Clearance of biofilms from dental unit waterlines through

the use of hydroperoxide ion-phase transfer catalysts

Paul A, Shepherd, DMD, MSVMaria A. Shojaei, DMD, MS^/Paul D, Eleazer, DDS, MSV
Arthur Van Stewart, DMD, PhDVRobert H. Staat, PhD, MS^

Objectives: The purpose of this research was to demonstrate the effectiveness of hydroperoxide
ion-phase transfer catalyst (HPI-PTC) cleaners and disinfectants for maintaining dental unit waterlines
free of planktonic organisms. Method and materials: Water samples were taken trom 117 sites, which
included a variety ot dental units and samples from the sink faucets ot most operatories. Samples were
plated on appropriate bactériologie media and incubated. The presence or absence oi biofilms was con-
firmed by scanning electron microscopy. Twenty-twc cf the dental units were retrctitted with independent
water systems; the cleaning procedure invclved an cvernight applicaticn otan HPI-PTC cleaner followed
by a 2-minute water rinse. Results: Water frcm both the air-water syringe and the high-speed handpiece
lines from all untreated units contained at least 6x10^ cclcny-forming units per milliliter of pianktonic or
froe-floating bacteria; the average was 1.4x10= CFU/mL. An initial 5% scluticn ot HPI-PTC successfully
cleared the lines of any apparent biofilm when applied for 3 ccnsecutive days. Thereafter, once weekly use
of the cleaner maintained the dental unit water supplies free cf significant numbers of planktonic organ-
isms. Conclusion: Routine weekly use of an HPI-PTC cleaner contrclled dental unit waterline biofilm and
reduced, with minimum effort, the microbial contamination level ct water used for patient treatment tc less
than 200 CFU/mL. fOti/n(essence int 2001:32:755-761)

Key words: biofilm, dental unit waterline, environmental bacteria, hydroperoxide ion cleaner, oral
streptccocci, phase transfer catalyst

t has been recognized for some time that dental unit

CLINICAL RELEVANCE: The American Dental Asso-
ciation has proposed a goal of no more than 200 colony-
forming units per milliliter of water in dental water supply
lines. A cieaner ccntaining the hydroperoxide ion coupled
wifh phase transfer catalysts was able to maintain bacter-
ial counts well below this level.
'Resident, Endodontics Program. School of Dentistry. University of
Louisville, Louisville. Kentucky.
=Resrdenl, Departmenl ot EnOodonlics, School of Derlistry, Case Western
Reserve University, Cleveland, Ohio.
^Professor and Chairman, Depanment of Endodontics and Pulp Biology,
Sctiool of Dentistry, University ot Alabama al Birmingham, Birmingham,
Alatiama.
•Professor, Department of Orthodonlic, Pedodontic and Geriatric Dentistry,
School of Dentistry. University of Louisville, Louisvilie. Kentucky.
^Professor and Director cf the Sterilizer Monitoring Program, Department of
Moiecular, Celluiar and Craniofacial Biology, Sctiool ot Dentistry,
University of Louisville, Louisville, Kentucky.
Reprint requests: Dr Robert H. Staat, Professor and Director of the
Sterilizer Monitoring Program, Department of fulclecular. Cellular and
Craniofaoial Biology, Schcoi of Dentistry. University of Lcuisvilie, Louisviile,
Kentucky 40292. E-mail: rhstaad ©gwise.louisviile.edij
Portions ol this artwle were presented at the 1997, 1998, and 1999 annual
meetings of the American Association for Dental Research/International
Association for Dental Research and as a table clinic at the 1997 annual
meeting ot the Amerrcan Dental Association.
I waterlines (DUWLs) usually harbor significant
numbers of bacteria. In 1995, the American Dental
Association (ADA) proposed a goal; By tbe year 2000,
all water coming from DUWLs should contain no
more than 200 colony-forming units (CFU) per 1 mL
of water.' Tbis level is similar to that found in munici-
pal water supplies, wbich bave guidelines for allow-
able beterotropbic bacterial loadings of 100 to 500
CFU/mL.2
The major source of tbe bacteria contaminating the
water coming from DUWLs is a biofilm formed by
coalescing microcolonies of bacteria that adbere and
tben develop on tbe inner walls of tbe DUWL. Once
tbe biofilm is sufficiently establisbed, bacteria are con-
tinually sloughed from the biofilm surface as plank-
tonic (free-living) organisms into the flow of water.
Dental unit waterlines are susceptible to biofilm for-
mation because they are made from a variety of plas-
tics; tbe common cbaracteristics are tbat the plastic
surfaces are bydrophobic and the plastic is not antag-
onistic to bacterial growtb,' Tbe hydropbobicity can
promote nonspecific bacterial attachment to tbe
DUWL surfaces.'' Tbe initial number of bacteria
required to establisb a biofilm can be very low;

