Infection Control

Bacterial contamination ofthe water supply in newly installed dental units

Henry N, Williams*/Alicia Johnson**/Jaqueline 1. Kel]ey***/Marcie L. Baer****/
Tracy S. King*****/Barbara Mitchell******/John F. Hasler*******

Bacterial contamination ofthe water stipply of newly installed dental units was investigated.
Water .samples were collected from water suppiy Unes to the dental operatories prior to
connection ofthe dental units. Within hours following connection, and continuing for up
to the 6 months ofthe study water samples were obtained from the air-water syringe ofthe
units. The samples were .•serially diiuted lO-foid and piated on cuiture media for qttantitative
analysis. The formation of bacterial biofiim in tiie dentai water stipply tubing was tnonitored
by scanning electron microscopy The results of these studies revealed that tiie buiiding's
water supply to the dental units was contaminated prior to connection to the units. The
water stipply from the air-water syringe was therefore contatninated as well. The number of
contaminant bacteria in the dentai unit water suppiy increased for several weeks and then
stabilized. The lumen of the dental tubing became progressively contaminated with bacterial
biofiim, which sttbsequently became the primar}- reservoirfor maintenance ofthe contamination
ofthe dentai unit water supply. (Quintessence Int 1995:26:331-337.)

Introduction addressed by several investigators.'"' However, the

It is weil established that the water supply of dental
units is grossly contaminated with bacteria. Some
potential sources of such contamination have been
* Associate Professor, Department ofMicrobioiogy. Utiiversity of
MarN'land at Baltimore. Baltimore College of Dental Surgery
Dental School. Baltimore. Maryland.
** Graduate Student, Department of Microbiology. University of
Maryland at Baltitnore, Baltimore College of Dental Surgery
Dental School. Baltimore. Mar>'land.
' " Research Assistant. Department of Microbiology. University of
Marj'land at Baltimore, Baltimore College of Dental Surgery
Dental School. Baltinrore, Maryland
***' Graduate Research Assistant, Department of Microbiology.
University of Maryland at Baltimore. Baltimore College of
Dental Surgery Dental Scliool. Baltimore. Maryland.
*•-*• Private Practice, Baltimore. Maryland.
***-'"' Private Practice. Fort Bennitig, Georgia.
- " * " * Associate Dean, Clinicai and Hospital Affairs, University of
Maryland at Baltimore, Baltimore College of Dental Surger>'
Dental School. Baltimore. Marylatid.
Reprint requests: Dr Henry N. Williams. Associate Professor, Depart-
ment of Microbiology. University of Maryland at Baltimore, Baltimore
College of Dental SLirgery Dental School. 666 West Baltimore Street,
Baltimore Maryland 21201.
original source and time of initial contatnination in
new units have not been reported.
Some ofthe earlier studies on the dental unit water
supply (DUWS) have reported the recovery of oral
bacterial species. However, more recent reports in
which the bacteria were identified revealed that the
predominant organisms recovered are nearly all aer-
obic or facultative Gram-negative bacilli of the type
conimonly found in water distribution systems.'' The
predominance of Gram-negative organisms suggests
that the bacteria contaminating the DUWS originate in
the community water source that supplies the dental
units. This is also suggested by resuits of a study in
which typical levels of bacterial contamination were
found in dental units that were used by students to
learn basic dental practices but had not been used with
patients.'
The DUWS has been reported to harbor as many as
1 million bacteria per mL of sample;'' some of these
bacteria have been implicated in opportunistic infec-
tions.' This is at least 1,000-fold greater than the
number recovered from potable water from fountains

