8050

Bacterial Bioluminescence

Approved by Standard Methods Committee, 2022. Joint Task Group: Ruth M. Sofield.

8050 A. Introduction

A bacterial bioluminescence test (BBT) is a metabolic inhibi- compartmentalization of internal functions, they provide many
tion test that uses luminescent bacteria to measure a substance’s target sites at or near the cell membrane for toxicants to exploit.
toxicity. This method provides a rapid, reliable, and convenient Also, the rate of respiration is 10 to 100 times greater in bac-
means of determining acute toxicity. The BBT has been validated teria than in mammalian cells, resulting in a dynamic metabolic
for various environmental applications (e.g., effluent monitoring; system that can be quantified easily and accurately by measuring
groundwater, drinking water, sediment,1,2 and hazardous waste the amount of light output from a bacterial suspension. Such sus-
testing; bioremediation-efficiency assessments; and general bio- pensions typically contain approximately 106 individual organ-
monitoring). isms. The light intensity is substantial (well within the operating
Luminescent bacteria possess several attributes useful for tox- range of common light sensors), and the large number of organ-
icity testing. Certain strains of luminescent bacteria divert up isms compensates for variations among individuals.
to 10% of their respiratory energy into the luciferase metabolic
pathway that converts chemical energy into visible light by the References
enzymatic catalysis of a chemical substrate. This pathway is
intrinsically tied to respiration; any change in cellular respiration 1. Puget Sound Estuary Program. Recommended guidelines for con-
or disruption of cell structures changes respiration and, therefore, ducting laboratory bioassays on Puget Sound sediments. Prepared
the amount of bioluminescence. for U.S. Environmental Protection Agency Region 10, Office of
In bacteria, many metabolic pathways are in or near the cell Puget Sound and Puget Sound Water Quality Authority. Olympia
membrane, including those pathways related to respiration, oxi- (WA): Water Quality Authority; 1995.
dative phosphorylation, osmotic stabilization, and transport of 2. Biological test method: Reference method for determining the tox-
icity of sediment using luminescent bacteria in a solid-phase test;
chemicals and protons into and out of the cell. The luciferase
Report EPS 1/RM/24. Ottawa (Ontario): Environment Canada; 2002.
enzymes, which shunt electrons directly to oxygen at the level of 3. Angell P, Langley D, Chamberlain AHL. Localisation of luciferase
reduced flavin mononucleotide, are also in the cell membrane.3 in luminous marine bacteria by gold immunocytochemical labelling.
Because luminescent bacteria are small (< 1 μm diam), have FEMS Microbiol Lett. 1989;65(1–2):177–181.
relatively simple morphology, and have no membrane-sided

8050 B. Bacterial Bioluminescence Test

1. General Principles half-maximal inhibitory concentration (IC50). Positive controls of

either standard phenol or zinc (Zn2+) prepared from zinc sulphate
In a BBT, analysts expose living bioluminescent bacteria, heptahydrate (ZnSO4 · 7H2O) solutions are used to assess ongo-
Aliivibrio fischeri, to a sample and then measure the sample’s ing method performance. Sample handling and preparation (e.g.,
effects on the bacteria as a change in bioluminescence. Both pH and salinity adjustments) may alter the sample toxicity, as can
the bacteria and the testing equipment are available commer- naturally occurring toxicants (e.g., sulfides).9 Consider the effects
cially. Manufacturer-recommended procedures and informa- of naturally occurring and artificially produced toxicity on test
tion are available for software and common testing methods.1–3 results when analyzing data.
Data reported in the literature were produced in tests using stan-
dard, commercially available, freeze-dried strains of the bacte- 2. Apparatus
rium, so test results from different laboratories can be compared
directly.4–8 Analysts measure the light output of rehydrated lyo- a. Luminometer.
philized bacteria after exposure to a specified dilution series of a b. Microplates or glass cuvettes, 2 ml minimum volume, one-
sample and then compare that data to the light output of a control time-use, with flat bottoms.
blank (Aliivibrio fischeri suspended in 2% NaCl diluent only). c. Pipettor, adjustable (200- to 1000-μL).
The assay’s premise is that a decrease in light production is due to d. Pipettor, with disposable 0.5-mL syringe tips.
metabolic inhibition of the bacteria, which is proportional to the e. Laboratory glassware, including sample bottles, 30- to
toxicant dose. Analysts determine a concentration–response curve 250-mL beakers, and volumetric flasks.
based on the test’s dilution series, which is used to estimate the