Quintessence internationai 755

 • Shepherd et al
increases in the biofílm mass are due primarily to bac-
terial replication. Growth of biofilm bacteria in
DUWLs is slow because of lower (room) temperatures
and nutrient limitation,'
Tbe organistns colonizing water supplies are gener-
ally nonpatbogenic environmental bacteria,^ altbough
patbogenic and/or opportunistic forms sucb as Le-
gionella^ Pseudomonas,^ and Mycobacterium^ species
bave been isolated from waterlines in bealtb care facil-
ities. Tbe opportunistic organisms may pose a threat
to immunocompromised individuals. Viruses specifíc
for tbe buman bost are not found in biofilms, because
tbey require living bost cells to replicate.
Tbe strengtb of attacbment of bacterial biofilms to
tbe interior surfaces of waterlines is sufficient to witb-
stand tbe sbear forces developed by water flow through
the lines.'* Consequently, a water purge of DUWL lines
bas little effect on ridding tbe lines of biofilm, altbougb
tbe number of planktonic bacteria may be reduced
sligbtly. Tbe reduction in DUWL planktonic bacteria
does not approacb tbe levels needed to meet tbe ADA'S
goals."-^^ In addition, tbere is virtually no residual chlo-
rine from tbe municipal water treatment in the DUWLs
to interfere with bacterial growtb.'^
Because Vess et aP demonstrated tbat bacteria
bound in biofílms attacbed to polyvinyl cbioride plas-
tic pipes were significantly more resistant to tbe effects
of disinfectants, it was felt tbat removing the biofilm
from DUWLs was preferable to killing tbe bacteria
witbin tbe film. In addition, if the biofilm bactena
were killed but not removed, tbe matrix would remain
and could provide a suitable structure for rapid recol-
onization of tbe DUWL.
Examination of the literature revealed a proprietary
product tbat is used in tbe food industry as an effec-
tive surface cleaner of biologic debris."*'' Tbe cleaner
is based on tbe powerful nucleopbilic bydroperoxide
ion (HPI) in conjunction witb pbase transfer catalysts
(PTC) and activators (Sterilex Ultra Dental Water Line
Cleaner, Sterilex). Tbe enhanced nucleopbilic activity
is ascribed to an alpba effect tbat imparts a high den-
sity of negative charge on the terminal oxygen of the
peroxide ion.'^ The net effect is tbe hydrolysis of bio-
logic molecules containing ester linkages such as lipids
and the amide linkages of proteins.
A major reason most proposed cleaners and disin-
fectants do not effectively remove biofilms is that both
the cleaner or disinfectant and the biofilm carry a net
negative charge, which results in repulsion or nonin-
teraction of the materials. The pbase transfer catalyst
in HPI-PTC eliminates this problem and is able to
penetrate the biofilm. Once inside the biofilm, the
hydroperoxide ion is released, where it hydrolyzes and
lifts tbe contaminating material away from the surface.
Tbe cleaner also has disinfecting properties.'''•'^•"
756
The level of toxicity for humans is low, as rep'^''''^°
in proprietary supporting documentation sul • ^°
tbe US Food and Drtig Administration and i ^^
Environmental Protection Agency for app. '^te
product registrations. Tbe present study évalua. • tbe
clinical effectiveness of the HPI-PTC cleaner for both
the initial removal of biofilms from DUWLs and for
subsequent maintenance of the lines.
METHOD AND MATERIALS
Sampiing sites
Samples from DUWLs were collected from nine oper-
atories within the University of Louisville Dental
School clinics and from 108 private practice operato-
ries located in both the Louisville metropolitan area
and other locations within the continental United
States. Tbe individual units all bad been in service for
at least 1 year. Some bad been used continually for
more than 20 years.
Water coilection
Samples were collected in sterile specimen cups from
tbe bigh-speed, air-water syringe, and, if present, tbe
dental assistant's cart air-water syringe waterlines. Tbe
lines were purged for 2 minutes prior to sampling only
if the unit had not been in service tbat day. Tap water
samples were also taken from most locations. A mini-
mum of 10 mL was obtained for eacb water sample.
All samples were received in tbe laboratory witbin 24
bours of collection, sent via surface or air courier,
depending on the location.
Microbioiogicai examination
Once received in the laboratory, samples were immedi-
ately processed by plating either a 10"' or 10"^ dilution
on the following media: R2A agar for total het-
erotrophic CFU/mL,'* Mltis-Salivarius (M-S) agar for
selective enumeration of oral streptococci,'^ and
DGVP Legionella Selective Agar for Legionella species
isolation (Becton-Dickinson Microbiology Systems).^"
The initial samples plated on R2A agar were incubated
at either 23''C to 26°C or 37°C for a minimum of 7
days. Those incubated at 23''C to 26°C produced about
a tbird more growth from the same water samples;
consequently, the ^I'C incubation set of R2A plates
was discontinued. The selective plates for streptococci
and Legionella were incubated at 37''C.
Unique colonies were selectively isolated from the
R2A agar plates and were given preliminary identifica-
tions using the RPI system of microbial characterization
Volume 32. Number 10. 200i