Q u i ntesaonoo .Intornatic Number 5/1995 331

 Infection Control
and sink faucets.'"' There is increasing agreement
among denial authorities that this represents an
unacceptable bacterial load to introduce into the
mouths of patients. This is supported by the fact that
bacteria in the DUWS have been reported to have been
a source of infection in immunocompromised pa-
tients.' Clearly, the aggressive pursuit ofthe study of aii
aspects of the contamination of the water supply of
dental units is warranted. This approach represents the
best prognosis for finding a solution to this problem.
During the complete renovation of all the dental
clinics at the Baltimore College of Dental Surgery' and
Dental School, the authors had the opportunity to
investigate the quality ofthe DUWS in newly installed
dental units. The results ofa preliminary investigation
in which water samples were collected from the
air-water (A/W) syringe and handpiece 1 month after
the installation ofthe first units revealed high levels of
bacterial contamination. On the basis of these results, a
more comprehensive study was designed to investigate
the source and initation of contamination of the
DUWS of newiy installed dental units in two clinics.
The study monitored bacterial contamination of water
fi'cm the inhouse water lines prior to connection to the
new dentai units and from the A/W syringe for 6
months foilowing installation of the new equipment.
Knowledge of the source and time of initiation of
contamination can enable clinicians to better under-
stand the nature ofthe contamination in their dental
units and guide researchers and manufacturers in
developing strategies to overcome the problem.
Method and materials
Prior to the connection of the new dental units to
newly installed building water lines in two clinics, the
lines were treated overnight for disinfection with a
bleach solution. This treatment is a standard practice
to reduce the number of microorganisms to a level
considered safe in accordance with public health
standards. Following treatment, the bleach was Hushed
from the lines. Water samples were collected from the
water supply lines within 24 hours after flushing and at
predetermined intervals up to the time of connection
to the new dental units. To collect these samples, the
water line shut-off valve, which extended into the
cut-out opening in the floor or wall, was turned on and
the water coming from the pipe was collected into
sterile 15 -mL centrifuge tubes. Samples collected prior
to installation of dental tinits were designated precon-
332
neetion samples. Following connection of the utitts.
water samples ( postconnection samples ) were collect-
ed from the dental A/W syringe for up to a 6-month
period. Collected samples were taken immediately to
the microbiology laboratory, where they were serially
diluted 10-fold and spread plated onto dilute peptone
medium.*'^ The plates were incubated for 4 weeks at
25°C. The number of colony-forming units (CFU)
were counted and the mean number for duplicate
plates was calculated. Although the general methods
used in collection and culturing of samples were as
described above, the specific protocols and method-
ology for each clinic are presented separately.
Chnic !
The disinfectant treatment of the water lines was
completed on February 21, 1991. The first sample was
collected on February 22 (day I). Samples were
collected daily through February 26 (day 5). The
remaining preconnection samples were usually collect-
ed at 2- to 4-day intervals during the next 24 days
before the new dental units were installed. The initial
postconnection sample was collected from the A/W
syringe within hours of installation ofthe new dentai
units on March 19 (day 26). Sampling ofthe water
delivered from the A/W syringe continued over the
next 35 days; the last sample was collected on April 23.
Two sampling protocols were used in this clinic. One
was designed to sample a single dental unit (unit 2)
continuously through the entire study period from the
first preconnection sample to the last postconnection
sample. The identical protocol was used for a second
unit (unit 11 ). with the exception that sampling ofthe
preconnection water line was initiated only 9 days
before connection. Postconnection sampling of the
unit continued for the duration of the study as
described for unit 2. Sampling the same unit offered
the advantage of monitoring the water quality from a
