation, filtration and disinfection and provide an indication of the cleanliness and integrity of the
distribution system. They can also be used to assess the suitabillty of a supply for the preparation of food and drink,
where the water supply should contain few micro-organisms to avoid contaminating the product with spoilage organisms.
The main value of colony counts lies in the detection of changes from those expected, based on frequent, long term
monitoring. Any sudden increase in the count can be an early warning of serious pollution and calls for immediate
investigation.
1 Scope
This European Standard specifies a method for the enumeration of culturable micro-organisms in water by counting the
colonies formed in a nutrient agar culture medium after aerobic incubation at 36 °C and 22 °C.
The method is intended to measure the operational efficiency of the treatment process of public drinking water supplies
and for general application to all types of water. It is particularly applicable to the examination of water
...