🌿 Vibrio fischeri luminescence inhibition test (freeze-dried bacteria)

Environmental PN-EN ISO 11348-3

Overview

STATUS (as of 02.11.2026). PN-EN ISO 11348-3:2008 — English version (Polish title in the PKN catalogue: “Jakość wody — Oznaczanie inhibicyjnego działania próbek wody na emisję światła przez Vibrio fischeri (badanie na bakteriach luminescencyjnych) — Część 3: Metoda z zastosowaniem liofilizowanych bakterii”), published on 15.12.2008, implements EN ISO 11348-3:2008 and ISO 11348-3:2007 [IDT]; it replaced PN-EN ISO 11348-3:2002 (Polish version); the amendment PN-EN ISO 11348-3:2008/A1:2019-03 (English version, 7.03.2019) implements EN ISO 11348-3:2008/A1:2018 and ISO 11348-3:2007/Amd 1:2018. The card has no withdrawal note — the standard is current. ISO 11348-3:2007 is the second edition, which after technical revision replaced ISO 11348-3:1998; in 2018 the amendment ISO 11348-3:2007/Amd 1:2018 was issued — it replaces the third item of the validity criteria (clause 11): for the dilutions determining the LID value the deviation from their mean shall not exceed 3 percentage points. The ISO 11348 series has three parts: 1 — freshly prepared bacteria, 2 — liquid-dried bacteria, 3 — freeze-dried bacteria. Trade names of test kits are not part of the standard — it requires the NRRL B-11177 strain from commercially available freeze-dried reagents.

WHY THREE PARTS (introduction). Standardization work showed that fresh, freeze-dried and liquid-dried bacteria may give different results, especially in the presence of heavy metals: the protective media used for preservation influence the bioavailability of toxicants and the light emission, and the user often does not know the composition of the preparations of different suppliers. Part 3 therefore describes a procedure with freeze-dried bacteria whose performance can be interpreted by the user in every detail; the choice of technique rests with the laboratory.

SCOPE (clause 1). Inhibition of the luminescence emitted by the marine bacterium Vibrio fischeri (NRRL B-11177) — waste water, aqueous extracts and leachates, fresh water (surface and ground water), sea and brackish water, eluates of sediment (freshwater, brackish and sea water), pore water, and single substances diluted in water.

INTERFERENCES (clause 4). The result may be affected by insoluble, slightly soluble or volatile substances or substances reacting with the dilution water or the suspension. Strong colour or turbidity absorbs or scatters light — settling, centrifugation or a double-chambered absorption correction tube help (Annex A). A high oxygen demand and readily biodegradable nutrients reduce luminescence independently of pollutants; pH outside the range 6.0–8.5 affects the luminescence. Salt-water samples often stimulate luminescence, which may mask inhibition (Annex D); salt concentrations above 30 g/l NaCl together with the required salt addition risk hyperosmotic effects — the resulting salinity should not exceed the osmolarity of a 35 g/l NaCl solution.

BACTERIA, SOLUTIONS AND APPARATUS (clauses 5–6). Suspensions are prepared from commercially available freeze-dried reagents stored at −18 °C to −20 °C; the bacteria glow immediately after reconstitution. The diluent is a 20 g/l NaCl solution, and the solution for freeze-dried bacteria contains 20 g NaCl, 2.035 g MgCl₂·6H₂O and 0.30 g KCl in 1 l. Reference substances, at about twice the expected EC50: zinc sulfate heptahydrate 19.34 mg/l, 3,5-dichlorophenol 6.8 mg/l and potassium dichromate 105.8 mg/l; each delivered batch of bacteria is tested with all three, and at least one is tested in parallel with each reconstituted vial. A thermoblock keeps the samples at (15 ± 1) °C, with a deviation within one test of at most ± 0.3 °C; the luminometer has a measuring cell at (15 ± 1) °C, and the stock suspension is kept at (4 ± 3) °C.

SAMPLES (clause 7). Containers according to ISO 5667-16 are filled completely; testing as soon as possible — up to 48 h at 2–5 °C in the dark, up to two months at −18 °C or lower, without chemical preservatives. Oxygen in the sample must exceed 3 mg/l (otherwise aeration or stirring); pH 6.0–8.5 usually needs no adjustment, if necessary it is adjusted to 7.0 ± 0.2 or to the limits 6.0 and 8.5. 20 g of NaCl per litre is added to the sample (for saline samples — enough to adjust the osmolarity to 20 g/l NaCl; at 20–50 g/l NaCl equivalents no salt is added). Strongly turbid samples are allowed to settle for 1 h, centrifuged (e.g. 10 min at 5 000 g) or filtered.