single source throughout the study period. However,
the disadvantage of this approach was that each time
the unit was activated and water was removed for
sampling, the environment ofthe water in the lines was
disturbed. To compensate for this, a second protocol
was used: a different unit was sampled on each
occasion for the first time without any prior disturb-
ance (with a single exception, unit 8). The sampling
schedule was similar, but not identical, to that de-
scribed for unit 2.
Clinic 2
Following overnight disinfection and flushing ofthe
Quintessence Internationai Volume 26. Niimhcr 5/1995
 Infection Control
water system, on July 9, 1992. 12 operatories were Table I Numbers of bacterial CFU (x \0\ except
randomly selected for study. On this date, samples where noted by *) per mL from clinic I
were taken from the in-house water lines ( preconnec-
tion samples) of six operatories. A sample was also
collected from the A/W syringe (postconnection Sample
samples) of one unit that had just been installed. day' Unit 2 Unit 11 Other units No.
Connection of remaining units was on July 10 (except 1* 15.0' 0.0'(1)
unit 8, which was connected on July 29). Sampling
from the A/W syringe of these units commenced on 2 0.0" 25.0'(3)
this date and continued daily through July 17. Subse- 3 15.0' 0.1 (4)
quently, samples were collected every 2 to 4 days 4 30.0' 0.0'(5)
through the end of July and on August 3, 10, 31, and 5 0.0' 260.0" (6)
September 4. A final sample was taken on Februaiy 1, 11 > 2.0
1993. The units were put in use for patient treatment 14 0.2 230.0'(9)
on July 29, 1992, Typically, all samples (5 mL) were 16 12.0 13.0 (10)
taken from the A/W syringe at the beginning ofthe day 17 0.2 15.0
and prior to use ofthe unit. Three sink faucets in the 26* 260,0 150.0 250.0 (19)
clinic were also routinely sampled in the same manner 640.0 (20)
to monitor the contamination level of water flowing 360.0 (8)
through the building's plumbing system. All samples 460,0 (17)
were processed and plated as described earlier. 28 11.0 260.0
30 980.0 510,0 420.0 (8)
In one dental unit from clinic 2, the lumen of tubing 33 520.0 310.0
supplying water to the A/W syringe was examined for 35 28.0 25.0
the colonization of bacteria hy scanning electron 40 800.0
microscopy (SEM). At 1 week, 2 weeks, 1 month, and 44 190.0 200-0 120.0 (8)
6 months postconnection ofthe unit, a section ( 10 cm) 47 220.0 160.0
ofthe water tubing was excised and cut into tour equal 50 560.0 140.0
pieces. Three of the pieces were placed in 2% 54 200.0 22.0
glutaraldehyde for examination by SEM. The fourth 61 260.0 680.0
piece was sliced in half lengthwise and scraped with a
sterile scalpel. The biofilm recovered on the blade was * Actual value.
suspended in water. The suspension was diluted and t After disinfection,
plated as described previously to determine the viabil- Í Preconnection samples obtained directly from the in-house
water lines prior to connection to dental units,
ity ofthe biofilm bacteria within the tube. The A/W
tj Posiconneclion samples obtained Trom the air-water syringe
syringe was replaced on the remaining tubing ofthe following connection of dental units to the in-house water
unit and normal usage was continued. system.
Tubing samples previously fixed in 2% glutaraldehyde
for electron microscopic examination were removed
from the fixative, cut in half lengthwise, and washed in
phosphate buffer solution for 10 to 15 minutes to
remove the fixative. The specimens were dehydrated
(for 10 minutes each) through a series of alcohol Results
washes (30%. 50%, 70%, 90%, and 100%) with a final Following treatment ofthe in-house water lines ofthe
treatment in hexamethyldisilazane (for 10 minutes) dental clinics with a disinfectant, the numbers of
and air dried overnight. The specimens were mounted bacterial contaminants were immediately reduced to
on SFM aluminum mounts and coated with gold- undetectable or low levels. However, this effect was
palladium in a Hummer VI Sputtering System (Tech- short lived, as the level of contamination increased
tiics) for 4 minutes. Examination by SEM (JSM-T200 rapidly within days (Tables 1 and 2). At the time of
Scanning Microscope. JEOL) was done at varying connection ofthe new dental units to the water lines,
magnifications and appropriate photographs were the water supply had already become contaminated
taken. with large numbers of bacterial colony-forming units.

