🌿 Microtox® test — Vibrio fischeri
In short
Biological toxicity test for water and wastewater based on inhibition of bioluminescence of Vibrio fischeri (Aliivibrio fischeri) bacteria. The test is performed according to PN-EN ISO 11348-3:2008; Test organism: Vibrio fischeri (Aliivibrio fischeri), NRRL B-11177. The procedure comprises 6 steps (about 1 h 20 min in total); it is used for: Industrial wastewater toxicity screening, Waste classification for ecotoxicity (H14/HP14), Assessment of aqueous extract toxicity from contaminated soils.
At a glance
- Standard: PN-EN ISO 11348-3:2008
- Category: Environmental
- Procedure steps: 6
- Total time of stages: 1 h 20 min
- Test organism: Vibrio fischeri (Aliivibrio fischeri), NRRL B-11177
- Exposure time: 15 and 30 min
- Endpoint: EC50 or % luminescence inhibition
Overview
The Microtox® test (or its equivalents) is the fastest and most commonly used bioassay for assessing toxicity of waters, wastewaters, and aqueous extracts from soils and wastes. The bacterium Vibrio fischeri (formerly Photobacterium phosphoreum) naturally emits light (bioluminescence) due to the enzyme luciferase. Toxic substances inhibit bacterial metabolism, causing a decrease in luminescence intensity.
The result is EC50 (concentration causing 50% luminescence decrease) after 15 and 30 minutes of exposure, or percent luminescence inhibition for environmental samples. The test is used in waste classification (toxicity H14), wastewater toxicity assessment, and water quality control.
Method principle
Freeze-dried Vibrio fischeri bacteria are reconstituted in salt solution (2% NaCl) and exposed to a series of sample dilutions. Luminescence is measured with a luminometer before sample addition (I₀) and after 5, 15, and 30 minutes of exposure (I_t). Percent inhibition = [1 − (I_t/I_control)] × 100. EC50 is calculated from dose-response curve. Test temperature: 15°C (optimal for V. fischeri).
Applications
- Industrial wastewater toxicity screening
- Waste classification for ecotoxicity (H14/HP14)
- Assessment of aqueous extract toxicity from contaminated soils
- Surface water toxicity monitoring
- Wastewater treatment process efficiency control
- Rapid chemical toxicity assessment (REACH)
Key parameters
| Parameter | Value |
|---|---|
| Test organism | Vibrio fischeri (Aliivibrio fischeri), NRRL B-11177 |
| Exposure time | 15 and 30 min |
| Endpoint | EC50 or % luminescence inhibition |
| Test temperature | 15 ± 1°C |
| Test time | 30–45 min (including preparation) |
Standard
- Standard number
- PN-EN ISO 11348-3:2008
- Title (PL)
- Jakość wody — Oznaczanie hamowania bioluminescencji Vibrio fischeri (test hamowania świecenia) — Część 3: Metoda z zastosowaniem bakterii liofilizowanych
- Title (EN)
- Water quality — Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) — Part 3: Method using freeze-dried bacteria
Step-by-step procedure
-
Sample preparation
Adjust sample pH to 6–8 (if necessary). Adjust salinity to 2% NaCl. Filter (0.45 µm) if containing suspension.
-
Bacteria reconstitution
Add 1 mL cooled reconstitution solution to lyophilized ampoule. Mix gently. Incubate 15 min at 4°C.
-
Dilution preparation
Prepare sample dilution series: 100, 50, 25, 12.5, 6.25% in 2% NaCl. Control = 2% NaCl.
-
Luminescence measurement I₀
Transfer bacteria to measurement vials (100 µL/vial). Measure initial luminescence I₀ in luminometer.
-
Sample addition
Add 900 µL sample/dilution to vials with bacteria. Mix gently.
-
Measurement after 15 and 30 min
Measure luminescence I₁₅ and I₃₀. Calculate % inhibition and EC50.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Microtox® luminometer | Modern Water Microtox M500, LUMIStox 300 (Hach) | 60,000–120,000 PLN |
| 15°C cooling block | Built into luminometer or external | built-in |
| Automatic pipettes | Eppendorf Research Plus 10–1000 µL | 800–2,000 PLN |
| Cuvettes/vials for luminometer | Borosilicate glass vials, pack of 500 pcs. | 500–1,000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Freeze-dried V. fischeri bacteria | — | Microtox Acute Reagent, ampoules (5 tests/ampoule), store at -20°C |
| Reconstitution solution | — | 2% NaCl, sterile, cooled to 4°C |
| Diluent solution | — | 2% NaCl, for sample dilution preparation |
| Reference substance | 7778-50-9 | Potassium dichromate K₂Cr₂O₇ or ZnSO₄, for bacterial sensitivity validation |
Health and safety (OHS)
- Potassium dichromate — carcinogenic, use in fume hood
- V. fischeri bacteria — BSL-1, low biological risk
- Samples may contain toxic substances — nitrile gloves
- Test waste — collect in biological waste containers
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 11348-3:2008 — “Water quality — Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) — Part 3: Method using freeze-dried bacteria”.
How does this method work?
Freeze-dried Vibrio fischeri bacteria are reconstituted in salt solution (2% NaCl) and exposed to a series of sample dilutions. Luminescence is measured with a luminometer before sample addition (I₀) and after 5, 15, and 30 minutes of exposure (I_t).
What is the measuring range and accuracy?
Test organism: Vibrio fischeri (Aliivibrio fischeri), NRRL B-11177; Exposure time: 15 and 30 min; Endpoint: EC50 or % luminescence inhibition; Test temperature: 15 ± 1°C.
How long does the test take?
The times given for the individual stages add up to approximately 1 h 20 min. The procedure comprises 6 steps.
What equipment is required?
Microtox® luminometer, 15°C cooling block, Automatic pipettes, Cuvettes/vials for luminometer. Indicative cost of the core instrument (Microtox® luminometer): 60,000–120,000 PLN.
Where is this test used?
Industrial wastewater toxicity screening; Waste classification for ecotoxicity (H14/HP14); Assessment of aqueous extract toxicity from contaminated soils; Surface water toxicity monitoring; Wastewater treatment process efficiency control; Rapid chemical toxicity assessment (REACH).
What safety precautions apply?
Potassium dichromate — carcinogenic, use in fume hood; V. fischeri bacteria — BSL-1, low biological risk; Samples may contain toxic substances — nitrile gloves; Test waste — collect in biological waste containers.
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 11348-3.