What is ATP?
ATP is an excellent indicator of microbial activity in industrial environments.
- ATP is the acronym for the compound adenosine triphosphate
- ATP is the primary energy-transferring molecule in all living cells.
- Metabolic processes that produce energy in a cell use ATP to transfer the energy to other cell reactions which require energy.
- While alive, cells produce ATP continuously.
- When processes for ATP production are inhibited, all available ATP is consumed.
- Without ATP, bacteria become dormant, and, unable to maintain cell integrity, eventually die.
- When cells are killed, their ATP rapidly decreases.
- Thus, ATP can be used to monitor levels of metabolically active organisms in a liquid or deposit sample.
Why use ATP?
- Detects all bacteria – aerobic and anaerobic. No bias from growth medium composition or conditions.
- Fast. Results are generated on-site in less than one minute.
- Portable. Small hand-held detector, all-in-one sampling device with built-in reagents.
- Excellent for bulk water and surface deposit samples.
Comparison with alternative methods
- Serial dilution vials (e.g., API bottles), agar plate counts, and dip slides require long incubation times (2 days – 2 weeks) selective growth media limits measurement of all microbes.
- Microscope direct counts are relatively fast and useful but laboratory, microscope, and skilled technician required.
- Immunoassay (e.g., SRB RapidChek II) is relatively fast and practical for on-site testing but not precise and not available for non-SRB organisms.
- Molecular methods (e.g., quantitative PCR) provide numbers and types of organisms but are expensive, time-consuming, and require the services of scientists with advanced instrumentation.
How is ATP measured quantitatively?
- ATP is easily and accurately measured by treating an ATP-containing sample with a reagent that reacts with ATP and produces light that can be measured in a luminometer. The amount of light produced is proportional to the amount of ATP in the sample. This reaction occurs naturally in fireflies to produce their characteristic light.
- In this reaction, a compound, luciferin, reacts with ATP and a molecule of oxygen with the help of an enzyme catalyst, luciferase to yield an oxidized form of luciferin, adenosine monophosphate (AMP), and pyrophosphate (PPi). Energy is also evolved in the form of light. The chemical equation is:
- A measured amount of water sample containing bacteria is collected for analysis.
- A reagent, known as a “lysing agent,” whose function is to break apart bacterial cell walls and membranes to release ATP into the bulk water, is added to the sample.
- ATP reacts with the luciferin-luciferase test reagents, producing light in quantities which can be measured by a sensitive luminescence detector.
- The sample containing the reagents is placed in the detector, and light is measured in Relative Light Units (RLU). The RLU reading from the instrument is directly proportional to the amount of ATP in the sample, which, in turn, is a function of the number of bacteria in the sample and their metabolic activity. The analysis can be conducted in a cuvette, with sample, lysing reagent and luciferin-luciferase reagents pipetted into the cuvette; the cuvette is then placed in the detector, where light measurement occurs. Alternatively, an ATP pen can be used. Sample (water or deposit) is collected on the swab, and the swab is returned to the pen, where pre-loaded reagents are released; the pen is then placed in the detector, where light measurement occurs.
Correlation of ATP with bacterial counts from solid or liquid media methods.
Can ATP be used to provide a direct measure of CFU/mL (Colony Forming Units per milliliter)?
- Very actively growing organisms will have relatively high levels of ATP per cell, compared to slow growing or dormant populations (e.g., populations in low-nutrient environments) which will have much lower levels of ATP per cell.
- In a laboratory setting, working with a single organism type, variations in ATP will reflect changes in population size (CFU/mL) and relative metabolic activity.
- However, in a natural or industrial environment there will be a number of different organism types present. Conditions of the environment will determine how diverse the associated microbial population is. In a diverse microbial population, cell size, and therefore cell volume can vary significantly. Under “normal” conditions, ATP per cell will vary roughly in proportion to cell volume. Hence, cell size/volume is the second factor which significantly affects the correlation between ATP and CFU in a series of samples.
- The difference in volume between a small and a large bacterium can easily be a factor of more than 100X.
- Changes in the numbers of large cells or changes in the metabolic activity of large cells will have a greater impact on total population ATP than comparable changes restricted to the small cells in the population.
- Hence, even though both a single small cell and a single large cell are sufficient to produce a single colony on a dip slide or agar plate, the impact on ATP RLU of increases or decreases in numbers of large cells will be significantly greater than comparable changes in the numbers of smaller cells.
- The presence of significant numbers of bacteria associated with particles – will have a significant impact on the correlation between ATP and CFU/mL. This is because ATP from 50 dispersed bacteria and 50 bacteria attached to a single particle are detected equally in an ATP analysis; however, while 50 dispersed bacteria would form 50 colonies on an agar plate, 50 bacteria attached to a single particle would form a single colony on the same agar plate.
- With this understanding of the impact of cell number, size and metabolic activity on the RLU measured for a bacterial population, it is important to note that many laboratory and field studies have demonstrated that for a specific system, ATP data (as RLU) correlate very well with other measures of microbial population size. ATP data can therefore be used to monitor changes in a system’s microbial population resulting from chemical treatments or changes in operating parameters. Read more about ATP in this Case Study.
- Click here to read more about the theoretical correlation of ATP vs CFU.