(bioMerieux Vitek). The colonies growing on tbe M-S
agar plates were identified based on colonial morpb-
ology, as seen tbrough a dissecting microscope," Initial
screening of colonies growing on tbe selective Le-
gionella agar was also perfonned with the dissecting
microscope.^"
Cleaning regimen for dental unit waterlines
A total of 24 dental units were used for the cleaning
trials. At the time of initial sampling, all units were
coupled directly to tbe municipal water supply, and,
tbus, tbere was no direct access to the lines.
Therefore, each unit was equipped with an indepen-
dent water supply system (A-dec). Some were fitted
witb a two-way valve so tbat either the independent
water supply or tbe municipal water supply could be
used.
The HPI-PTC dental waterline cleaner was pre-
pared freshly as eitber a 5% or 7% solution by adding
tbe powder to tap water and then shaking the bottle.
On occasion, a small amount of undissolved material
settled to the bottom of the bottle, but it had no dele-
terious effects on the cleaning process. The freshly
prepared cleaner was tben introduced into tbe
DUWL, Red dental disclosing dye is part of tbe for-
mulation of the cleaner, so that it is easy to deter-
mine when the entire Une has been filled witb
cleaner.
The HPI-PTC remained in tbe lines ovemigbt and
was then rinsed out with at least 6 volumes of water.
Tests with cleaner- and disinfectant-detecting Indicon
swabs (Sterilex) indicated that there was less than fOO
ppm of HPf-PTC (10 ppm quaternary ammonium
compound} in the lines following the rinses. There
have been no reports of anyone experiencing a bitter
taste, which is indicative of residual cleaner, following
adequate rinsing, A 2-minute flush yielded at least a
10-volume flush for all lines tested, Aiter initial testing,
all lines were maintained with weekly treatments of
5% HPI-PTC,
The procedure took approximately 3 to 5 minutes
at the close of the day to complete, and it took
another 3 to 5 minutes to rinse the lines the following
morning.
Scanning eiectron microscopy
Approximately 1-cm lengths of DUWL tubing were
cut from existing units before and after treatment with
HPI-PTC. Tbe dried tubing was cut to expose the
interior surfaces, anchored to scanning electron
microscope (SEM) studs, and the surfaces were gold
coated. A Phillips 300 SEM was used to view the
specimens.
Quintessence International
Shepherd et al •
TABLE 1 Pretreatment recovery of planktonic
bacteria (CFU/mL) (rom dental unit waterlines at
different locations
Average recovered
Location of denial unit No, of units pretreatmenf
Private practice offices 108 1,44X10=
University of Louisviile 9 1,01X10=
Totai 117 1,41X10=
(mean)
•Data are expressed as CFU/mL detected on R2A medium incubated
aefobicaiiy at 23°C lo J6°C,
RESULTS
Of the 117 sites tested, the total bacteria recovered on
R2A agar ranged from 0 to more than 1X10« CFU/
mL; the average was 1,4x10' CFU/mL. Four sites of
117 met tbe ADA's goal of less than 200 CFU/mL per
mL, and three of these had 0 CFU/mL, Three of the
four sites were already using HFI-PTC on a weekly
hasis; one dentist used a daily flush of chlorhexidine-
based mouthrinse. No untreated DUWLs met tbe
ADA'S goal. Table 1 presents a summary of tbe quanti-
tative microbiologie data for tbe initial sampling of
DUWLs,
The types of bacteria recovered on the R2A agar
were typical of the environmental bacteria associated
witb water supplies and were predominately gram-
negative rods (Table 2), The only readily identifiable
bacteria with pathogenic potential for immunocompe-
tent individuals were opportunistic Pseudomonas
species. Examination of tfie different waterlines within
each operatory indicated that the composition of the
microbial flora was relatively uniform among tbe dif-
ferent lines and that the level of contamination was
relatively constant.
An unexpected finding was that approximately 80%
of the DUWLs tested harbored streptococci typical of
tbose found in the oral cavity. The average count for
streptococci recovered on M-S agar was 1x10^ CFU/
mL. Table 3 lists the predominant streptococcal types,
based on descriptions of colonial morphology. There
was an absence of culturable levels of Legionella
species from any DUWL sample. Analysis of the tap
water in each operatory revealed that most contained
less than 100 CFU/mL, although counts as high as
2x10'' have been detected in tap water from municipal
lines surveyed in this study. The source of these piank-
tonic bacteria was not determined, althougb it is sus-
pected tbat tbere is a plumbing fixture problem.
757
 • Shepiierd el al

