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Year : 2006  |  Volume : 9  |  Issue : 3  |  Page : 93-98
Evaluation of bacterial contamination of dental unit water Lines and the efficacy of a commercially available disinfectant

Department of Conservative Dentistry & Endodontics, Meenakshi Ammal Dental College & Hospital, Chennai, India

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Evaluation of Bacterial Contamination of Dental Unit Water Lines and the Efficacy of a Commercially Available Disinfectant (Sterilex Ultra® )
Key words: dental unit waterlines, disinfection. biofilm, hypochlorite. Microbial colonies that adhere to solid surfaces wherever there is sufficient moisture are referred to as the biofilm. The microbes acquire a slimy covering called glycocalyx, which protects them, in a moist environment. Biofilms formed in dental unit water lines can act as a source of cross infection.
Aim: A study was undertaken to evaluate the bacterial contamination of dental unit water lines and to evaluate the efficacy of a commercial disinfectant (Sterilex Ultra® ) in eliminating biofilms from dental unit water lines.
Materials and Methods: To begin with, random water samples were collected from water booster, air-­turbine, air water syringe of three dental units and were subjected to bacteriological analysis. A commercially available disinfectant. Sterilex Ultra® , was used to treat the dental unit water lines. Water samples from different parts of dental unit water lines were collected on the third, fifth and seventh day following treatment with the reagent and the samples were sent for bacteriological examination. One inch tubing from the outlet of the booster, air turbine and air/water syringe was also sectioned and processed for bacteriological examination.
Results: Bacterial isolates were observed before treatment with Sterilex Ultra® and but there were no bacterial isolates after treatment with the reagent for a period of six days.
Conclusion: Usage of disinfectant was found to be very effective for a period of six days. For maintenance of sterility of dental unit water lines it is essential to have a good source of water. an effective disinfectant and the use of an anti-retraction valve.

How to cite this article:
Venkatesh VK, Vidyashree NV, Velmurugan, Parameswaran A, Kandaswamy D. Evaluation of bacterial contamination of dental unit water Lines and the efficacy of a commercially available disinfectant. J Conserv Dent 2006;9:93-8

How to cite this URL:
Venkatesh VK, Vidyashree NV, Velmurugan, Parameswaran A, Kandaswamy D. Evaluation of bacterial contamination of dental unit water Lines and the efficacy of a commercially available disinfectant. J Conserv Dent [serial online] 2006 [cited 2023 Oct 3];9:93-8. Available from:

   Introduction Top

Microbial colonies that adhere to solid surfaces wherever there is sufficient moisture are referred to as the biofilm. It is composed of a variety of life forms which include bacteria, fungi, parasites, nematodes etc. These biofilms adhere to any inviting surface like a rock, tooth surface, a dental implant, removable dental prosthesis or the hull of a boat. Biofilms are also formed in dental unit water lines and they can act as a source of cross infection. In such an environment, they form a slimy covering, called glycocalyx, which protects them. The microbes in the biofilm produce a protective polysaccharide matrix that provides a mechanism for surface attachment and retention to the waterline. The glycocalyx which is 30 - 50m thick is a filamentous network of carbohydrate rich molecules that coats cells. The glycocalyx protects organisms against external assaults, desiccation, chemical insults, antimicrobial agents etc; they unite individual cells to form large multicellular aggregates. A bacterium residing in a biofilm is hidden from predators and is five hundred times more resistant to antibiotics than free floating planktonic forms. Biofilms are seen in oral soft tissues and teeth, middle ear, gastrointestinal tract, urogenital tract, heart valves, hip and joint prosthetic replacements, catheters, intrauterine devices, contact lens and dental unit water lines.

