Bioluminescence
Bioluminescence is the production and emission of light by a living organism as the result of a chemical reaction during whichchemical energy isconverted to light energy. Itsname is a Hybrid word, originating from the Greek bios for "living" and the Latin lumen "light". It is generated by an enzyme-catalyzed chemoluminescence reaction, wherein the pigment luciferin is oxidized by the enzyme luciferase.
How QwikLite Utilizes Bioluminescence
Bioluminescence is the total light emitted from the marine plankton (called dinoflagellates) and compared to a standard or control sample in each test.
During the stimulation process, the optical and electrical components measure bioluminescence of all the test samples, capturing emitted light at specific wavelengths. Toxicity is determined by measuring the light reduction from the bioluminescent marine dinoflagellates after they have been exposed to possible toxicants. The light emitted is inversely related to toxic stress and decreases rapidly when the sensitive, thin walled organisms are in water with contaminants (usually within 4 hours).
Biological testing with marine plankton can detect toxicity in the environment since natural bioluminescent light reduction is directly related to the toxicity of the tested sample. The greater the level of toxin– the lower the level of light emitted. This is a direct measure that the organism has been severely stressed or has a toxic response from the exposure.
Pyrocystis lunula
Pyrocystis lunula is a crescent or half-moon shaped dinoflagellate species that belongs to the oceanic plankton of tropical and subtropical seas. It is a single-celled alga (round nucleus) and reproduces asexually. This species is non-toxic, easily available, hardy, and grows well at room temperature.
Dinoflagellates can be characterized as microscopic organisms or marine phytoplankton that reside at the surface of the ocean (commonly regarded as “algae”). Neither plant nor animal, many dinoflagellates are unicellular (single cell) and exhibit a great diversity of form including the following characteristics:
- They are planktonic. 90% of all dinoflagellates are marine plankton, which passively float, drift or aggregate in a body of water, primarily comprising microscopic algae and protozoa.
- They are small. Although many of them are microscopic and range from 15 to 40 microns in size, the largest, Noctiluca, may be as large as 2 mm in diameter.
- They are motile. Many dinoflagellates swim by means of flagella, movable strands that propel the cell through water (P lunula does not).
- Many are photosynthetic. They manufacture their own food using the energy from sunlight, and provide a food source for other organisms.
- Some dinoflagellates are bioluminescent, capable of producing their own visible light. This light is emitted when they are agitated or disturbed. Dinoflagellates are the most common sources of bioluminescence at the surface of the ocean.
Handling of the Live Test Organisms
Storage Temperature
Place the live organisms in an area where the temperature range is between 18˚C (65˚F) and 29˚C (85˚F). For brief periods, this range can be expanded, but temperatures outside these ranges are harmful.
Circadian Rhythms
A circadian rhythm is a roughly-24-hour cycle in that is used by thousands of living things. Circadian rhythms can guide or control physiological processes. Based on its circadian rhythm to the day and night cycle in nature, the biological test species used in the QwikLite 200 Biosensor System (Pyrocystis lunula) maximize their natural bioluminescence approximately 2 to 4 hours after dark, when stimulated, this is when they will react (glow) the best. Therefore, this is the optimal time for performing the tests.
Light Cycling
Immediately Upon Receipt – Open the shipment and expose the cells to indirect light (since they have been in a dark environment during shipment).
Do NOT place the cultures in direct sunlight – expose the plankton cells to moderate indirect and/or artificial light only.
Upon receipt, the new shipment of sensitive plankton need to remain in the light for 4 to 12 hours. This is called a Light cycle. After the initial Light cycle, the cultures need rest and enter the Dark cycle. In general, the Light and the Dark cycles should approximately be 12 hours, but this can vary 1 to 2 hours.
NOTE: the plankton must complete a full Light/Dark cycle prior to any testing!
Repeat the Light/Dark Cycle: Throughout the time you have your plankton; from receipt through final testing; it is important to establish a Light/Dark cycle so that the sensitive cells remain healthy and ready to be used in bioluminescence testing.
Best Time to Dose: Typical experimental designs involves a 24 hour exposure cycle of the organism to the water sample. For best bioluminescence, the ideal testing time is 3 hours after start of the Dark cycle, when they are the brightest. So when you consider when to “dose” your samples, it is best to determine when you want to perform your final testing, and then dose 24 hours earlier.
Best Time to Test: Approximately 3 hours after start of the Dark cycle. Bioluminescent response in the test culture is optimized approximately 3 hours after they are in the darkness.
Simply calculate backwards from the time you want to test, and this will determine when you will want the Light/Dark cycle to be established.
Light/Dark cycling and testing example: If you would like to perform a 24 hour exposure test on Friday at 12noon, you need to dose your samples on Thursday at 12 noon.
This testing plan would have this appropriate Light/Dark schedule:
- Tuesday – Receive a shipment of live cultures at 2 pm, provide light and air immediately
- Wednesday 9 am – start Dark cycle (12 hours)
- Wednesday 9 pm – start Light cycle (12 hours)
- Thursday 9 am – start Dark cycle (12 hours)
- Thursday 12 noon – Dose samples and put back into Dark cycle
- Thursday 9 pm – start Light cycle (12 hours)
- Friday 9 am – start Dark cycle (12 hours)
- Friday 12 noon – Test (this is optimum: 3 hours after the Dark cycle has started)