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 8050 Bacterial Bioluminescence - B. Bacterial Bioluminescence Test

f. Spatulas, stainless steel. as described. After the IC50 is calculated, determine if the con-
g. Weigh boats, plastic. centration equivalent to the IC50 has visible color. If so, a color-
h. Water bottles, 250- to 500-mL, glass, polyethylene, or poly- correction procedure is needed.10
propylene. Luminescent bacteria are adversely affected by pH < 6.0 or
i. Incubator, capable of maintaining 15 º ± 1 ºC. > 8.0, so neutralize the sample’s pH to between 6.5 and 7.5 with
j. Freezer, to store bacteria (not self-defrosting). either NaOH or HCl, as needed. If over-titration occurs, discard
k. pH meter and probe. the sample and start over.
l. Refractometer. If the salt content is < 2% as determined using a refractometer,
m. Analytical balance. osmotically adjust the samples to a final concentration of 2.0%
n. Air compressor. NaCl (mass/mass). If the sample has a higher salt content, make
a diluent substitute to match the sample’s salinity with ultrapure
3. Reagents and Materials water and NaCl. The diluent substitute is used in place of the dilu-
ent. If the sample has 0% NaCl, pour 50 g of well-mixed sam-
a. Bacteria, Aliivibrio fischeri, freeze-dried. ple into a 100-mL beaker. Tare the sample-filled beaker and add
b. Water, ultrapure (lacking trace impurities). 1.00 g solid analytical reagent (AR)-grade NaCl crystals to the
c. Sodium chloride (NaCl), solid, reagent-grade. beaker. Stir with a disposable glass pipet until all crystals have
d. Diluent: Make up a 2% w/v NaCl solution with ultrapure completely dissolved.
water. Use to dilute sample and bacteria as necessary. Luminescent bacteria are sensitive to chlorine, so sample
e. Acetic acid, glacial, 50% aqueous. collection before chlorination is preferred. If it is not possible,
f. Sodium analyzed stock solution: Dissolve 1.575 g Na2SO3 in dechlorinate the samples with sodium sulfite. Determine the
1000 mL ultrapure water. Prepare daily as needed. amount of sulfite solution required on a 100- to 1000-mL sub­
g. Phenol, reagent-grade, crystalline. sample of well-mixed, neutralized sample that will not be used in
h. Zinc (ZnSO4 · 7H2O), reagent-grade. the test by sequentially adding 10 mL of 50% acetic acid, 10 mL
i. Hydrochloric acid (HCl) and sodium hydroxide (NaOH), 1 M KI solution, and 10 mL starch indicator solution per 1000 mL of
each, for pH adjustment. sample, mixing between additions. If the sample becomes blue-
j. Potassium iodide (KI). Dissolve 10 g KI in 100 mL ultrapure black, chlorine is present. Titrate with Na2SO3 solution slowly
water. to the starch-iodide endpoint (blue color is discharged to a clear
k. Starch indicator solution. Either use a commercial product solution). To the remaining neutralized sample, add the propor-
or prepare as follows: to 5 g starch (potato, arrowroot, or soluble), tional volume of Na2SO3 solution determined by the above resid-
add a little cold water and grind to a thin paste in a mortar. Pour ual test, mix, and then retest a portion for residual chlorine as
into 1 L boiling water, stir, and let settle overnight. Use the clear described above after 10 to 20 min.
supernate, preserving with 1.25 g salicylic acid and 4 g zinc chlo- b. Prepare positive control: As part of the basic test protocol,
ride, or a combination of 4 g sodium propionate and 2 g sodium include positive controls of either phenol or Zn2+ and analyze
azide per liter. them at least once a day (frequency depends on the number of
l. Parafilm. samples to be tested). Phenol ordinarily has an IC50 between
m. Disposable wipes. 13 and 26 mg/L after 5 min exposure, while Zn2+ has an IC50
n. Dissolved oxygen (DO) meter. between 0.6 and 2.2 mg/L after 15 min exposure. (If available,
o. Oil-free compressed air and disposable glass pipet: May be consult Bacterial Certificates of Performance, which report IC50
required based on DO and BOD of sample. values for phenol and Zn2+, for each lot number of bacteria.) Pre-
pare a 100-mg/L stock solution of either reagent-grade phenol or
4. Procedure ZnSO4 · 7H2O by accurately weighing and transferring 100 mg of
chemical into a clean, rinsed (with dilution water), 1000-mL volu-
a. Sample collection, handling, and treatment: This method is metric flask. Protect the phenol standard from light and prepare in
suitable for measuring the toxicity of samples that include chemi- an amber-colored or aluminum-foil-wrapped flask or equivalent.
cals, receiving waters, effluents, elutriates, and leachates. Collect Although both standards will last for 3 to 4 months when stored
250 to 500 mL sample in glass, polyethylene, or polypropylene at 2 to 8 °C, preferably prepare standards on each day of testing.
jars or bottles, leaving no airspace. Test the sample as soon as Alternatively, analyze standards on the testing day to determine
possible, preferably within 24 h of collection. Before use, store actual concentration.
the sample in the dark and refrigerated (≤10 °C but not frozen). c. Prepare bacteria (Aliivibrio fischeri): If cuvettes are used to
Prompt testing avoids possible changes in sample toxicity. contain bacteria and sample, hold them near the open end so light
If significant turbidity is present, results may be affected by measurements are not affected by finger- or handprints.
light scattering. Suspended matter may be reduced by settling for Add 1000 μL ultrapure water at 15 º ± 1 ºC to the freeze-dried
a few hours in storage conditions or by centrifugation. Transfer bacteria. Add it quickly because slow reconstitution causes bacte-
the cleared sample to a new container. (Note: The material caus- rial lysis and low bacterial light levels. Swirl the bacterial vial only
ing turbidity may also have toxic effects.) If turbid samples must 3 or 4 times to avoid overmixing, which warms the bacteria. Use
be analyzed as received, other tests are available (i.e., solid-phase the 500-μL pipettor to mix the reconstituted bacteria by filling and
testing procedures3) or a correction can be applied.10 Color dispensing the pipettor 20 times for uniform dispersal of bacteria.
may also interfere with measurements by absorbing light. If an Maintain the reconstituted bacteria at 15 º ± 1 ºC for 20 minutes.
osmotically adjusted sample has visible color, conduct the BBT d. Prepare samples: Generally, as long as DO is >1 mg/L (10%
saturation at the test temperature of 15 º ± 1 ºC) there is no effect