Dental unit waterlines are an integral part of dental surgery equipment, supplying water as a coolant for air-turbines and ultrasonic scalers. The handpiece is attached by flexible plastic lines to the dental unit that controls air and water supplied to the handpiece. Dental unit waterlines are susceptible to biofilm formation because they are made from a variety of plastic materials. The plastic surfaces promote growth of bacteria in the biofilm. Growth is generally slow, due to lowered oxygen supply and limitation of nutrients. The predominant bacteria isolated from biofilms arc Grain -ve organisms like Pseudomonas, E. Coli, Legionella, Non-tuberculous mycobacterium, others like Staphylococcus, Bacillus, Actinobacter, Xanthomonas maltophilia, Achromobacter xyloxidans, Pasteurella haemolytica, Klebsiella pneumoniae, Serratia marcenscens, Micrococcus and Flavobacterium. The predominant organisms have little potential to cause disease in immuno-competent hosts. The powerful endotoxins form immune complexes which may produce mild inflammation to serious toxic shock [1] .

With the emergence of opportunistic infections in the last few decades, many of these organisms typically recovered from dental unit water systems could prove harmful. People considered to be at risk are elderly people and immuno-compromised persons. Immuno compromised persons would comprise of HIV/AIDS patients, patients with chronic autoimmune diseases/organ transplant recipients (patients on steroidal therapy), patients on prolonged radiotherapy, patients with multiple blood transfusions, patients exposed to immunosuppressive agents, etc.

The colonization and proliferation of many and varied species of microorganisms is therefore a well documented fact. Several methods have been advocated by which the contamination of dental unit waterlines can be minimized. Contamination of dental unit water lines can be minimized by incorporation of anti retraction values, flushing of dental unit water lines at the beginning and end of patient treatment and chemical disinfection [2] . Sodium hypochlorite was the most commonly used disinfectant for dental unit waterlines. But research has proved that they give away by-products like trihalomethanes, which have hazardous effects on the human tissues. Recent research provides us with a new material in the form of Sterilex Ultra® , which contains sodium bicarbonate, sodium percarbonate, alkyl benzyl dimethyl ammonium chloride and tetra sodium ethylene diamine tetra acetic acid, for use as a cleaning agent in dental unit waterlines. Present study was undertaken to study the efficacy of this disinfectant (Sterilex Ultra® ) in dental practice.

   Methodology Top

The study was undertaken in the department of Conservative Dentistry and Endodontics, Meenakshi Animal Dental College and Hospital, Chennai. Three dental units were used for the study. Random water samples (20ml) were collected in sterile containers from water booster, air-turbines and air-water syringe of dental units and subjected to microbiological analysis. The samples were centrifuged at 3000rpm for 5 minutes, supernatant was discarded and centrifuged deposit was used for cultures. The collected samples were subjected to both aerobic and anaerobic cultures. Viability count study was made using pour plate and most probable number technique (M PN).

Commercially available disinfectant (Sterilex Ultra® ) a specialty cleaner powder was used on three dental units and its efficacy in bringing down the bacterial count and removing biofilm was evaluated. The disinfectant (Sterilex Ultra® ) was prepared according to the manufacturer's instructions.

Method of application of commercially available disinfectant (Sterilex Ultra® )

  1. The prepared solution was added to the booster of the unit. The prepared disinfectant (Sterilex Ultra® ) solution was made to run through the system until the pink solution appeared at the end of the air/ water syringe and hand-piece lines. The air turbine was always removed. Booster used was a standard glass bottle supplied by the chair manufacturer. Plastic bottle could not be used because hydrogen peroxide liberated during the action of commercially available disinfectant (Sterilex Ultra® ) could burst the plastic.
  2. The disinfectant (Sterilex Ultra® ) solution was allowed to sit in the unit overnight. The ends of waterlines were placed into a sink in case any pink solution dripped overnight.
  3. At the beginning of the next workday, remaining disinfectant (Sterilex Ultra® ) solution was discarded and the external water bottle was rinsed with hot water.
  4. The bottle was filled with hot water, each of the dental unit water lines (air/ water syringe, hand­piece lines) were flushed till froth disappeared and clear water could be seen. Water from Aquaguard was made to run through the tubing.

