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Inside Dentistry
August 2015
Volume 11, Issue 8

The Vital Basics of Dental Unit Waterline Testing

Patient care begins with protection against waterborne infections

Nancy (Andrews) Dewhirst, RDH, BS

Dental unit waterline (DUWL) contamination presents a confirmed but limited risk to immunocompetent individuals, but may pose a significant hazard for immunocompromised patients and dental professionals. In the past 20 years, scientists have provided evidence that organisms found in unmanaged DUWLs may include pathogens in sufficient numbers to infect susceptible people. A recent case published in The Lancet validated the concern that contaminated waterlines may be deadly under specific circumstances. In 2012, an elderly Italian woman died after acquiring Legionella pneumophila subtype 1 from her dentist’s waterlines. The case was carefully researched, and no doubt remains that her death resulted from a dental exposure.1 Though rare and extreme, this case validates the need for the American Dental Association (ADA) and Centers for Disease Control and Prevention (CDC) recommendations stating that: 1) water used in nonsurgical dental care must meet quality standards for treatment (≤200 CFU/mL) and irrigation (≤500 CFU/mL, which is also the potable drinking water standard) and 2) surgical standards require the use of sterile water delivered through sterile devices.2-4 All offices should be using one of the available systems to manage DUWL safety. However, many may not be testing their waterlines and cannot be sure how effective their efforts are. The only way to verify that a dental water treatment regimen is working is to monitor the water flowing from the dental unit. So, what is the best advice about waterline testing? Who should be testing, how should it be performed, and how often?

Testing untreated waterlines is not useful or recommended because they are known biofilm incubators and the water is presumed to be contaminated. However, the ADA and CDC suggest anyone who is treating waterlines should test the output water to assess the efficacy of the practice’s DUWL maintenance protocol.

Testing dental output water can be costly and time consuming. It makes sense to correct obvious problems before testing. Analyze the system for weak links to confirm that manufacturers’ directions are being followed and to make corrections before testing waterlines. Consult both the equipment manufacturer’s and the DUWL anti-biofilm product manufacturer’s recommendations and correct obvious problems. Many offices will find simple mistakes are being made, such as not using the correct dilution of an active ingredient, utilizing incorrect sequencing or frequency of water treatment, failing to shock the system periodically, not emptying water bottles as recommended, failing to change internal waterline filters when they become a source of in-line contamination, using the wrong source water in the reservoirs, and failing to flush waterlines in the morning, at the end of the day, and between patients. Other common mistakes are confusing “flushing” with “shock” treatments or omitting the strong chemical “shock” treatments that are required weekly if no chemical is continually present in the waterlines; it is usually recommended monthly if daily chemical tablets or drops are added to the treatment water.The above examples illustrate how biofilm promotion and the introduction of pathogens in waterlines can occur. More than one person in the dental office should review all the factors because people tend to interpret written directions differently, possibly leading to poor DUWL quality. The great value of waterline testing is to confirm that the combined product selection, equipment maintenance, planned protocol, technique, and compliance are correct.

Testing Basics

Biofilm, the multispecies microbial community within a glycocalyx (slime layer) attached to the inner surfaces of DUWLs, releases planktonic organisms into the alternately stagnant and flowing dental water as it passes into the dental handpieces and ports. Routine waterline testing captures representative organisms that indirectly indicate the presence and amount of biofilm contamination. Some testing media select for representative microbial species such as Pseudomonas aeruginosa, Escherichia coli, or Staphylococcus aureus as a way to detect total biofilm contamination. Routine testing for other organisms of concern such as Legionella is not performed unless outbreaks are being investigated because these organisms are difficult and expensive to isolate and grow.2 DUWL test results are expressed in a count of total colony-forming units per milliliter (CFU/mL) of aerobic mesophilic heterotrophic bacteria. Safe levels are based on the Environmental Protection Agency’s Safe Drinking Water Act that allows a maximum of 500 CFU/mL. The culturing media and analysis methods are designed to capture stressed organisms typically found in waterlines that are being maintained by commercially available antibiofilm products, including those that add a chemical to the water. These tests are most accurate when water quality approaches or is better than 500 CFU/mL. When untreated or heavily contaminated waterlines are tested, the results are often too numerous to count—CFU/mL > 200,000. Another factor is total dissolved solids (TDS) present in source water, particularly municipal water. TDS is a total weight of the mineral, salt, and metal ions that encourage biofilm growth in waterlines, and values range from 120 ppm to >350 ppm.4 High TDS levels challenge DUWL treatment products and may exhaust active ingredients before the chemicals can neutralize biofilm microbes. In areas where groundwater has high TDS levels, or is “hard,” alternative sources such as distilled water can greatly improve the effectiveness of DUWL cleaning /antimicrobial products. Manufacturers of waterline products should advise users about source-water requirements.

