🧫 Total Microbial Count 36°C
In short
Determination of total colony count of microorganisms capable of growth at 36±2°C after 44±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 20 min in total); it is used for: Control of drinking water quality (indicator parameter per MoH Regulation), Assessment of sanitary risk — detection of mesophilic bacteria, Monitoring of water disinfection effectiveness (chlorination, UV).
At a glance
- Standard: PN-EN ISO 6222:2004
- Category: Microbiology
- Procedure steps: 10
- Total time of stages: 5 h 20 min
- Result: cfu/1 mL (colony forming units)
- Criterion (drinking water): No abnormal changes (practically: <20 cfu/mL)
- Incubation temperature: 36 ± 2°C
Overview
Total microbial count at 36°C (HPC 36 — Heterotrophic Plate Count at 36°C) is a complementary parameter to determination at 22°C and provides different diagnostic information. Microorganisms growing at temperature 36±2°C (close to body temperature) are organisms potentially of human or animal origin, which better adapt to warm-blooded host conditions. Their presence in drinking water has greater sanitary significance than microorganisms at 22°C.
Elevated colony count at 36°C compared to 22°C may indicate fresh water contamination with biological material of human or animal origin, ineffective disinfection or contamination of water supply network. The result is interpreted together with determination at 22°C — the ratio HPC 36°C / HPC 22°C provides valuable information about the nature of contamination.
The analytical procedure is identical to determination at 22°C — pour plate method on YEA agar — with the difference that incubation is conducted at temperature 36±2°C for 44±4 hours (approx. 2 days). Shorter incubation time results from faster growth of mesophilic bacteria at their optimal temperature.
The parameter is mandatory in drinking water testing per MoH Regulation. Normative value: "no abnormal changes". In laboratory practice reference value: <20 cfu/mL in treated water, up to 100 cfu/mL in network.
Method principle
Identical principle as in determination at 22°C (pour plate): 1 mL water sample is measured into a sterile Petri dish, poured with 15–20 mL melted and cooled to 44–47°C yeast extract agar (YEA). After solidification incubated at 36±2°C for 44±4 h. Temperature 36°C is close to human body temperature, which favors growth of mesophilic bacteria — potentially pathogenic or indicative. Colonies are counted on surface and inside agar.
Applications
- Control of drinking water quality (indicator parameter per MoH Regulation)
- Assessment of sanitary risk — detection of mesophilic bacteria
- Monitoring of water disinfection effectiveness (chlorination, UV)
- Control of bottled water — changes during storage
- Testing of water in hospitals and healthcare facilities
- Monitoring of pool water
- Comparison with HPC 36°C / HPC 22°C ratio — diagnostics of contamination source
Key parameters
| Parameter | Value |
|---|---|
| Result | cfu/1 mL (colony forming units) |
| Criterion (drinking water) | No abnormal changes (practically: <20 cfu/mL) |
| Incubation temperature | 36 ± 2°C |
| Incubation time | 44 ± 4 hours (approx. 2 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
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YEA medium preparation
Dissolve YEA agar according to manufacturer's instructions. Sterilize in autoclave 121°C/15 min. Maintain in water bath 44–47°C (max 4 hours).
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Sample preparation
Mix sample gently (25 bottle inversions). Neutralize chlorine with sodium thiosulfate if present.
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Pour plate plating
Measure 1 mL sample with pipette into sterile Petri dish (duplicate). For samples with suspected high contamination: prepare dilutions 10⁻¹, 10⁻².
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Pouring with YEA agar
Immediately pour with 15–20 mL melted YEA (44–47°C). Mix by gentle rotary motion (figure-eight movement). Do not create bubbles.
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Solidification
Leave dishes on flat surface until solidification (15–20 min). Do not move dishes before solidification.
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Incubation 36°C
Place inverted dishes in incubator 36±2°C. Incubate for 44±4 hours (approx. 2 days).
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Result reading
Count all colonies (surface and subsurface) using magnifier or automatic counter. Optimal range: 15–300 colonies per dish.
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Result calculation
Result = mean from duplicates × dilution factor. Report in cfu/1 mL. Round to 2 significant figures. E.g., 127 → 130 cfu/mL.
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Diagnostic interpretation
Compare result with HPC at 22°C. Ratio HPC 36°C / HPC 22°C > 1 suggests fresh contamination from outside. HPC 36°C / HPC 22°C < 1 — typical for multiplication of natural water flora.
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Quality control and disposal
Negative control: dish with agar only. Temperature control: recorder in incubator. Autoclave dishes after reading 121°C/30 min.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Incubator 36°C | Binder BD 56, Memmert IN30, Pol-Eko CLN 32 | 5,000–15,000 PLN |
| Water bath 44–47°C | Memmert WNB 7, Julabo TW2 | 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, 1.8% tablets |
| Buffered peptone water | — | For diluting samples, sterile 9 mL tubes, 0.1% peptone + 0.85% NaCl |
Health and safety (OHS)
- Water samples may contain mesophilic pathogens — nitrile gloves, lab coat, safety goggles
- Melted agar (44–47°C) — risk of burns when pouring dishes
- Mesophilic colonies may contain opportunistic pathogens — autoclave before disposal
- Caution when working with Bunsen burner — open flame in laboratory
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?
Identical principle as in determination at 22°C (pour plate): 1 mL water sample is measured into a sterile Petri dish, poured with 15–20 mL melted and cooled to 44–47°C yeast extract agar (YEA). After solidification incubated at 36±2°C for 44±4 h.
What is the measuring range and accuracy?
Result: cfu/1 mL (colony forming units); Criterion (drinking water): No abnormal changes (practically: <20 cfu/mL); Incubation temperature: 36 ± 2°C; Incubation time: 44 ± 4 hours (approx. 2 days).
How long does the test take?
The times given for the individual stages add up to approximately 5 h 20 min. The procedure comprises 10 steps.
What equipment is required?
Incubator 36°C, Water bath 44–47°C, Autoclave, Automatic pipettes 1 mL, Sterile Petri dishes 90 mm, Magnifier / colony counter. Indicative cost of the core instrument (Incubator 36°C): 5,000–15,000 PLN.
Where is this test used?
Control of drinking water quality (indicator parameter per MoH Regulation); Assessment of sanitary risk — detection of mesophilic bacteria; Monitoring of water disinfection effectiveness (chlorination, UV); Control of bottled water — changes during storage; Testing of water in hospitals and healthcare facilities; Monitoring of pool water; Comparison with HPC 36°C / HPC 22°C ratio — diagnostics of contamination source.
What safety precautions apply?
Water samples may contain mesophilic pathogens — nitrile gloves, lab coat, safety goggles; Melted agar (44–47°C) — risk of burns when pouring dishes; Mesophilic colonies may contain opportunistic pathogens — autoclave before disposal; Caution when working with Bunsen burner — open flame in laboratory.
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.