PROCEDURE (clause 8). The vial of freeze-dried culture (contents for up to 100 measurements) is taken from the freezer immediately before reconstitution: 1 ml of distilled water cooled to (4 ± 3) °C is poured into the vial at once, all of it, without a pipette. The stock suspension contains about 10⁸ cells per millilitre and is used for testing as long as it meets the validity criteria. After at least 10 min the test suspensions are prepared: in variant A 10 µl of stock suspension is added to 500 µl of the solution for freeze-dried bacteria in tubes at (15 ± 1) °C, in variant B 1 volume of stock suspension is mixed in a conical flask with 50 volumes of that solution at (4 ± 3) °C and 500 µl portions are pipetted into tubes at (15 ± 1) °C — in both variants at the same time intervals (5–20 s) as the later measurements. Because equal volumes of sample and suspension are combined, the highest sample concentration in the test is as a rule 50 %; testing nearly undiluted samples (80 %) needs an extra control. Luminescence is measured after 15 min or 30 min (optionally after 5 min), with the correction factor fkt for the change in intensity of the control samples during exposure (clause 3).

WHAT WE DO NOT GIVE HERE. The detailed course of the measurement, the calculation formulae (clauses 9–10), the other validity criteria (clause 11) and precision (clause 12, Annex C) are outside the available text of the 2007 edition.

Method principle

Specified volumes of the sample or its dilutions are combined in tubes with a suspension of Vibrio fischeri and their light emission is measured with a luminometer at (15 ± 1) °C — after 15 min or 30 min of contact, optionally also after 5 min.

The inhibition of light emission is calculated against control samples with sodium chloride solution, with the correction factor fkt for the change in their light emission during exposure; from the dilution series the lowest ineffective dilution (LID) or the EC20 and EC50 values are determined (clause 3).

Applications

• Waste water (clause 1)
• Aqueous extracts and leachates (clause 1)
• Fresh surface and ground waters; sea and brackish waters — taking the stimulation of luminescence by salt into account (clause 1, Annex D)
• Eluates of freshwater, brackish and marine sediments; pore water (clause 1)
• Single substances diluted in water (clause 1)

Key parameters

Standard

Standard number

PN-EN ISO 11348-3:2008 + A1:2019-03

Title (PL)

Jakość wody — Oznaczanie inhibicyjnego działania próbek wody na emisję światła przez Vibrio fischeri (badanie na bakteriach luminescencyjnych) — Część 3: Metoda z zastosowaniem liofilizowanych bakterii

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

1. Sample

   Containers according to ISO 5667-16, filled completely; testing as soon as possible — up to 48 h at 2–5 °C in the dark, up to two months at −18 °C or lower; no preservatives (7.1).

2. Sample preparation

   Oxygen above 3 mg/l, aerate if necessary; pH 6.0–8.5 usually without adjustment, if necessary to 7.0 ± 0.2; add 20 g of NaCl per litre; turbid samples — allow to settle for 1 h, centrifuge (e.g. 10 min, 5 000 g) or filter (7.2).

3. Series and controls

   Dilution series (Annex B), reference samples and controls with NaCl solution in tubes at (15 ± 1) °C; a new batch of bacteria — all three reference substances, each vial — at least one (8.1).

4. Stock suspension

   Pour 1 ml of distilled water cooled to (4 ± 3) °C into the vial of freeze-dried bacteria at once, all of it, without a pipette; about 10⁸ cells per millilitre; storage at (4 ± 3) °C (8.2).

5. Test suspensions

   After at least 10 min — in variant A 10 µl of stock suspension to 500 µl of the solution for freeze-dried bacteria at (15 ± 1) °C; in variant B 1 volume of stock suspension to 50 volumes of the solution at (4 ± 3) °C in a conical flask, then 500 µl portions into the tubes; at the same time intervals (5–20 s) as the later measurements (8.3).

6. Measurement and result

   Light emission after 15 min or 30 min (optionally after 5 min), with the correction factor fkt from the control samples; result as LID or EC20 and EC50 (clause 3).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• The standard states that the test requires familiarity with normal laboratory practice and suitably trained staff, and that safety practices are established by the user (warning of the standard)
• Potassium dichromate is a carcinogenic substance, toxic via inhalation; the standard allows ready-made preparations of defined concentration (5.6, note; ISO 6341, 6.4)

Frequently asked questions