Methods of Testing

In-office waterline tests are those that a practice purchases, uses, and processes on-site. Some can be processed at room temperature, while others require incubators to control the temperature during sample growth. Similar to sterilizer monitoring, the in-office testing systems place the burden of proof of reliability on the office. Three important variables must be managed: first, dental personnel rather than trained laboratory technicians will be drawing and incubating the samples and reading the results. Simple requirements such as ordering supplies, following directions, using an aseptic technique, adhering to scheduled testing dates, and reading results at the optimal time may be difficult for employees. Second, the samples must be kept under the controlled conditions, including temperature, to provide reliable results. Samples should be drawn early in the week so processing occurs when the staff is at work and can read test results. Incubation times may vary from 24 hours to more than 72 hours. Lastly, recordkeeping and follow-up on test results must be done. Any office that decides to do in-office monitoring must have trained dedicated personnel and a well-designed written protocol. Examples of on-site waterline testing products include small paddles, plates, or vials of culture medium to which sample water is added. Each system requires specific techniques such as dilution of the sample, adding the water for a specific time and then removing it, incubating the sample in controlled temperature units, controlling light and position of the sample, and isolation of the sample. Aseptic technique is vital. If paddles and plates are used, visual counting of colonies is the usual technique for assessing results. Some vials change color to show positive or negative growth. However, one study compared three in-office testing systems, Aquasafe™(Patterson Dental,, 3M™ Petrifilm™ (3M,, and heterotrophic plate count sampler, with R2A plating methodology (considered the gold standard for enumerating heterotrophic bacteria in potable water). The authors reported, “These in-office test systems should not be used for assessing compliance with the ADA and CDC standard for acceptable heterotrophic bacterial counts in DUWLs (≤500 CFUs/mL).”6 If in-office testing is desired, product manufacturers should provide documentation of reliability.Professional dental water testing services employ trained technicians who work under controlled laboratory conditions, reducing variables that might impact the test results. Sterile vials are provided, along with shipping containers and cold packs. Shipping conditions and timing are important variables, and samples must be drawn so that they can be sent and received overnight; lab processing must be started at about 24 hours from sample capture. Testing services may vary in the time needed to incubate samples and typically provide results in about a week. As a rule, samples should not be sent late in the week because technicians may not work on the weekends, possibly delaying processing and skewing results. Universities such as Loma Linda University or commercial services such as ProEdge and Aqua-Dent provide excellent analyses and reports.3

The Bottom Line

DUWL contamination is now an accepted and validated infection-control challenge in dentistry. Because recommendations exist and products are available to meet those recommendations, DUWL management and assessment are important and required. Water testing is the only way to validate successful DUWL management. In-office and mail-in water testing options are available.


1. Ricci ML, Fontana S, Pinci F, et al. Pneumonia associated with a dental unit waterline. Lancet. 2012;379(9816):684.

2. Kohn WG, Collins AS, Cleveland JL, et al. Guidelines for infection control in dental health-care settings. CDC MMWR Recommendations and Reports Accessed June 29, 2015.

3. Dental unit waterlines. ADA website. Accessed June 29, 2015.

4. Gruninger S. Disease transmission through dental unit water: an update. ADA Professional Product Review. 2014;9(2):8.

5. Sarrett DC. A laboratory evaluation of dental unit water treatment systems. ADA Professional Product Review. 2014;9(2):9-17.

6. Momeni SS, Tomline N, Ruby JD, Dasanayake AP. Evaluation of in-office dental unit waterline testing. Gen Dent. 2012;60(3):e142-e147.

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