lb). Biofilm debris released after initial treattiien* ran

TABLE 2 Major planktonic bacterial types
recovered from dental unit wateriines on R2A
appeared as small particles in the water as '" ¡es
medium prior to cleaning were flushed; however, on occasion intact sei .of
biofilm were sloughed from the DUWL (Fig 2)
Cell morphology Genus Evaluation of the cleaner and the recomí, ded
G ram-negative, rod Actinobacler procedures was then extended to 14 private practice
G ram-negative, rod Alcaligines operatories that had heen retrofitted with independent
Gram-negative, rod Flavobaclerium water supplies. The office staff were instructed on the
G ram-negative, rod Pseudomonas use of the HPI-PTC cleaner and a 6-week supply was
G ram-negative, rod Sphingomonas left with each office. Water samples were gathered hy
G ram-negative, rod Xantfiomonas the University of Louisville laboratory staff on a
Gram-positive, rod Bacilius weekly basis. Results of this study (Table 5) suggested
Gram-positive, coccus Streptococcus
that the one-time initial treatment did not always ren-
der the DUWLs free of biofilm. The noncompliant
lines were treated for three consecutive days by the
dental office staff, which brought most DUWLs to
TABLE 3 Predominant streptococci* isolated as
acceptable levels.
planktonic bacteria from dental unit wateriines There were three dentai units that appeared to be
resistant to the initial cleaning procedure; however, the
Species Typical location in the mouth dental staff was questioned about the product use, and
S sanguis Dentai piaque an inventory of the product was taken, it was con-
S mutans/sobrinus Dental plaque cluded that the DUWLs had not been treated in accor-
S intermedlus Dental plaque: mucosal tissues dance with the instructions. Once the lines were treated
S mitis Dental plaque: mucosal tissues for 3 consecutive days, they were well within the ADA's
S saiivarius Tongue: saiiva goals. No evidence oí biofilm re-formation in any
•These microorganisms are considered to be normal oral microflora tor DUWL that was treated consistently on a weekly
most people.
schedule was found over the period of this study.

