Overview
ISO 6222:1999 specifies a standardized laboratory method for the enumeration of culturable micro-organisms in water by colony count using a nutrient agar culture medium. Commonly cited as the “colony count by inoculation in a nutrient agar culture medium” method, it defines the pour-plate procedure, incubation conditions, media preparation and reporting conventions for results expressed as colony‑forming units per millilitre (cfu/ml).
Key topics and technical requirements
- Scope: Enumeration of aerobic bacteria, yeasts and moulds able to form colonies under the test conditions; applicable to all water types including drinking water and bottled natural mineral waters.
- Sampling & handling: Follow EN ISO 5667‑3 and EN 25667‑2 for sampling, preservation and handling. Bottled waters should be examined within 12 hours of bottling, stored at (5 ± 3) °C.
- Culture medium: Yeast extract agar recipe provided (e.g., tryptone 6.0 g, yeast extract 3.0 g, agar 10–20 g per 1 000 ml). Adjust pH to 7.2 ± 0.2 after sterilization. Use high-quality water (EN ISO 3696).
- Diluent: Use peptone diluent as specified in ISO 8199 and ISO 6887 for dilution preparation.
- Inoculation (pour‑plate method): Place ≤ 2 ml sample or dilution into a Petri dish, add 15–20 ml molten medium (maintained at 45 ± 1 °C), mix and allow to set. Time between sample addition and molten medium must not exceed 15 minutes.
- Incubation: Two sets of plates - one at (36 ± 2) °C for (44 ± 4) h and one at (22 ± 2) °C for (68 ± 4) h. Invert plates during incubation.
- Counting & reporting: Count colonies per ISO 8199 and express results in cfu/ml. Report “not detected in 1 ml” where no colonies appear on undiluted plates; report “> 300” where plate counts exceed 300 colonies. Include method details, temperatures, media and sample identification in test reports.
Applications and users
- Practical uses: Monitoring operational efficiency of water treatment (coagulation, filtration, disinfection), assessing distribution system integrity, quality control for bottled water and suitability of water for food and beverage preparation.
- Who uses it: Water testing laboratories, public health authorities, water utilities, beverage and food manufacturers, and regulatory bodies performing routine surveillance or troubleshooting sudden changes in microbial counts.
- Value: Provides an early warning of contamination events and a baseline for long‑term monitoring of water quality using culturable counts.
Related standards
- ISO 8199 – General guide to enumeration of micro-organisms by culture
- ISO 6887 – Preparation of dilutions for microbiological examinations
- EN ISO 3696 – Water for analytical laboratory use
- EN ISO 5667‑3 / EN 25667‑2 – Sampling and sample handling guidance
Keywords: ISO 6222:1999, water quality, colony count, culturable micro‑organisms, nutrient agar, pour‑plate method, cfu/ml.
Open preview (PDF)Water quality — Enumeration of culturable micro-organisms — Colony count by inoculation in a nutrient agar culture medium