Nine water samples (20m1 each) were collected from the water booster, outlet of air/ water syringe and air turbine handpiece of the three dental chairs after treatment with commercially available disinfectant (Sterilex Ultra® ). Samples were collected on the third day, fifth day and seventh day following treatment with the reagent. All the samples were sent for bacteriological examination.

One day after treating the dental unit waterline with the disinfectant (Sterilex Ultra® ), one inch of the tubing from the outlet of the booster within the control box, second at the point of the attachment of air­-turbine coupling and the third at the point of attachment of air/ water syringe were sectioned, processed and cultured from the three dental units to examine the presence or absence of biofilm.

   Results Top

The results of the study showed that before application of the disinfectant, Pseudomonas formed 40% of the bacteria followed by Streptococci 20%, Coagulase ve Staphylococci and Micrococci were 13.3% each and Candida and Staphylococci aureus formed 6.7% each of the total count. After the application of the disinfectant third, fifth day samples showed zero bacterial growth. The seventh day sample showed some bacterial growth.

   Discussion Top

Biofilms can be prevented by periodical flushing of the waterlines, use of sterile water, temperature controlled water supply and by the use of biocides. The biocides used are sodium hypochlorite, chlorhexidine gluconate, hydrogenperoxide based solution and citric acid based products. Sterilex Ultra® , a hydroperoxide ion-phase transfer catalyst, is one of the latest waterline cleansers .

Sterilex Ultra® was granted seal of acceptance by the ADA in 2000. Since the year 2000, several studies have been conducted in various countries although the agent is yet to be tested in India. The present study was aimed at evaluating the efficacy of speciality cleaner power/ disinfectant (Sterilex Ultra® ), on the microbiological water quality in dental unit waterlines.

In the first part of the study, prior to the use of disinfectant (Sterilex Ultra® ) for flushing dental unit waterlines, water samples were collected at random intervals. These samples were sent to the laboratory for microbiological analysis. Almost all the samples showed different isolates of microorganisms such as Pseudomonas, Streptococci, Coagulase -ve staphylococcus aureus, Micrococci and Candida. Preliminary analysis of water revealed that the viable bacterial count found in booster was 774 colonies forming units per ml; air turbine water showed 495 colonies forming units per ml, air/water syringe showed 171 colonies forming units per ml before application of disinfectant (Sterilex Ultra® ). Water samples collected from three dental units after microbiological analysis revealed 2054 colonies forming units per ml in booster, 639 colonies forming units per ml in three-way syringe and 1343 colonies forming units per ml in air turbine.

One inch of the tubing cut from three sources before application of disinfectant (Sterilex Ultra® ), showed that effluent water booster tubing contained 1350 colonies forming units per ml, dental unit waterlines before entering air turbine showed 1226 colonies forming units per ml and dental unit waterline before entering air/water syringe was found to be 1107 colonies forming units per ml.

After allowing the disinfectant (Sterilex Ultra® ) to remain in dental unit waterlines overnight, random specimens were collected on third, fifth and seventh day and subjected to microbial examination. It was found that no bacterial isolate was encountered. It was also observed that the sterility was maintained for a period of six days. The flushing of dental unit waterlines over night would enhance the efficacy of the reagent to 100%.

During the study it was found that Legionella species which generally predominate the bacterial contamination of dental unit waterlines was absent. Hence, we can deduce that legionella, one of the primary contaminants of waterlines and species with pathogenic potential, as found by other studies4, 5, 6, was not a factor under the conditions in which the present study was conducted.

