The technique has a detection limit of 1 pg ATP which is equivalent to approximately 1000 bacterial cells.

The use of bioluminescent ATP detection for hygiene monitoring offers:

  • Speed (minutes, much faster than seeding and colony counting)
  • Convenience
  • Measurement of total hygiene

Principle of ATP testing for hygiene monitoring

For hygiene testing the total ATP content of the sample is determined. This will include eukaryotic and microbial ATP. Caution must be exercised to avoid contamination of the sample or material as ATP is found on many surfaces.

An ATP-free swab is provided pre-moistened or is moistened by the user with an ATP-free buffer, water or extractant. The use of extractant can help with sampling as it is effective at releasing ATP from the surface. Using portable luminometers the testing of the swab is usually done immediately. However, with some products the swabs are stable for several hours allowing the user to return to the instrument at a "workstation" if preferred.

To determine the microbial ATP level selective extraction is used. First, non-microbial ATP is extracted with a non-ionic detergent (Triton X-100) and then destroyed by treating with a high level of potato ATPase for 5 minutes. Subsequently the microbial ATP is extracted using trichloroacetic acid (5%), an organic solvent (ethanol, acetone or chloroform) which will require subsequent dilution to avoid luciferase inhibition, or cationic detergents. In the last step, luciferase and luciferin are added and luminescence is measured using a luminometer. Careful timing of mixing and reading is required to allow for luciferase inhibition (Simpson & Hammond, 1991). Since the level of ATP in eukaryotic cells is three orders of magnitude higher than in bacterial cells it is sometimes difficult to quantify separately eukaryotic and microbial ATP with accuracy.

Many manufacturers offer all needed reagents and materials as ready-to-use kits to make the protocol easier and faster.

Interpretation of results

Generally, clean surfaces show low levels of total ATP. Therefore, light output greater than 2 to 3 times background of the clean surface indicates that the area tested is contaminated with biological material. This method is very sensitive and in practice a threshold of 10 times background can be accepted. Nevertheless, whichever product is used there is a need for some preliminary work to establish the relevant Pass/Fail limits for the test. This is usually done by collecting reference data according to normal cleaning procedures. The level set will depend on type and condition of the surface and the method of cleaning used.

Recommended instruments for Hygiene Monitoring

Portable luminometers are usually the instrument of choice for hygiene monitoring as the measurement is commonly performed on site. Cheap hand-held luminometers based on photodiodes are sometimes enough for qualitative analysis (contaminated/not contaminated). PMT-based instruments such as the Junior LB 9509 offer higher sensitivity and dynamic range. This is the best option for quantitative measurements.