🧫 Total Microbial Count 36°C

Microbiology PN-EN ISO 6222

Overview

STATUS (as of 29.10.2026). PN-EN ISO 6222:2004 — Polish version, published on 19.07.2004, adopts EN ISO 6222:1999 [IDT] and ISO 6222:1999 [IDT]; it replaced the English version PN-EN ISO 6222:2002. The standard is current — no withdrawal note. ISO 6222:1999 replaced the 1988 edition and has the status of a current standard in the catalogue.

SCOPE (clause 1). 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 of public drinking water supplies and is suitable for all types of water, particularly for water intended for human consumption, including water in closed containers and natural mineral waters.

DEFINITION (clause 3). Culturable micro-organisms are all aerobic bacteria, yeasts and moulds capable of forming colonies in the specified medium under the test conditions.

SIGNIFICANCE (introduction). Waters of all kinds contain micro-organisms derived from various sources, such as soil and vegetation. Colony counts are useful for assessing the integrity of groundwater sources and the efficiency of treatment (coagulation, filtration, disinfection), indicate the cleanliness and integrity of the distribution system, and also help to assess the suitability of water for the preparation of food and drink. The main value of the determination lies in detecting deviations from values expected on the basis of frequent, long-term monitoring — a sudden increase in the colony count can be an early warning of serious pollution and calls for immediate investigation. According to the British official method (The Microbiology of Drinking Water, part 7, 2020), the colony count at 37 °C is particularly useful for assessing the quality of relatively unpolluted groundwaters and can provide an early indication of more serious pollution.

REGULATIONS. Water intended for human consumption: colony count at (36 ± 2) °C — at most 100 in 1 ml in water in tankers and storage tanks of land, water and air transport (Journal of Laws 2026, item 748, Annex 1 Part A Tables 2–3); PN-EN ISO 6222 is the reference method for the colony count at 22 °C and at 36 °C (Annex 4 Part A). Swimming pools: total count of micro-organisms at (36 ± 2) °C after 48 h — at most 20 in 1 ml of water supplied to the basin and 100 in 1 ml of water in the basin; the requirement does not apply to outdoor pools (Journal of Laws 2022, item 1230, Annex 1). Natural mineral waters and spring waters: at the source, the total bacterial count at 37 °C after 24 h — not more than 5 in 1 ml; in bottled water — not more than 20 in 1 ml at 37 °C after 24 h, measured within 12 h of bottling, with storage at 4 °C (Journal of Laws 2011, No 85, item 466, § 4(3) point 2 and (4)). The incubation conditions in the last two regulations (48 h; 37 °C and 24 h) differ from those of the standard.

WHAT WE DO NOT GIVE HERE. The text of the standard is available in full. For sample preparation, dilutions and the rules for counting colonies and calculating the result, the standard refers to ISO 8199 and ISO 6887 — we do not summarize those standards here.

Method principle

Measured volumes of the sample or its dilutions are mixed in Petri dishes with molten yeast extract agar (pour plate). One set of plates is incubated at 36 °C for 44 h, the other at 22 °C for 68 h, and the number of colony-forming units (cfu) per 1 ml of sample is calculated from the number of colonies formed in the medium (clause 4); tolerances: (36 ± 2) °C and (44 ± 4) h and (22 ± 2) °C and (68 ± 4) h (8.2).

Applications

• Water intended for human consumption, including water in closed containers and natural mineral waters (clause 1)
• Measuring the efficiency of treatment of public drinking water supplies — coagulation, filtration, disinfection (clause 1, introduction)
• Assessing the integrity of groundwater sources and the cleanliness and integrity of the distribution system (introduction)
• Assessing the suitability of water for the preparation of food and drink (introduction)
• Water intended for human consumption in tankers and tanks of means of transport — at most 100 colonies in 1 ml at (36 ± 2) °C (Journal of Laws 2026, item 748, Annex 1 Part A)
• Swimming pools — at most 20 in 1 ml of water supplied to the basin and 100 in 1 ml of water in the basin, at (36 ± 2) °C after 48 h (Journal of Laws 2022, item 1230, Annex 1)

Key parameters

Standard

Standard number

PN-EN ISO 6222:2004

Title (PL)

Jakość wody — Oznaczanie ilościowe mikroorganizmów zdolnych do wzrostu — Określanie ogólnej liczby kolonii metodą posiewu na agarze odżywczym

Title (EN)

Water quality — Enumeration of culturable micro-organisms — Colony count by inoculation in a nutrient agar culture medium

Step-by-step procedure

1. Sampling

   According to EN 25667-2 and EN ISO 5667-3. Water in closed containers, including natural mineral waters, is examined within 12 h of bottling and kept at (5 ± 3) °C during this period (clause 6).

2. Preparing the medium

   Melt the yeast extract agar, cool it and maintain it in a water bath at (45 ± 1) °C; not longer than 4 h is recommended, after which the medium is discarded (7.3).

   🌡 Temperature: 45 ± 1 °C

3. Sample and dilutions

   Sample preparation, dilutions and inoculation according to ISO 8199, EN ISO 5667-3 and ISO 6887 (clause 8).

4. Pour plate

   Place not more than 2 ml of sample or dilution in a Petri dish, add 15–20 ml of molten medium, mix carefully by gentle rotation and allow to set. No more than 15 min may pass between adding the sample and adding the medium. Inoculate at least one plate for each incubation temperature (clause 8).

5. Incubation

   Invert the plates; incubate one set at (36 ± 2) °C for (44 ± 4) h and the other at (22 ± 2) °C for (68 ± 4) h (8.2).

   🌡 Temperature: 36 ± 2 °C

6. Reading

   Examine the plates as soon as they are removed from the incubator; if this is not possible, store them at (5 ± 3) °C and examine within 48 h. Reject plates with confluent growth (8.2).

7. Counting and calculation

   For each temperature count the colonies on each plate and calculate the number of colony-forming units in 1 ml of sample according to the rules of ISO 8199 (8.3).

8. Expression of the result

   Result in cfu/ml for each temperature. If there are no colonies on the plates with the undiluted sample — “not detected in 1 ml”; if there are more than 300 colonies on the plates with the highest dilution — “> 300” or an approximate result. The test report gives, among others, the technique (pour plate), the medium, and the time and temperature of incubation (clauses 9–10).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Media, reagents and bacteria require a risk assessment before the tests are started; standard laboratory microbiology safety procedures apply (British official method The Microbiology of Drinking Water, part 7, 2020)
• Tests should be carried out by competent, trained analysts in properly equipped laboratories (as above)

Frequently asked questions