ATP Testing in Health Care: What It Can and Cannot Tell Us

Measuring the effectiveness of cleaning and disinfection remains one of the most critical yet persistent challenges in health care today. Tools such as fluorescent markers and adenosine triphosphate (ATP) testing are often adopted to provide quick, quantifiable insights into environmental hygiene. However, while these methods can support staff training and quality improvement, their results are frequently misunderstood—and sometimes misused—as indicators of microbial contamination or disinfectant performance. Understanding what these tools measure and their inherent limitations is critical for applying them appropriately in health care settings.

Environmental Contamination and Infection Risk

Environmental contamination has been conclusively demonstrated to increase the risk of acquiring organisms (and therefore the risk of infection) for newly admitted patients in acute care.¹ Preintervention performance indicators for thoroughness of cleaning and disinfection can be abysmally low (eg, 48%)² and yet quality improvement programs can show dramatic improvement in both cleaning thoroughness (59% increased to 93.6%)³ and decreased infection rates (such as Clostridioides difficile rates decreasing by 50 to 75%)^4,5 with improvement performance sustained for up to 6 years.⁴

While performance improvement in environmental hygiene can take many forms (eg, re-education, training), most successful programs incorporate some form of near-real-time auditing and feedback. Although direct observation may be one such monitoring approach, like hand hygiene audits, it can be resource-intensive and is susceptible to the Hawthorne Effect (temporary performance improvement when being observed). Therefore, the most commonly used methods today involve surrogate, post-cleaning assessments such as fluorescent surface marking or ATP testing.

Fluorescent Marking: Proxy for Cleanliness, Not Disinfection

Fluorescent marking methodology is a common, relatively easy, and inexpensive method of evaluating surface cleaning and disinfection. It utilizes a fluorescent marker that is less visible under normal light but fluoresces under UV black light. An individual, often an EVS supervisor or infection preventionist, applies it to surfaces, either in a room or on equipment, before it is expected to be cleaned or disinfected, either as part of a daily clean or terminal discharge. The person responsible for cleaning and disinfecting surfaces preferably unknowingly performs the task, and the same index individual or applicator of the marker returns to the setting, uses a handheld fluorescent light or flashlight, and observes whether the mark has been removed.

It is important to remember that fluorescent markers are only a proxy for surface contamination. They do not represent bacteria but are simply removable marks used to track cleaning. It is effective in indicating whether surface contamination, such as dirt or body fluids, has been removed, but it is not a reliable indicator of disinfection. Furthermore, the removal of the marker is contingent on the surface itself, and the substrate used, and some substrates will perform better (new microfiber cloth) than others (eg, cloth, paper towel, disposable wipe) when equal force is applied.

ATP Testing: Origins and Health Care Adoption

ATP is the primary form of biochemical energy storage and is generated through cellular respiration in the mitochondrial matrix. ATP testing was first proposed and developed by the National Aeronautics and Space Administration (NASA) in 1974 as a method for identifying extraterrestrial life.⁵ Over the next 3 decades, it found commercial use in the food industry as an indicator of cleanliness via the removal of organic matter.

Beginning in 2003, health care experiences using ATP testing as a method of evaluating environmental hygiene were described in the literature, and the adoption of this technology as a strategy for measuring health care cleaning and disinfection performance has since been widely adopted.⁶ Yet, what is often misunderstood is that much like the aforementioned fluorescent marking system, the ATP testing results are a proxy for surface contamination and are not an indicator of viable microorganisms. This misconception is understandable: ATP monitors have the appearance of scientific instruments rather than magic markers, but it is critically important to understand what they measure and what they do not.

All living material generates ATP, but this compound can remain behind long after that material is dead (hence the NASA proposal). It is analogous to running an X-ray over a graveyard; just because there are bones does not mean anyone is out walking around.

Why ATP Cannot Measure Disinfectant Effectiveness

What about using ATP meters to measure the effectiveness of disinfectants? The short answer is no. Multiple peer-reviewed studies have examined the relationship between ATP readings and actual microbial contamination.

• Drs Rutala, Weber, and colleagues demonstrated that relative light units (the measurement value of ATP testing) had virtually no correlation with colony-forming units (CFUs) of viable microorganisms (R2 for pre- and post-cleaning of 0.013 and 0.008, respectively).⁷ In other words, higher ATP values did not mean more (or any) organisms were on the surface.
• Formerly, per the FDA (no longer available), now only available from CMS, “An ATP test only gives a sense of how well something has been cleaned. For example, a prep table in a food processing facility could show high levels of ATP (ie, an unclean surface) while providing a negative result on a series of environmental pathogen tests. This is because ATP tests will register any organic matter on a surface, even if none of that organic matter is specifically pathogenic.”⁸
• Van Arkel and colleagues assessed this as well, concluding that "ATP measurement can be used to give a quantifiable outcome for the rating of cleanliness in health care facilities; however, the results cannot be translated into the level of microbial contamination."⁹
• Lastly, Omidbakhsh and colleagues demonstrated that disinfectant effectiveness cannot be accurately measured by ATP results, concluding that “introducing ATP meters to health care facilities, as a disinfection validation tool, is not a reliable choice.” ¹⁰

In other words, ATP testing is a more quantifiable, less subjective, and therefore potentially more reliable indicator of cleanliness, superseding direct observations or fluorescent marking programs. However, what it is not is an indicator of microbial contamination or the effectiveness of disinfectants.