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 8050 Bacterial Bioluminescence - B. Bacterial Bioluminescence Test

on test results,11 however, there is some concern that samples high Immediately after the last transfer, record the time shown on
in biological or chemical oxygen demand could strip the oxygen the timer to note the time elapsed since the last I0 reading. This is
fast enough to affect results. (Peter Adolphson, Senior Environ- the transfer time and is used to schedule It measurements (where
mental Toxicologist, Washington State Department of Ecology. t = 5 or 15 min from when a sample was added to diluted bacteria)
Personal communication, Nov. 10, 2014.) to ensure that all exposure times are equal. Measure biolumines-
Therefore, if DO is < 40% saturation once the sample is at test cence in each container, starting with the negative control and
temperature, side-by-side tests with and without aeration are rec- ending with the highest sample concentration at I5 and I15. The
ommended, especially if high oxygen demand is suspected. The final sample concentrations are calculated by including solution
sample must be aerated if DO is < 10% at the test temperature. To added for osmotic adjustment, the sample dilutions from step e,
aerate samples, dispense oil-free compressed air through a glass the additional dilutions in step g (500 μL into 510 μL diluted bac-
pipet. Use a minimal flow rate to obtain effective aeration. Per- teria), and the Na2SO3 solution if added for dechlorination. The
form aeration until 40% saturation is obtained in ≤ 20 min. Then composition, volume, and example percentage of sample in each
perform the following: cuvette or equivalent is shown in Table 8050:2.
• Create serial dilutions of the osmotically adjusted sample
with the diluent. Include a negative control (diluent only) and 5. Data Reduction, Reporting, and Interpretation
at least 4 samples at different dilutions in a geometric series.
• Mix the diluent and the sample or diluted samples before Once testing is complete, the data recorded for each sample con-
transfer to the next dilution by aspirating the sample with a sist of the final sample concentration (as a percentage), I0, I5, I15,
pipettor. Each solution must be at least 1000 μl. and gamma (Γ). Gamma is the ratio of light lost to light remaining
• Wait 5 minutes for temperature equilibration to 15 º ± 1 ºC. after the diluted bacterial suspension was exposed to the diluted
e. Add bacteria: Using a repeat pipettor, add 10 μL reconsti- sample. It is calculated for each concentration as follows:
tuted and mixed bacteria (or an equivalent volume to achieve
about 106 bacteria) into 500 μL of 15 ºC diluent to create a diluted
bacterial solution. Prepare the same number of diluted bacterial Γt = (CR × I 0 ) ÷ I t  −1
solutions as there are diluted samples in the dilution series. When
dispensing reconstituted bacteria, be sure to dispense the recon- where:
stituted bacteria into the cuvette diluent.
CR = control ratio (negative control It ÷ negative control I0), which
After adding reconstituted bacteria to diluent, mix each dilu-
corrects for natural light loss over time,
tion by gently tapping the bottom of the container 2 to 3 times. It = light level t minutes from when the sample was added to
Wait 15 min to let the diluted bacteria stabilize, while maintaining diluted bacteria
the temperature at 15 ± 1 ºC. If using recording software attached I0 = initial light level
to the luminometer, set up the recording software per the manu-
facturer’s instructions. The components, volumes, and example Using the Γt for each diluted sample as the response data
concentrations of test samples are shown in Table 8050:1. (dependent variable) and the final sample concentration as the
f. Measure light emission: Once the diluted bacteria have sta- concentration data (independent variable), estimate the IC50 with
bilized, calibrate the luminometer by measuring the initial light a 95% confidence range using appropriate regression models
level (I0) for one of the samples with diluent and diluted bacteria and statistical approaches for toxicity data. A narrow confidence
(the bacterial control) according to the instrument manufacturer’s range means more accurate results. By definition, IC50 occurs
instructions. All subsequent readings are expressed as values on a when the Γ value = 1. An IC10 or IC20 may also be calculated.
comparative scale, based on this reading (i.e., the bacteria’s actual Collectively, the data are expected to show a concentration–
performance). Then, measure I0 for each diluted bacterial sample. response relationship, meaning that Γ increases as the sample
After all I0 readings have been acquired, begin a timer to track concentration increases (unless the sample does not inhibit biolu-
sample additions to each diluted bacterial sample and perform the minescence at any concentration). A partial effect is a Γt greater
following steps immediately. than 0, where a 0 means there was no reduction in biolumines-
g. Transfer diluted sample to diluted bacteria: Complete this cence at that concentration and does not have an It = 0, indicat-
step as quickly as possible. Using a pipettor adjusted to deliver ing there was complete reduction of bioluminescence. Not every
500 μL, transfer 500 μL from the serial dilutions to a diluted concentration produces a Γt with a partial effect. At least 3 Γt
bacteria container (cuvette or equivalent). Start with the negative values with a partial effect are needed to make final calculations.
control and end with the dilution with the highest sample concen- If all light output data at time t in the diluted samples = 0, then
tration. Gently mix each diluted sample with diluted bacteria by retest the sample at lower concentrations. If light output data are
repeatedly drawing up and delivering the mixture with the pipettor. similar for a concentration at different times, (i.e., no significant

