Bioluminescent ATP Assay
ATP (adenosine triphosphate) is the chemical compound in which energy is stored in all living cells. In the ATP-luminometric test the firefly enzyme (luciferase) in the presence of its substrate, luciferin, oxygen and magnesium ions catalyzes conversion of chemical energy of ATP into light through oxidation-reduction reaction. The quantity of light generated is directly proportional to the amount of ATP present, thus, the light units can be used to estimate the biomass of cells in a sample. With state of the art equipment, and highly purified reagents, it is possible to detect amounts of ATP corresponding to approximately 100 bacterial cells, although in practice it is usually nearer to 103-104. Quantification of intracellular microbial ATP can be conveniently accomplished using rapid and simplified extraction and assay procedures. The light emitted by this process can be monitored by a variety of luminometers. Supplying companies provide customers with test kits with all necessary reagents. The reagents are injected into the instruments and readout is reported in relative light units (RLUs). By knowing the number of microorganisms responsible for generating known RLUs, one can estimate the number of microorganisms in the (food) sample. The ATP method has been used to evaluate microbial loads in e.g. meat, milk, water, fruit juice samples in a winery and a brewery, and sweeteners and syrups (FUNG, 1997). Much interest has been developed also in using ATP estimation not for total viable cell counts but as a sanitation check including also the verification of somatic cells presence on a surface.
This version of the ATP bioluminescence method based on detecting all ATP on a surface provides an indication of cleanliness detecting also ATP of somatic origin that the traditional plate count method does not detect, instead of only of microbial origin (CHEN, 2000; ILLSLEY et al., 2000; QUINN et al., 2002; PAEZ et al., 2003). Samples for assessing surface hygiene by this method can be obtained by swabbing the surface, or by taking aliquots of the rinse water (KISS et al., 1999). Reading of the bio-luminometers may be assessed as “acceptable” or “unacceptable”. The procedure can be easily performed by almost anyone, with little training. The preparation and measuring time takes only several minutes. Portable luminometer reading units have test swabs with pre-packaged reagents. The user swabs the surface to be tested, activates the swab by placing it into the solution of reagents then inserts it into the chamber of the luminometer to obtain the measurement.