The Case of Hydrogen Peroxide-Based Disinfectants

The limitations of ATP testing are particularly significant when evaluating oxidative disinfectants such as hydrogen peroxide formulations. The reason lies in the chemistry of the assay. ATP detection depends on the luciferase enzyme. Oxidative compounds, including hydrogen peroxide, can denature this enzyme, preventing it from catalyzing the bioluminescent reaction. Enzymes are generally more sensitive to oxidative damage than intact microorganisms.

When a surface is treated with hydrogen peroxide—especially at higher concentrations—the residual chemistry can inactivate luciferase present during testing. This results in lower ATP readings, not because more pathogens were eliminated, but because the test mechanism itself was impaired. In practice, a product with nearly 3 times the hydrogen peroxide concentration may appear to produce a “cleaner” surface according to ATP, when, in fact, the difference is due to enzyme interference rather than superior microbial kill.¹⁰

This phenomenon highlights a core risk of misinterpretation: ATP results in such cases may reflect chemical interference rather than a reduction in infection risk.

Additional Complications in ATP Interpretation

Beyond enzyme interference, there are other important caveats to ATP interpretation:

• Nonspecificity to pathogens: ATP testing is not specific to pathogenic microorganisms. The fact that ATP is on the surface simply signifies the presence of biomaterial, which can include things such as skin or other tissue cells.
• Persistence of ATP after kill: ATP can remain reactive on the surface from recently killed organisms, even if they no longer pose an infection risk. While it typically degrades quickly, if you perform the test immediately following disinfection, you can still generate some false positives.
• False sense of cleanliness: A low ATP reading does not necessarily mean a surface is disinfected; it may simply mean fewer organic residues or that residues interfered with the test.

These factors make ATP unsuitable for head-to-head disinfectant performance comparisons—particularly when different active chemistries are involved.

EPA-Validated Methods: The Gold Standard for Proving Kill Claims

While ATP and fluorescent markers can help environmental services teams measure cleaning activity, they cannot confirm microbial inactivation. The only reliable way to validate disinfectant efficacy is through EPA-validated microbiological methods, which directly measure pathogen kill. These standardized assays are required for disinfectant product registration and are designed to demonstrate reductions against pathogens of critical concern. Unlike ATP testing, which measures residual organic matter, EPA protocols quantify actual reductions in viable organisms under standardized laboratory conditions.

Understanding the purpose and limitations of different testing methods is essential for health care settings. ATP testing can be a useful tool for monitoring cleanliness, but it should not be used to verify disinfection or pathogen removal. Effective disinfection in health care comes from evidence-based disinfection protocols, supported by staff training and consistent application. By aligning the right tools with the right purpose, facilities can ensure safer environments for both patients and staff.

References

1. Mitchell BG, Dancer SJ, Anderson M, Dehn E. Risk of organism acquisition from prior room occupants: a systematic review and meta-analysis. J Hosp Infect. 2015 Nov;91(3):211-7. doi: 10.1016/j.jhin.2015.08.005. Epub 2015 Aug 22. PMID: 26365827.
2. Carling PC, Parry MM, Rupp ME, Po JL, Dick B, Von Beheren S; Healthcare Environmental Hygiene Study Group. Improving cleaning of the environment surrounding patients in 36 acute care hospitals. Infect Control Hosp Epidemiol. 2008 Nov;29(11):1035-41. doi: 10.1086/591940. PMID: 18851687.
3. Carling PC, et al. (2023). Mitigating hospital-onset Clostridioides difficile: The impact of an optimized environmental hygiene program in eight hospitals. Infection Control & Hospital Epidemiology, 44: 440–446, https://doi.org/10.1017/ice.2022.84
4. Parry MF, Sestovic M, Renz C, Pangan A, Grant B, Shah AK. Environmental cleaning and disinfection: Sustaining changed practice and improving quality in the community hospital. Antimicrob Steward Healthc Epidemiol. 2022 Jul 12;2(1):e113. doi: 10.1017/ash.2022.257. PMID: 36483421; PMCID: PMC9726550.
5. https://ntrs.nasa.gov/api/citations/19740014615/downloads/19740014615.pdf(Accessed August 6, 2025)
6. Nante N, Ceriale E, Messina G, Lenzi D, Manzi P. Effectiveness of ATP bioluminescence to assess hospital cleaning: a review. J Prev Med Hyg. 2017 Jun;58(2):E177-E183. PMID: 28900359; PMCID: PMC5584088
7. Rutala, Kanamori, Gergen, Sickbert-Bennett, Huslage, Weber. APIC Annual Conference 2017.
8. CMS Facebook page post accessed August 6, 2025: https://www.facebook.com/CMSLLCMD/#:~:text=WHAT%20DOES%20AN%20ATP%20TEST,organic%20matter%20is%20specifically%20pathogenic.
9. van Arkel A, Willemsen I, Kluytmans J. The correlation between ATP measurement and microbial contamination of inanimate surfaces. Antimicrob Resist Infect Control. 2021 Aug 6;10(1):116. doi: 10.1186/s13756-021-00981-0. PMID: 34362450; PMCID: PMC8349058.
10. Omidbakhsh N, Ahmadpour F, Kenny N. How reliable are ATP bioluminescence meters in assessing decontamination of environmental surfaces in healthcare settings? PLoS One. 2014 Jun 18;9(6):e99951. doi: 10.1371/journal.pone.0099951. PMID: 24940751; PMCID: PMC4062432.