August 23, 2022

Complete Guide to Biofilm in Dental Unit Waterlines

Waterline-based bacteria and biofilm floating in dental water

We spend a great deal of time around here talking about biofilm (it might be an obsession, but we are okay with that). Our efforts to educate individuals like you and other dental professionals really start with understanding biofilm and the role it can play in dental infections. While not all biofilms are inherently harmful, they form the perfect environment for dangerous pathogens.

So, we believe it’s important to understand how bacteria and the biofilms they form can impact your patients. In this article we will discuss:

  • What are heterotrophic bacteria?
  • What is a colony-forming unit (CFU)?
  • What is biofilm?
  • How do biofilms form?
  • Are there biofilms in my dental waterlines?
  • How do I clean biofilm from my dental unit waterlines?

What are heterotrophic bacteria?

Bacteria is to biofilm what a single lego piece is to an epic replica of the Death Star–it’s a single piece that contributes to a much larger, more complex whole. From a scientific perspective, bacteria are single-celled organisms with a single motivation: to survive and multiply. And they have gotten pretty darn good at doing just that.

If you’ve read about contamination in dental unit waterlines, you have probably heard the term heterotrophic. Heterotrophic bacteria are natural inhabitants of animals but are not necessarily indicators of pathogenic conditions (Amanidaz N, Zafarzadeh A, Mahvi AH, 2015). But, when they form into colonies and then into biofilms, there is a greater concern about what might be lurking inside the growth.

What is a colony-forming unit (CFU)?

Jackie Reynolds, a Professor of Biology at Richland College, describes a colony as “a visible mass of microorganisms all originating from a single mother cell that grows on solid medium” (Reynolds, 2021), like dental unit waterline tubing. In layman’s terms, a colony is a group of bacteria.

A colony-forming unit or CFU is described by Revive of the Global Antibiotic Research and Development Partnership as the “unit that is used in microbiology to estimate the number of viable bacteria or fungi in a sample.” In the context of waterline testing, CFUs have long been the standard for measuring the presence of bacteria in dental unit water.

What is biofilm?

So, how does all of this relate to biofilm? Well, Alfred B. Cunningham, John E. Lennox, and Rockford J. Ross explain in their textbook Biofilms: The Hypertextbook that, “a biofilm forms when certain microorganisms (for example, some types of bacteria) adhere to the surface of some object in a moist environment and begin to reproduce. The microorganisms form an attachment to the surface of the object by secreting a slimy, glue-like substance.”

This glue-like substance, or biofilm, becomes the city walls that surround a complex ecosystem of colonies where bacteria can transfer information between cells uninterrupted. These walls are challenging to penetrate, which leads to the need for a treatment protocol in waterlines.

While there is a lot of complexity in this topic, you are far more acquainted with biofilms than you might think. Plaque is an example of a biofilm. As you know, the build-up of plaque can be challenging to remove without a lot of elbow grease. Imagine the same build-up being in dental waterlines. Attempting to clear away this stubborn material simply by flushing your lines will, understandably, be unsuccessful.

You can see this process in action below where dental plaque that has formed on top of the teeth, then acid, produced by the bacteria, dissolves the tooth enamel leading to infections and dental caries (Hollmann, Perkins, Walsh).

Biofilm decaying tooth enamel

In this instance, biofilm can cause a lot of harm and requires a dental professional like yourself to dive in there and remove it. So, now that we’ve talked about what biofilm can look like in various environments, let’s talk about how this growth cycle presents itself in waterlines.

How do biofilms form?

Biofilm, like all living organisms, goes through a lifecycle. Birte Hollmann, Mark Perkins, Dean Walsh Centre of Biomolecular Sciences, University of Nottingham describe this cycle in their publication in British Society for Immunology, Biofilms and their role in pathogenesis, as such:

Biofilm growth cycle

Figure 1. Adapted from Vasudevan, 2014, J Microbiol Exp 1(3): 00014. DOI: 10.15406/jmen.2014.01.00014.

  1. Bacteria attach to the surface
  2. Bacteria multiply and form a slimy biofilm layer
  3. A microcolony is formed
  4. Colonies form a mushroom structure
  5. Bacteria detach and the biofilm disperses to form new colonies

Bacteria’s only goal is to grow and multiply. As you can see, they are experts at this. The cycle shown above occurs regularly in dental waterlines and can only be disrupted by a consistent, effective waterline maintenance protocol. Because of how quickly a single bacteria cell can multiply, regular shocking and continuous treatment are required to keep bacteria from making themselves at home in your waterlines. As we have seen in Barbeau’s study, Multiparametric Analysis of Waterline Contamination in Dental Units, within just five days, bacteria counts can grow to 200,000 CFU/mL.