TABLE 4 Recovery of planktonic bacteria
(CFU/mL) from University of Louisville dental unit
wateriines before and after cleaning'
Prior tc t D post- 7 D post-
HPI-PTC No. of units cleaning cleaning cieaning
7.0% 0-tO 0-ao
5.0% 0-tO 0-100
'Hydropercxide tor-phase transfer catalyst (HPI-PTC} Ireatment took
plaoe overnight and was followed by a 2 minute water flusli in the
morning.
Disassembly and scrubbing of the tap aeration devices
often reduced the bacterial count, and different taps
within the same office were not equally contaminated.
The first attempts to clean DUWLs were made in
the institutional setting of the University of Louisville
Dental School, This allowed for close supervision of
the procedures. Table 4 presents before and after data
regarding treatment of the DUWLs with HPI-PTC,
Scanning electron microscopic examination of sec-
tions cut from the wateriines revealed a dramatic dif-
ference foiiowing the cleaning procedures (Figs la and
DISCUSSION
Dental unit waterline biofilm is a mixture oí hving
bacteria, extracellular carbohydrates, and biologic
debris that adheres to the surfaces of plastic tubing.
The biofilm is conceptually similar to dental plaque on
the surfaces of teeth. The predominant planktonic
bacteria sloughed from the DUWL biofilms are typical
of the environmental bacteria encountered in normal
municipal water systems.^ This was confirmed by com-
paring the general bacterial population obtained from
the DUWLs with those isolated from the tap water
samples. This strongly suggests that the DUWL biofilm
originates with the supply water.
The fact that oral streptococci were isolated from
80% of the DUWLs indicates that contamination from
patient-derived bacteria can occur from the functional
end of the line also, Bagga et aP' presented data to
indicate that oral bacteria are sucked back into the
high-speed handpiece when there is no check valve in
the water retraction device. The majority of the dental
units tested in this study were equipped with antire-
traction valves. The reason for the regression of oral
bacteria into these wateriines is not clear. It must be
assumed that the oral streptococci were established in
the biofilm, because fhey were never detected in the

758 Volume 32, Number 10, 2001

 Shepherd et al •

Fig 18 Scanning electron micrograph demonstrating Ihe typical

microbJal biofilm formed on the inner walls of unlreaied dental imii
waterline tubing Ihai hau been in service for approximately 10
years. (Original magnificalion x 1,000.)
Fig l b Scanning electron micrograph of dental unit waterline
tubing taken from the same dental unit used for Fig la, foiiowing
treatment with hydroperoxide ton-phase transfer catalyst oieaner
The smooth surface is the plastic surface of tho tubing (Original
magnification X10,000, )

Fig 2 Intact piece of dental unit waterline biofilm

that was recovered from the rinse wafer following
overnight cieaning of the dental unit wateriine with
the hydroperoxide ion-phase transfer oatalyst
cleaner. The piece measured approximately 17
om in length.

TABLE 5 Recovery of planktonic bacteria (CFU/mL) from private practice dental unit
waterlines foilowing prescribed cleaning treatment*

Clinic

Test period 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Week 1 0 0 3000 6000 0 0 2900 32000 0 1700 0 0 17000 0
Week 2 0 0 0 0 0 0 0 0 0 0 200 350 14000 16000
Week 3 0 0 0 0 0 0 3000 0 0 0 0 0 15000t 300001
Week 4 0 0 0 0 0 0 2800' 15000t 0 4000t 0 0 0 0
Week 5 0 0 0 0 0 0 0 200 0 0 0 0 0 0
Week 6 0 0 0 0 0 0 —5 0 0

•All clinic personnel were instructed ¡n the use of the cleaner pnorto week 1, Those with detectable counts for the veek 1 s m pies were instructed to use the
cleaner for 3 consecutive days followed by 1 weekly treatment.
'Additional reinforcement of cleaning procedures was given to the clinic staff by University of Louisvllte personnel,
^Dashes indicafe waterlines were nof fested during that week.