Table 8050:1. Sample Components and Example Percent Concentration of Bioluminescence Test Sample After Adding Bacteria (8050 B.4e)
Sample Number
Row a
1 2 3 4 5 6 Sample Composition
1 0.00 3.125 6.25 12.5 25.0 50 1000 μL total volume comprising test sample, diluent, and
solutions used for osmotic adjustment and dechlorination
2 0.00 0.00 0.00 0.00 0.00   0.00 510 μL sample; contains diluent and bacterial suspension
a
Row refers to a series of 6 bioluminescence test cuvettes.

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 8050 Bacterial Bioluminescence - B. Bacterial Bioluminescence Test

Table 8050:2. Sample Components and Example Percent Concentration of Bioluminescence Test Sample After Transferring Sample to Diluted Bacteriaa
(8050 B.4g)
Sample Number
Rowb 1 2 3 4 5 6 Sample Composition
1 0.00 3.125 6.25 12.5 25.0 50 500 μL total volume comprising test sample, diluent, and
solutions used for osmotic adjustment and dechlorination
2 0.00 1.55 3.09 6.19 12.38 24.75 1010 μL total volume comprising test sample, diluent, solutions
for osmotic adjustment and dechlorination, and reconstituted
bacteria
a
Assumes 500 μl was transferred from Row 1 to Row 2.
b
Row refers to a series of 6 bioluminescence test cuvettes.

light decrease), flag those data points with an asterisk. If multiple 11. Biological test method: Reference method for determining the tox-
data points are flagged, a more concentrated dilution series can be icity of sediment using luminescent bacteria in a solid-phase test;
created by adding 10 μl reconstituted and mixed bacteria directly Report EPS 1/RM/24. Ottawa (Ontario): Environment Canada;
into 500 μl serially diluted samples. Then, conduct the luminom- 2002.
eter calibration described in step f on the negative control after the
bacteria are added. Bibliography

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Published Online: August 27, 2018

Revised: April 12, 2022
https://doi.org/10.2105/SMWW.2882.153 4