The study also proved beyond doubt the efficacy of disinfectant (Sterilex Ultra® ) as a cleaning agent of the dental unit waterlines and in eliminating the biofilm formation. The material when used according to the manufacturer's instructions was found to be effective for a period of 6 days. The effectiveness of tested disinfectant (Sterilex Ultra® ) could be attributed to the individual action of each of its constituents. The constituents were sodium bicarbonate, sodium percarbonate, alkyl benzylammonium chloride and tetra sodium ethylene diamine tetra acetic acid. Sodium bicarbonate possesses effervescent action while sodium percarbonate was an excellent detergent, bleaching agent with a strong fungicidal effect and stain removing and deodorizing capabilities. Alkyl benzyl dimethylammonium chloride, a medical disinfectant and tetra sodium ethylene diamine tetra acetic acid acted as a chelating agent.

An analysis of the results of this study showed that the use of purified water is mandatory to begin with. To continue maintaining the sterility of the dental unit waterlines and to complete the infection control measures adopted in the dental clinics, anti-retraction valves could be incorporated into the handpiece and a suitable disinfectant like Sterilex Ultra® was a must.

In the recent years, biofilm has been recognized as a primary source of contamination of water delivered by dental units. Biofilm formation is an excellent source for growth and multiplication of various types of biofilm originates from small number of bacteria present in the main water source to contaminate other areas. The contaminates adhere to luminal walls of the small bore tubing in the dental units, and over a period of weeks or months, become a dense accumulation of bioflm resulting in dense growth of microorganisms [7] . Microorganisms suspended in an aqueous medium become attached to an available surface and multiply, forming micro colonies. Dental unit waterline biofilm is a mixture of living bacteria, extracellular bacteria, carbohydrates and biological debris. Once established, bacteria are continuously released from the biofilm into water flowing through or standing in the tubing lumen. This results in gross contamination of the water supply, especially when the unit is not being operated and the fluid is stagnant.

Biofilm contamination of the dental unit water systems is a universal problem. Pseudomonas, Pasteurella, Achromobacter, Klebsiella, Serratia, Nocardia, Streptococcus, Micrococcus, Flavobacterium, Stapyloccus, Legionella, Alcaligenes, Bacillus, Acentobacter, Ochromobaterium Species'. Penicillum, Cladosporium, Alternaria and Scopulariopsis are some of the organisms identified in dental unit waterlines. The relationship between biofilm organisms is often symbiotic with one species providing key cofactors required by another. Some species communicate with each other in the biofilm community using a variety of compounds. Depending upon the environmental conditions, these chemical signals can initiate slime formation or order the break up biofilm.

Biofilm formation is reversible in the early stages if adhesion to the surface is not strong. The inner surfaces of the tubing are first conditioned by absorbed macromolecules and low molecular weight hydrophilic molecules from the water mains which enhances the efficacy of bacterial adhesion.

As water moves through the tubes of a working dental unit, the water flow is faster at the center of the lumen and more slowly away from the center as a result of friction with the walls. It is this part of the tubing where water is virtually stagnant, allows bacteria to colonize the internal surface. As the diameter of the waterline decreases, an increasingly larger surface area relative to volume becomes available for colonization allowing the bacteria to be in contact with the water line surface for a longer period of time.

Majority of the organisms removed from dental unit water system are Gram -ve bacteria. They synthesize powerful endotoxins which can cause mild inflammation to serious toxic shock. Majority of illness are contracted through mucosal tissue, eye and open tissue exposure, as well as by breathing and swallowing organisms.

Dental practitioners exposed to waterline aerosols should be aware about this exposure [9] .If slime encased bacteria biofilm becomes established inside our bodies. It is very difficult to detect and treat the infection. Unless it is prevented the sloughed biofilm will enter the mouth. lung and blood stream of patients and will enter the mouth, lung and blood stream of patient and will be cross infecting workers.

This contamination may lead to serious sinus problem and other type of infections. Two fatal cases of legionellosis due to dental water contamination have been reported in literature [9].