Quintessence International Volume is. Number 5/1995 333

Í
 Infection Control

Table 2 Numbers of bacterial CFU (X IÛ\ except where noted by *) per mL from in-house water lines and newly
installed dental units from clinic 2

Sample Unit Number

day
4 6 8 12 14 17 22 25 28 30 34 39
1+ -í
-
— _
-
0,7 > 1,0* > o,r 0,2' 0,2' - > 3.0 - > 3,0
2^ 2,6 > 3,0 0.2 > 3.0 > 3.0 2,4 > 3,0 > 3.0 0,4 > 3.0
3 ;> 3,0 0.7 - > 3,0 0,6 1.8 > 3,0 > 3,0 > 3.0 > 3.0 > 3,0 > 3.0
4 ;> 3.0 0.2 - > 3.0 1.3 > 3.0 > 3.0 > 3.0 > 3,0 > 3,0 > 3,0 > 3,0
5 ;> 3.0 0,7 - > 3,0 > 3,0 > 3,0 > 3,0 > 3.0 > 3,0 > 3,0 > 3,0 > 3.0
6 ;> 3,0 > 3,0 - > 3.0 > 3,0 > 3,0 > 3,0 > 3.0 > 3,0 > 3,0 > 3.0 > 3,0
7 ;> 3,0 > 3,0 - > 3,0 > 3,0 > 3,0 > 3,0 > 3.0 > 3.0 > 3.0 > 3.0 > 3,0
8 ;> 3,0 > 3,0 - > 3.0 > 3,0 > 3,0 > 3,0 > 3,0 > 3,0 > 3,0 > 3,0 > 3,0
9 ;> 3.0 > 3,0 - > 3,0 > 3,0 > 3,0 > 3,0 > 3.0 > 3.0 > 3.0 > 3,0 > 3,0
12 82,0 47,0 - 47,0 95,0 150,0 94,0 100,0 200,0 270,0 260,0 240.0
14 42,0 13,0 - 25,0 48,0 45.0 22,0 66,0 22,0 92,0 87,0 90,0
16 33,0 110,0 - 20,0 120,0 58,0 47.0 36,0 55,0 130,0 110,0 76.0
19 26,0 170,0 - 54,0 130.0 68.0 26.0 18,0 62,0 75,0 38,0 50,0
23 96,0 74,0 1,3 38.0 35,0 45,0 14,0 48,0 46,0 74.0 72.0 80.0
26 46,0 220.0 7,6 44,0 120.0 110.0 48,0 46,0 42.0 150,0 180,0 60,0
33 28,0 92,0 19.0 37.0 37,0 78,0 43,0 42,0 9.5 74,0 54.0 44,0
40 58,0 22.0 91,0 82.0 62.0 150.0 54,0 48,0 42.0 62,0 41,0 38,0
54 36,0 120,0 52,0 100,0 46,0 37,0 52,0 31.0 66,0 66,0 34,0 56,0
58 24,0 65,0 54,0 15,0 36.0 42,0 59,0 41,0 66.0 34.0 23,0 57,0
206 8,2 12,0 30,0 300,0 30,0 10,0 30,0 110,0 13,0 30,0 11.0 12.0

t Preconnection samples,
% Sample not taken,
S AU samples from day 2 through day 206 were pnstconnection samples.

The first water samples from the new dental units,
collected within hours of installation, were observed in
all cases to be grossly contaminated.
In clitiic 1. following the disinfection treatment, the
level of contamination of water samples obtained
directly from the water lines remained at undetectable
to low levels for the first 2 weeks. However, the
number had increased to 2.000 CFU/mL in unit 2 and
to 150,000 CFU/mL in unit 11 by day 17. when the
last sample was taken before connection was made to
the new dental units (Table 1). The pattern and level of
contamination of units 2 and 11, which were disturbed
by continuous samphng, were remarkably similar to
those ofthe other units, which were typically sampled
only once and therefore remained undisturbed (until
the units were placed in routine use}. In units 2 and 11.
the counts appeared to stabilize somewhat within 4
days postconnection, although unusually high and low
counts were sporadically recovered.
In clinic 2, the number of colony-forming units
recovered from water from the plumbing system had
exceeded 1,000 CFU/mL for most units by 1 day
following disinfection. The counts had exceeded
300,000 CFU/mL in the majority of units by 2 days
postconnection. Such high counts so soon following
disinfection exceeded expectations (based on the
previous results of ciinic 1 ) and the dilutions made of
the samples. This caused the number of colony-
forming units on the plates ofthe greatest dilution to be
too many to count, or greater than 300, The results
from these samples are expressed in Table 2 as > 3,0
(x 10') CFU/mL, Peak counts were recovered for the
majority of units between days 12 and 26. At the end of
54 days in clinic 2, the level of contamination in most
units had stabilized, typically to within a range of 3,1 to