Quintessence International 759

 Shepherd el ai
lines after cleaning with HPi-i . .tamma-
tion was strictly transient, coming .'.'.¡m mi; previous
patient, the oral bacteria would have been readily
detected regardless of the cleaning procedures.
No isolates of Legionelia were detected in this
study, either because tbey were not present or because
tbe metbods used were not sufficiently sensitive to
detect very iow levels.^-" The samples were not con-
centrated, nor were molecular detection tecbniques
employed. An alternative explanation is that the
Legionelia, if present, had formed somnicells, which
are cells that are still viable but cannot be cultured on
routine media." Walker et aF' recently reported simi-
lar results on the lack of Legioneila isolates from
DUWLs.
Reduction of tbe planktonic bacteria in DUWLs to
iess tban 200 CFU/mL by tbe year 2000 is a stated
goal of the ADA.' Numerous approaches have been
suggested, including disinfection of tbe lines with
hypocblorite-containing products (bleacb)^^ or otber
disinfectants, inciuding glutaraldebydes and iodo-
pbores. None of these alternate disinfection
approaches has formal approval for use in DUWLs. In
addition, most disinfectants have significant draw-
backs, including damage to the equipment and the
potential for residual toxicity to the patient. Further-
more, disinfectants do not readily remove the biofilm
matrix from plastic surfaces; consequently tbe matrix is
available to trap and protect newly introduced bacter-
ial contamination. Antibacterial moutbrinses bave
been used to reduce tbe level of planktonic bacteria
and bave been successful, aitbougb tbe time to reacb
satisfactory levels is a matter of weeks." Submicron fii-
ters will adequately remove bacteria from DUWLs, but
fail to remove bacterial by-products and can clog
quickly wben placed in heavily contaminated lines.
It is our position that the best approacb to control-
ling tbe release of planktonic bacteria is the removal
of tbe biofiim. Scanning electron micrographs of
DUWLs treated witb HPI-PTC readily sbow tbat tbe
biofilm is removed from the plastic surfaces. Tbe
microbiologie data confirm the lack of significant
numbers of planktonic bacteria in tbe treated DUWLs.
Independent studies by botb A-dec and tbe Sterilex
Corp of tbe effects of the HPI-PTC on typical dental
unit parts demonstrated no significant deleterious
effects on or loss of material from the parts. Parts were
made of different metals, plastics, and rubbers. Some
discoioration of macbincd brass surfaces was noted.
The parts were exposed to a scries of different applica-
tions of the HPI-PTC solutions for 29-day periods (E.
deLaubenfels, personal communication, June, 1999).
Application of the HPI-PTC to DUWL was
straightforward in the defined setting of tbe dental
scbool with laboratory personnel tending to tbe
760
details. However, wben tbe cleaner was supr''' '"'^
private offices, the reliability of applicatio -,
office staff came into question. The private ^]^^
offices tbat did not meet the ADA's goals afte ^^""^^
treatment were asked to repeat the treatme lor 3
consecutive days. One group was still not in compli-
ance with the guidelines, and further questioning sug-
gested that the lines had yet to be cleaned. The lines
were treated under University of Louisville staff super-
vision and quickly came into compliance. Cooperation
of office personnel appears to be key factor in main-
taining DUWLs.
An overriding question is wbether DUWL biofilms
pose a bealtb risk to tbe dental patient.^' Tbere is
anecdotal evidence tbat seems to suggest tbat some ill-
ness has been linked to tbe bacteria in DUWL.'
Dentists bave a significantly elevated antibody titer to
Legionella compared to the general population,^^'^"
and there is a strong probability that at least one den-
tist has contracted Legionella through his practice.' In
addition, several opportunistic bacteria routinely iso-
lated from various dental water supplies can cause
serious disease in immunocompromised individuals. It
is appropriate for healtb care facilities to reduce
planktonic organisms in DUWLs to at least tbe levels
proposed by tbe ADA and to use sterile water for sur-
gical procedures. Tbe data in tbis paper indicate tbat
tbe typical dental practice can meet tbe goal of less
tban 200 CFU/mL in all DUWLs with a minimum of
time expenditure by the dental staff.
A commercial form of HPI-PTC (Sterilex Ultra
Dental Unit Water Line Cleaner, Sterilex) was issued a
510K approval by tbe FDA in 1999 for use as a
DUWL cleaner. On July 17, 2000, the ADA Council on
Scientific Affairs granted a Seal of Acceptance for
Sterilex Ultra "as being safe and effective for cleaning
deposits and controlling aerobic, mesophilic, bet-
erotropbic bacterial contamination in DUWL when
used as directed."
CONCLUSION
1. Essentially all dental unit waterlines harbor bacteria
tbat develop into a biofilm, and significant numbers
of bacteria are continually released from the biofilm
into the water flow passing througb the line.
2. The majority of bacteria are typical environmental
microbes, aitbougb, oral streptococci were isolated
from the majority of units tested.
3. Dental unit waterlines can be effectively cleared of
biofilms by application of bydroperoxide ions cou-
pled to phase transfer catalysts. Weekly ap|:i[|ca-
tions are sufficient to keep tbe lines witbir he
ADA'S goal of less tban 200 CFU/mL.
Voiume 32. Number 10.

4. Tbe performance of tbe dental practice staff must
be monitored to ensure that routine weekly clean-
ing procedures are carried out to prevent reestab-
lishment of tbe biofilm.
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