People with compromised immune systems are most susceptible to biofilms. Research shows that bacterial counts above 200 colony forming units/ml resulted in rapid colonization of hemo-dialysis units. The same is likely happen to dental unit also. Hence the safe drinking water act sets a standard for noncoliform bacteria in drinking and recreational water at 200 colony forming units/ml[10]. By comparison dental unit waterline contamination in untreated systems often exceeds 1000 colony forming units per ml. Until the emergence of opportunistic infections during the last several decades, many of the bacteria typically recovered from dental unit water system were thought to be harmless. Recent studies have proven that those who are undergoing multiple blood transfusions, organ transplant recipients, patients exposed to prolonged radiation or steroidal therapy and those suffering from chronic autoimmune disease and infected with HIV/AIDS are susceptible.

Retraction of disease factors is a known problem with dental equipment. Some units retract water into the tubing every time the water is stopped, pulling contaminants from the patient in to the water lines and releasing them later to other patients. This normally occurs because of the complex design of dental water chair equipment lines where bacteria can proliferate.

Most of the cleaners and disinfectants do not effectively remove biofilm because the biofilms carry a net +ve charge which results in repulsion or non­interaction of materials. A variety of commercial cleansing agents are available over the counter. Commonly used hypochlorite agent can damage dental equipment by causing corrosion or deterioration of parts and release of by products like trihalomethanes. Several microfiltration membrane filters are available to remove bacteria.

   Conclusion Top

From the results we can infer that most of the identified microorganisms comprise of Gram-ve Pseudomonas predominating up to 40% of the total isolates while Legionella was not found in any of the tested samples. Usage of disinfectant (Sterilex Ultra® ) was found to be very effective for a period of six days. To maintain the sterility of dental unit water lines it is essential to have a good water source, an effective disinfectant and the use of an anti-retraction valves.[Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5]

   References Top

1.Smith AJ, Hood J, Bagg J, BurkeFT. Water, water everywhere but not a drop to drink? Br Dent J 1999; 186:12-20.  Back to cited text no. 1    
2.Fayle SA, Pollard MA. Decontamination of dental unit water systems: a review of current recommendations. Br Dent J 1996; 181:369-372.  Back to cited text no. 2  [PUBMED]  
3.Shepherd PA, Shojaei MA, Elcazer PD. Stewart AV, Staat RH. Clearance of biofilms from dental unit water lines through the use of hydrogen peroxide ion-phase transfer catalysts. Quintessence Int 2001; 32:755-761.  Back to cited text no. 3    
4.Challacombe, Path, Fernandes. Detecting legionella pneumophilia in water systems. J Am DentAssocl995; 126:603-608.  Back to cited text no. 4    
5.Williams H, Paszko-Kolva C, Palmer C, Kelley J. Molecular techniques reveal high prevalence of legionella in dental units. J Am Dent Assoc 1996; 127:1188-1193.  Back to cited text no. 5    
6.Meiller T, DePaola L, Kelley J. Dental unit water lines: Biofilms, Disinfection and Recurrence. J Am Dent Assoc 1999; 130:65-71.  Back to cited text no. 6    
7.Shorman HA, Nabaa LA, Coulter WA, Pankhurst CL, Lynch E. Management of dental unit water lines. Dental Update 2002; 29:292-298.  Back to cited text no. 7    
8.Williams FJ, Johnston AM, Johnson B, Huntington MK, Mackenzie CD. Microbial contamination of dental unit water lines. Prevalence, intensity and microbiological characteristics. J Am Dent Assoc 1993; 124: 59-65.  Back to cited text no. 8    
9.Shannon E Mills. The dental unit water line controversy: defusing the myths, defining the solutions. J Am Dent Assoc 2000; 131: 1427-1440.  Back to cited text no. 9    
10.ADA Council on Scientific Affairs. Dental unit waiter lines: Approaching the year 2000. J Am DentAssoc 1999; 130:1653-1664.  Back to cited text no. 10    

Correspondence Address:
Vijay K Venkatesh
Department of Conservative Dentistry & Endodontics, Meenakshi Ammal Dental College & Hospital, Chennai
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-0707.42329

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

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