EN ISO 6222:1999 PDF

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.