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ATP-Bioluminescence
can someone please tell me what that does?
TIA
TIA
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ATP bioluminescence is a test used to detect the presence of microorganisms. ATP stands for adenosine triphosphate, which is the basic energy module present in all living things.
When the enzyme luciferase comes into contact with ATP, it emits a glow under ultra-violet light (it is naturally present in fireflies, which is why they glow at night).
By spraying a surface, for example, with a luciferase-based formula, and then exposing it to an ultra-violet light source (a luminometer), it is possible to tell how many living organisms are present on the surface. The amount of light is expressed as Relative Light Units (RLU). The higher the level of RLU, the more microorganisms that are present.
This process is called ATP bioluminescence, and is generally used in laboratory settings to check the effectiveness of disinfectants and other cleaning materials. Hope that helps?
When the enzyme luciferase comes into contact with ATP, it emits a glow under ultra-violet light (it is naturally present in fireflies, which is why they glow at night).
By spraying a surface, for example, with a luciferase-based formula, and then exposing it to an ultra-violet light source (a luminometer), it is possible to tell how many living organisms are present on the surface. The amount of light is expressed as Relative Light Units (RLU). The higher the level of RLU, the more microorganisms that are present.
This process is called ATP bioluminescence, and is generally used in laboratory settings to check the effectiveness of disinfectants and other cleaning materials. Hope that helps?
Adenosine triphosphate is a universal energy transfer
molecule that is found in all living cells. It is a nucleotide
identical to the molecule found in RNA. The phosphate bonds of the molecule are the major source of energy release. It is typically used for synthesis of amino acids, protein synthesis, active transport systems, etc. Research has suggested that the level of ATP in a sample could be used to measure biomass. However, several assumptions are made if this were applied to bacteria:
1. All living organisms contain ATP.
2. ATP is neither associated with dead cells nor
absorbed onto surfaces.
3. The level of ATP among taxa is fairly consistent given a
set environmental conditions and metabolic activity.
Quantifying the level of intracellular ATP in a sample gives an indirect measurement of the number of cells in that sample. An easy method to quantify ATP levels is to rely on the production of light from the bioluminescence assay.
(continued)
molecule that is found in all living cells. It is a nucleotide
identical to the molecule found in RNA. The phosphate bonds of the molecule are the major source of energy release. It is typically used for synthesis of amino acids, protein synthesis, active transport systems, etc. Research has suggested that the level of ATP in a sample could be used to measure biomass. However, several assumptions are made if this were applied to bacteria:
1. All living organisms contain ATP.
2. ATP is neither associated with dead cells nor
absorbed onto surfaces.
3. The level of ATP among taxa is fairly consistent given a
set environmental conditions and metabolic activity.
Quantifying the level of intracellular ATP in a sample gives an indirect measurement of the number of cells in that sample. An easy method to quantify ATP levels is to rely on the production of light from the bioluminescence assay.
(continued)
Bioluminescence is the biological production of light from various animals and fungi. A common occurrence in nature is the intermittent glow from the American domestic firefly (Photinus pyralis).
Research has shown that ATP is a major constituent of the bioluminescence reaction from the firefly and that the evolution of light is directly proportional to the amount of ATP present.
In the firefly, light production is a catalytic reaction between luciferin and luciferase which is fuelled by ATP. In more detail, luciferase combines with luciferin to form an unstable enzyme-bound luciferyl adenylate molecule. The molecule will react with oxygen to form oxyluciferin, carbon dioxide, water and light at 600nm. The reaction is stoichiometric (i.e. 1 ATP molecule yields 1 photon of light), enabling evolution of light to be used as an index of ATP level.
(continued)
Research has shown that ATP is a major constituent of the bioluminescence reaction from the firefly and that the evolution of light is directly proportional to the amount of ATP present.
In the firefly, light production is a catalytic reaction between luciferin and luciferase which is fuelled by ATP. In more detail, luciferase combines with luciferin to form an unstable enzyme-bound luciferyl adenylate molecule. The molecule will react with oxygen to form oxyluciferin, carbon dioxide, water and light at 600nm. The reaction is stoichiometric (i.e. 1 ATP molecule yields 1 photon of light), enabling evolution of light to be used as an index of ATP level.
(continued)
Now to the practical side of ATP-bioluminescence.
A means of quantifying microorganisms in a sample
can be developed based on the assumptions concerning
the ATP content in microorganisms and the stoichiometry of bioluminescence reaction.
The ATP content of microorganisms can be used as a fuel source for the luciferase enzyme in place of the ATP found in firefly tails. Therefore, the light output generated from this bioluminescence reaction should be proportional to the total ATP found in the microbial population. The reaction would be instantaneous and can be easily monitored by a light-measuring device such as a luminometer. However extraction of microbial ATP from food samples may prove more of a challenge since most food products will contain a certain amount of non-microbial ATP.
Earlier research showed that determination of microbial
content in food was difficult because of interference by background ATP from food products. This was especially true for meat products. Background ATP concentrations can be equivalent to ATP levels found in bacterial populations of 1 x l0-5 colony forming units (cfu) per millilitre or more. Therefore, it is imperative that the background ATP must be minimized in food samples to increase the sensitivity of the ATP bioluminescence assay.
(continued)
A means of quantifying microorganisms in a sample
can be developed based on the assumptions concerning
the ATP content in microorganisms and the stoichiometry of bioluminescence reaction.
The ATP content of microorganisms can be used as a fuel source for the luciferase enzyme in place of the ATP found in firefly tails. Therefore, the light output generated from this bioluminescence reaction should be proportional to the total ATP found in the microbial population. The reaction would be instantaneous and can be easily monitored by a light-measuring device such as a luminometer. However extraction of microbial ATP from food samples may prove more of a challenge since most food products will contain a certain amount of non-microbial ATP.
Earlier research showed that determination of microbial
content in food was difficult because of interference by background ATP from food products. This was especially true for meat products. Background ATP concentrations can be equivalent to ATP levels found in bacterial populations of 1 x l0-5 colony forming units (cfu) per millilitre or more. Therefore, it is imperative that the background ATP must be minimized in food samples to increase the sensitivity of the ATP bioluminescence assay.
(continued)
Most ATP bioluminescence assay kits for food purposes
employ some system of minimizing non-microbial
ATP. This may include a non-ionic detergent to break open somatic cells, sonication, low-speed centrifugation or chromatographic techniques (e.g. ion exchange resins).
In some protocols, filtration or an apyrase (an ATP hydrolysing enzyme) may be used to ensure reduction of background ATP from the sample.
After concentration of cells, bacteria can be combined
with acids (e.g. trichloroacetic acid), organic
solvents, strong cationic detergents or boiling buffers
to release intracellular ATP from microorganisms.
employ some system of minimizing non-microbial
ATP. This may include a non-ionic detergent to break open somatic cells, sonication, low-speed centrifugation or chromatographic techniques (e.g. ion exchange resins).
In some protocols, filtration or an apyrase (an ATP hydrolysing enzyme) may be used to ensure reduction of background ATP from the sample.
After concentration of cells, bacteria can be combined
with acids (e.g. trichloroacetic acid), organic
solvents, strong cationic detergents or boiling buffers
to release intracellular ATP from microorganisms.
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