🧫 Total Microbial Count 22°C
In short
Determination of total colony count of microorganisms capable of growth at 22±2°C after 68±4 h by pour plate method in yeast extract agar. The test is performed according to PN-EN ISO 6222:2004; Result: cfu/1 mL (colony forming units). The procedure comprises 10 steps (about 5 h 19 min in total); it is used for: Control of drinking water quality (indicator parameter per MoH Regulation), Monitoring of water supply network — detection of secondary contamination, Assessment of water treatment process effectiveness.
At a glance
- Standard: PN-EN ISO 6222:2004
- Category: Microbiology
- Procedure steps: 10
- Total time of stages: 5 h 19 min
- Result: cfu/1 mL (colony forming units)
- Criterion (drinking water): No abnormal changes (practically: <100 cfu/mL)
- Incubation temperature: 22 ± 2°C
Overview
Total microbial count at 22°C (HPC 22 — Heterotrophic Plate Count) is a basic microbiological parameter of drinking water, reflecting the general sanitary condition of water and effectiveness of treatment processes. The result is expressed as the number of colony forming units (cfu) in 1 mL sample.
Microorganisms growing at temperature 22±2°C after 68±4 hours are mainly environmental (autochthonous) bacteria, naturally occurring in water and soil. These include Pseudomonas, Aeromonas, Flavobacterium, Caulobacter, Arthrobacter and others. Their presence in drinking water in moderate amounts is natural, however a sudden increase in numbers may indicate: secondary network contamination, water stagnation, pipe damage, biofilm formation or ineffective disinfection.
The method according to PN-EN ISO 6222:2004 consists of pour plate plating — a measured volume of sample (1 mL) is introduced into a sterile Petri dish, poured with melted and cooled yeast extract agar (YEA — Yeast Extract Agar), mixed gently and after solidification incubated at 22±2°C for 68±4 hours. Colonies grow both on the surface and inside the agar.
Normative value for drinking water: no numerical requirements in MoH Regulation, but "no abnormal changes" is required. In practice: <100 cfu/mL is considered acceptable, >200 cfu/mL — warning signal.
Method principle
Pour plate method: 1 mL water sample is measured with a pipette into a sterile Petri dish, then poured with 15–20 mL melted yeast extract agar (Yeast Extract Agar — YEA) cooled to 44–47°C. The sample is mixed with agar by gentle rotary motion. After solidification of the medium, incubated at 22±2°C for 68±4 hours. Microorganisms capable of growth under these conditions form colonies visible to the naked eye, which are counted using a magnifier or automatic counter.
Applications
- Control of drinking water quality (indicator parameter per MoH Regulation)
- Monitoring of water supply network — detection of secondary contamination
- Assessment of water treatment process effectiveness
- Control of bottled and spring water
- Testing of water in swimming pools
- Monitoring of groundwater intakes
- Assessment of water stagnation in internal installations
Key parameters
| Parameter | Value |
|---|---|
| Result | cfu/1 mL (colony forming units) |
| Criterion (drinking water) | No abnormal changes (practically: <100 cfu/mL) |
| Incubation temperature | 22 ± 2°C |
| Incubation time | 68 ± 4 hours (approx. 3 days) |
| Sample volume | 1 mL (pour plate) |
| Medium | Yeast extract agar (YEA) |
Standard
- Standard number
- PN-EN ISO 6222:2004
- Title (PL)
- Jakość wody — Oznaczanie ilościowe mikroorganizmów zdolnych do wzrostu — Oznaczanie ogólnej liczby kolonii metodą posiewu w 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
-
YEA medium preparation
Dissolve YEA agar in distilled water. Sterilize in autoclave 121°C/15 min. After sterilization maintain in water bath 44–47°C (max 4 hours).
-
Sample preparation
Mix water sample gently (inverting bottle 25 times). If contains chlorine, use thiosulfate tablet.
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Pour plate plating
Measure 1 mL sample with pipette into sterile Petri dish. For dilutions: 1 mL to 9 mL peptone water, repeat.
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Pouring with agar
Pour sample with 15–20 mL melted YEA cooled to 44–47°C. Immediately mix by gentle rotary motion (figure-eight). Avoid bubbles.