Why does biofilm grow so well in dental unit waterlines?

As a dental professional, it might seem like the mouth is the number one environment for microbial growth, but dental unit waterlines are a close contender. Not only do dental waterlines support growth, but they also feed it.

The 4 main reasons biofilm grows so well in dental unit waterlines are:

  1. The material used to make dental unit waterlines
  2. The slow flow rate of water through dental unit waterlines
  3. The low volume of water required/use
  4. The spread of bacteria caused by water use


Let’s take a closer look at each of these:

The material used to make dental unit waterlines

Dental unit waterlines are often made up of porous rubber or plastic. Unlike municipal lines made of copper (which is an antimicrobial), dental lines contain carbon, which is one of bacteria’s favorite food. So, while the material is perfect for a dental pro moving their equipment around a chair, it is not great for keeping bacteria from turning into full-grown biofilms.

The slow flow rate of water through dental unit waterlines

Most of us would probably picture the water in dental waterlines flowing like a river, but infrequent use and long periods of stagnation cause water to flow much more like a pond with a small drain. In comparison, your municipal lines (think faucet or sink), flow at 5L/minute. Dental unit waterlines flow over 1,000 times slower (R-Tech Dental of MN, LLC)!

Because there isn’t much movement within the lines, bacteria have the time to multiply without being disturbed. This creates a perfect breeding ground for biofilm.

The low volume of water required/use

Consider this, water remains stagnant inside of your dental unit waterlines for over 22 hours, even on a busy day. When you use your waterlines, it is usually just for a few seconds and then it continues to sit in between, and long after, patient visits. Not to mention closures, weekends, and overnights. This stagnation allows biofilm to build up in thick layers.

The breakoff caused by water usage

When the air-water syringe, handpiece, or scaler is engaged, bacteria take the opportunity to break off and multiply further down the line. We often describe the situation like this: every time your handpiece is engaged, it’s like blowing on a dandelion, spreading the seeds down the line to develop roots, grow strong, and do it all over again. And while the colonies build up reinforcements, new bacteria is entering the waterline all the time.

Additionally, with each new patient that sits in any one of your chairs, there is potential for handpieces to retract liquid (and the bacteria in it) from the patient’s mouth. This is why flushing between patients isn’t just best practice, it’s essential!

Remember, bacteria never quits.

That is why it is important to develop a waterlines maintenance protocol that is consistently evaluating your water quality through testing and eliminating biofilm through shocking and treating.

Is there biofilm in my dental waterlines?

The only sure way to know if your lines have biofilm is to test them (you can check out our Complete Guide to Testing Dental Unit Waterlines).

While there are various types of tests (like QuickPass, R2A, or Flo), the results from any of these tests should reveal if your lines fall outside the CDCof less than 500 CFU/mL or not. If your waterline fails a water test, it is reasonable to assume that the environment is ripe for biofilm formation and the affected operatory needs immediate attention.

How do I clean biofilm from my dental unit waterlines?

While this all may feel very intimidating and receiving the results of a failed dental waterline test is not ideal, you are not alone. We talk to dental offices every day who are in the same situation as you. The most important thing to do is take action. Don’t push it down the to-do list or ignore it. We recommend cleaning your dental waterlines using the simple 3 Steps to Safe Water.

The first step is to shock your lines with a chemical that can break down biofilm, such as a diluted bleach protocol. Don’t worry, we have a Complete Guide to Shocking Dental Unit Waterlines to help you through the process.

The next step is to treat your lines. This may be something you’ve already been doing in your dental office with waterline treatments. However, these alone are not enough to remove stubborn biofilm in your lines, rather treatment product was intended to maintain already clean lines. This is why pairing your treatment solution with a shock protocol is so important! In our Complete Guide to Treating Dental Unit Waterlines, we will walk you through what options are the best for your office and for your staff.

Lastly, you want to test your waterlines to ensure that what you are doing is effectively removing biofilm and keeping your water safe. You can check out our Complete Guide to Testing Dental Unit Waterlines to see what options are available for your practice.

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Publish Date:

August 23, 2022