334 Quintessence Int

 Infection Control

6.6 X 10'^ CFL)/mL, At the 6-month sampling interval, Figs la to 1c Sequentially developing biofilm within the
tlie range for most units was 1,0 to 3,0 « 10' CFU/mL- déniai tubing (Original magnification X 1,600 )
By contrast, the mean number of bacterial colony-
forming units recovered from three sink faucets prior
to routine use ofthe clinics was 2,6 >* 10^ CFU/mL,
Following the opening of the chnics and start of
routine use, the counts from the faucets decreased to a
mean of 5.0 >i lO^* CFU/mL,
Examination by SEM of the lumen walls of the
tubing from a dental unit in clinic 2 revealed the
presence of attached individual bacterial cells and
microcolonies in samples obtained in the first weeks
following installation of the units and of developed
biofilm in samples collected 6 months postconnection
(Figs la to lc). The mean numbers of bacterial colonv-
forming units recovered from biofilm scrapings re- Fig la Biofilm at 2 weeks.
moved from the lumen ofthe tubing from one unit at 1
week, 2 weeks, 1 month, and 6 months were 4,1 x 10%
1,1 X 10\ 3.6 X 1O^ and 3.6 x lO" CFU/mL, respec-
tively.

Discussion
Bacterial contatttination of water delivered by the
dental A/W syringe and handpiece is a persistent and
universal problem for dental units connected to
community water supply systems. In this study, the
contamination of newly installed dental units was
investigated. The results revealed that the in-house
water lines supplying water to the dental uitits became
heavily contaminated, with microorganisms even after
bleach treatment, before connection to the new units.
The water going into the units following connection
immediately introduced hundreds of thousands of Fig 1b Biotilm at 1 month.
bacteria throughout the dental unit water delivery
system. Some of these bacteria undoubtedly attached
to the luminal walls ofthe plastic tubing within the
dental units, ultimately establishing a biofilm.
The development ofa biofilm on the lumen walls
over a period of 6 months was observed by SEM.
Durhig that time, the biofilm developed from a few
individual cells and microcolonies scattered along the
lumen walls after I or 2 weeks to a mature biofilm with
a dense population of coalescing microcolonies. Sev-
eral reports have implicated bacterial biofilms as the
primary source of microbiai contaminants in the
DLTWS,''^''"" This has been confirmed by recent
studies in our laboratory (unpublished data). Once
formed, these biofilms shed bacteria into the water
occupying the tubing. Unless treated with special
agents for its removal, the biofilm will persist as long as
it is exposed to an aqueous environment. For this Fig lc Biof im at 6 monttis.