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Solidification
Leave dishes on level surface until complete solidification of agar (approx. 15–20 min). Perform duplicate for each sample.
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Incubation 22°C
Place inverted dishes (bottom up) in incubator 22±2°C. Incubate for 68±4 hours (approx. 3 days). Do not open incubator unnecessarily.
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Result reading
Count all visible colonies (surface and subsurface) using magnifier with illumination or automatic counter. Optimal count: 15–300 colonies per dish.
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Result calculation
Result = arithmetic mean from duplicates × dilution factor. Report in cfu/1 mL. Round to two significant figures.
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Quality control
Negative control: dish with agar only (medium sterility). Duplicate every 10 samples. Incubator temperature control (recorder).
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Disposal
Autoclave dishes after reading 121°C/30 min. Dispose as infectious waste.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Incubator 22°C | Binder KB 53, Memmert IPP 110, Pol-Eko CLW 53 (with cooling) | 8,000–20,000 PLN |
| Water bath 45–47°C | Memmert WNB 7, Julabo TW2 — for holding melted agar | 3,000–8,000 PLN |
| Autoclave | Tuttnauer ELV 2540, Systec VX-40 | 20,000–45,000 PLN |
| Automatic pipettes 1 mL | Eppendorf Research Plus 100–1000 µL, Gilson Pipetman L | 800–2,500 PLN/pc |
| Sterile Petri dishes 90 mm | Polystyrene, disposable, sterile. Citotest, Gosselin | 150–300 PLN/pack (500 pcs) |
| Magnifier / colony counter | Stuart SC6+, Interscience Scan 500 | 1,500–25,000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Yeast extract agar (YEA) | — | Yeast Extract Agar: peptone 5 g/L, yeast extract 2.5 g/L, glucose 1 g/L, agar 9–18 g/L. Manufacturers: Merck, Oxoid, Biokar |
| Sodium thiosulfate (Na₂S₂O₃) | 7772-98-7 | For neutralizing chlorine in tap water samples |
| Buffered peptone water | — | For diluting samples, sterile 9 mL tubes |
Health and safety (OHS)
- Water samples may contain pathogens — nitrile gloves, lab coat, safety goggles
- Melted agar (44–47°C) — risk of burns. Use heat-resistant gloves
- Colonies on plates treat as potentially infectious — autoclave before disposal
- Work with Bunsen burner — exercise caution, do not leave unattended
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 6222:2004 — “Water quality — Enumeration of culturable micro-organisms — Colony count by inoculation in a nutrient agar culture medium”.
How does this method work?
Pour plate method: 1 mL water sample is measured with a pipette into a sterile Petri dish, then poured with 15–20 mL melted yeast extract agar (Yeast Extract Agar — YEA) cooled to 44–47°C. The sample is mixed with agar by gentle rotary motion.
What is the measuring range and accuracy?
Result: cfu/1 mL (colony forming units); Criterion (drinking water): No abnormal changes (practically: <100 cfu/mL); Incubation temperature: 22 ± 2°C; Incubation time: 68 ± 4 hours (approx. 3 days).
How long does the test take?
The times given for the individual stages add up to approximately 5 h 19 min. The procedure comprises 10 steps.
What equipment is required?
Incubator 22°C, Water bath 45–47°C, Autoclave, Automatic pipettes 1 mL, Sterile Petri dishes 90 mm, Magnifier / colony counter. Indicative cost of the core instrument (Incubator 22°C): 8,000–20,000 PLN.
Where is this test used?
Control of drinking water quality (indicator parameter per MoH Regulation); Monitoring of water supply network — detection of secondary contamination; Assessment of water treatment process effectiveness; Control of bottled and spring water; Testing of water in swimming pools; Monitoring of groundwater intakes; Assessment of water stagnation in internal installations.
What safety precautions apply?
Water samples may contain pathogens — nitrile gloves, lab coat, safety goggles; Melted agar (44–47°C) — risk of burns. Use heat-resistant gloves; Colonies on plates treat as potentially infectious — autoclave before disposal; Work with Bunsen burner — exercise caution, do not leave unattended.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-EN ISO 6222.