SHyilUti llllöfliütlonal Number 5/1995 335

Infection Control
reason, it has been neariy impossible to eliminate or
reduce bacterial contamination of the DUWS in
conventional dental units. The larger numbers of
bacteria recovered fromthedental units in the days and
weeks following connection to the water lines in this
study were a direct result ofthe biofiim formation and
tiie shedding of biotllm oi^anisms into the water
supply. The difference between the relatively low
number of colony-forming units recovered from the
sink faucets and the large numbers from the A/W
syringe of dental units is tlirther evidence that the
source of contamination of the DUWS is within the
dentai units. The dental units contained built-in
antiretraction valves to prevent the backflow of con-
taminated oral lluid from the A/W syringe or hand-
piece during use.
Although not a long-term remedy to the develop-
ment of bioHim In dental unit tubing, the numbers of
bacteria transported by the building's water supply into
newly installed units can be nearly eiiminated by
proper disinfection and flushing ofthe water supply
lines just before the connection to dental units is made.
Tliis was observed in chnic 1. in which the bacterial
counts remained low for at least 2 weeks. The reason
for the results in clinic 2, in which liigh levels of
contamination were observed within a day following
disinfection, are not known. It may indicate that
improper disinfection procedures were used. How-
ever, preconnection treatment will not eliminate the
eventual contamination ofthe tubing in the dental unit
with bacterial biofiim and the perpetual problem of
contamination of the DUWS. Until there is some
means of preventing the development of biofiim in the
tubing of dental units, there is little hope of effectively
reducing or eh mina ting the associated bacteria! con-
tamination of dental units that receive nonsterile water.
The higher numbers recovered in this study from the
sink faucets, particuiariy at the beginning ofthe study
prior to use (more than 200,000 CFU/mL), was sur-
prising, because previously water from these sources
typically contained fewer than 100 CFU/mL and
rarely in excess of 500 CFU/mL of heterotrophic
bacteria. A representative sample ofthe water from the
dental units was tested and confirmed by a commercial
laboratory to be free (fewer than 1 per 100 mL) of
detectable coliform bacteria and, by this standard, to
be safe for human consumption. However, evidence
suggests that other bacteria in the DUWS, such as the
pseudomonades, may be a source of infection, espe-
cially in immunocompromised individuals.
Dental professionals should not remain comfortable
336
with the use of a water supply that contains thousands
and millions of contaminant bacteria. To ensure the
delivery of water that is essentially ^''^'^ '^^ bacteria,
some manufacturers of dental equipment have devel-
oped dental units thai are designed to have a sterile
water supply. In combination with a regular chemical
flush, this provides the units with the capacity to
deliver water that is essentially free of detectible
bacteria. Several filtration apparatuses have also been
described. Dentists are encouraged to consider use of
such systems. In the interim, further study on the
effects of flushing are warranted to resolve the flush
time controversy and establish uniform recommenda-
tions.
Summary
The results of this first such study on new dental units
revealed that the source of inital bacterial contamina-
tion of the water delivered by newly installed new
dental units was the public water supply. Further, the
contamination occurred immediately when the incom-
ing water supply, which carries large numbers of
colony-forming units, was introduced into the new
dental units. Individual bacterial cells adhered to the
lumen walls ofthe tubing, where subsequent growth
resulted in formation of a biofiim. This biofiim became
the primary source and sustaining agent for the
bacterial contamination ofthe DUWS. Within days or
weeks following connection to the building's water
lines, the level of contamination in the new units was
typical ofthat observed in used dental units.
Recognizing this, dental clinicians are encouraged
to fiush the water lines of new dental units for at least 2
minutes, just as recommended for used units, at the
start of each business day and between patients.
However, fiushing, which is currently the best availa-
ble and most practicai procedure to reduce micro-
organisms, should not be considered an acceptable
solution to the problem of DUWS contamination.
Even after flushing, high numbers (more than 1,000
CFU/mL) of bacteria often remain. Also, the optimal
duration of fiushing is somewhat conti"oversial, based
on widely disparate results that have ranged from 2
minutes' to 20 minutes.' To provide water that
contains few, if any, potential pathogens, dentists are
encouraged to use equipment that provides water of
similar quality to that which comes from water taps,
typically fewer than 500 CFU/mL.
Quintessence International Voliirpfj •iiimliBX'"''1O3::
 Infection Control

Acknowledgments Prosthetic
The aiilhors express appréciât ion lo Ms Judy Pennington for assistance in
the preparation ofttiis manuscript. Ms Eleanor B, V^de for tiie eiectron
tnicroscopic work, and Mr Vernon Leciincr for leehnical assislance.
Rehabilitation
KctUi F.
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8. MakiJS. L.aCrtjis SJ, Hopkins BS, StaleyJT. Recovery and diversity
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C|b

Contents include:
Psytholügi<;al Considerations; Pationi Assessment^
Inipi-essiori aiit! Facial Materials; Impression Teeh-
niques; Ear PTO.'ÍI iiesps; Nasal Prostlicscs; Orliital
Pfostheses; Partial Prostheses; Color Mati;liing;
Alternative Method;, of I'^biation and Ri'tciition;
Direct Adhesive Fixation Lsinj; Aillit-sivfs aitd
Removers; Tissue Conditioinn'i; Killing the
Piii>dieiis; Os.scoitt1.egrated Implonls^, liiiniakes;
Pinlili'iiid.; Glossary;, Skin Grafts, Fliips, and
Tissue [v\pansion: and Anatomy.

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QuintgAënce Intemaiionäl Volume '¿ä. Number 5/1995