The disinfection of impression materials

by using microwave irradiation and

hydrogen peroxide
Yu-Ri Choi, PhD,a Kyoung-Nam Kim, DDS, PhD,b and
Kwang-Mahn Kim, DDS, PhDc
Yonsei University College of Dentistry, Seoul, Republic of Korea
Statement of problem. Microwave irradiation and immersion in solutions have been recommended for denture disinfection.
However, the effect of dry conditions and impression materials has not been completely evaluated.

Purpose. The purpose of this study was to evaluate the effectiveness of microwave irradiation and hydrogen peroxide for the
disinfection of dental impression materials.

Material and methods. Specimens (diameter 10 mm, thickness 2 mm) were made with polyvinyl siloxane. Experimental
groups were treated with hydrogen peroxide (group H), microwave irradiation (group M), and a combination of both
hydrogen peroxide and microwave irradiation (group MH) for 1 minute, 2 minutes, and 3 minutes. The control group was
untreated. The total sample size was 120. The specimens were divided into 2 groups, those exposed to Streptococcus mutans and
those exposed to Escherichia coli. The disinfection effect and physical properties (contact angle, compatibility with gypsum,
strain in compression, tear strength) were evaluated.

Results. All 3 groups (H, M, MH) were effective in reducing the number of colony forming units (CFU) per unit volume (mL)
for both S mutans and E coli compared with the control. The most significant reduction in the CFU/mL of both bacteria
was noted in the MH group and was used to compare either treatment alone (P<.05). No statistically significant difference
was noted between the control and treatment groups in terms of all of the physical properties tested (P>.05).

Conclusions. Microwave irradiation was identified as a useful disinfection method against S mutans and E coli, especially
when combined with H2O2, without adversely affecting the physical properties of dental impression materials. (J Prosthet
Dent 2014;112:981-987)

Clinical Implications
Microwave irradiation combined with hydrogen peroxide is suggested as
a useful method of disinfecting dental impression materials.

Dental impressions are often con-
taminated by exposure to saliva, blood,
or both.1,2 To minimize contamination,
impressions are washed with flowing
water to remove these fluids.3-5 How-
ever, this method cannot completely
remove bacteria and microorganisms;
impressions must be disinfected before
being sent to a dental laboratory.6,7
Chemical treatment (with, for example,
glutaraldehyde, sodium hypochlorite,
or povidone iodine) is the most com-
monly used method of disinfecting
dental impressions. However, glutaral-
dehyde induces inhalation toxicity and
eye irritation, sodium hypochlorite in-
duces the bleaching of dental materials,
and povidone iodine induces loss of
pigmentation.8,9
Microwave ovens are simple to use,
are low in cost, and can provide disin-
fection effect.10,11 The disinfection of
dentures with microwave irradiation
has been attempted11 and has been
found to be better than the use of so-
dium hypochlorite.12 When denture
base resin was microwaved at 650 W
for 2 minutes, a disinfection effect on
Candida albicans and Streptococcus mutans
bacteria was observed.13 When the
denture resin was microwaved at 650 W
for 5 minutes, a disinfection effect on
Staphylococcus aureus and Pseudomonas

a
Research Scientist, Department and Research Institute of Dental Biomaterials and Bioengineering, Yonsei University College of
Dentistry, and Brain Korea 21 PLUS Project.
b
Professor, Department and Research Institute of Dental Biomaterials and Bioengineering, Yonsei University College of Dentistry, and
Brain Korea 21 PLUS Project.
c
Professor, Department and Research Institute of Dental Biomaterials and Bioengineering, Yonsei University College of Dentistry, and
Brain Korea 21 PLUS Project.

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982
aeruginosa was also observed.9 However,
no research is available on the disin-
fection of polyvinyl siloxane dental im-
pressions when treated with microwave
irradiation.
Although microwave irradiation has
been suggested as a useful method
of disinfecting dental materials, in pre-
vious studies, dental materials have
typically been immersed in distilled
water during microwave irradiation14;
thus, in such cases, disinfection was
probably due to thermal effects rather
than microwave irradiation. Moreover,
immersion in water can alter the struc-
tural stability of dental impressions.15,16
In this study, the effectiveness of mi-
crowave irradiation on dry specimens
was investigated.
Hydrogen peroxide (H2O2) is a well-
known and commonly used disinfec-
tant. Its effects are known to be caused
by reactive oxygen molecules. Such ef-
fects will therefore be enhanced if
hydrogen peroxide is in the gaseous
phase. When microwave irradiation
and certain concentrations of H2O2
were applied to sewage sludge at the
same time, the formation of OH

caused by the microwave irradiation
splitting the H2O2 confirmed that this
combination destroyed bacterial cell
walls and therefore provided more
effective disinfection.17
In this study, the disinfection effect
and the effect of microwave irradiation
and hydrogen peroxide on the physical
properties of polyvinyl siloxane impres-
sions were evaluated for microwave
irradiation durations of 1, 2, and 3
minutes, with and without hydrogen
peroxide. The null hypothesis was that
there would be no difference between
experimental group and control group
dental impression materials, in terms of
disinfection effect.
MATERIAL AND METHODS
A household microwave oven was
used as the source of radiation (MR-
173W; LG). The frequency of this de-
vice was 2.45 GHz, and the output was
620 W. A 3% hydrogen peroxide solu-
tion (hydrogen peroxide; Songkwang)
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was used. The polyvinyl siloxane impres-
sion material investigated (Imprint II;
3M ESPE) is a Type 3 material according
to ISO 4823.18
Gram-positive Streptococcus mutans
(S mutans; ATCC 25175) and gram-
negative Escherichia coli (E coli; ATCC
87739) were acquired from the Korean
Culture Center of Microorganisms. S
mutans bacteria were cultured on brain-
heart infusion agar (Brain Heart Infu-
sion agar; Becton, Dickinson and Co)
at 37 C, and E coli bacteria were
cultured on nutrient agar (Nutrient
agar; Becton, Dickinson and Co) at
37 C. The microorganism-containing
solution was diluted to a concentra-
tion of 2  106 colony forming units
per milliliter (CFU/mL) with selective
media for S mutans and E coli.
The specimens were prepared by
placing a polytetrafluoroethylene mold
with a diameter of 10 mm and thickness
of 2 mm on a glass plate. The impression
material was placed inside the
mold and pressed with another glass
plate on top to produce disk-
shaped specimens (diameter 10 mm 
height 2 mm).
The specimens were placed in the
middle of a tissue culture net (Embed-
ding-cassette; Hyunil Lab-mate Co.) on
a glass dish with a diameter of 90 mm
for microwave treatment, as shown in
Figure 1. The specimens were placed
on the tissue culture net, and 100 mL
(2  105) of bacteria were seeded on
the specimens. The glass cell culture
1 Tissue culture net, with specimen on top, was placed at
center of sterile glass dish.
Volume 112 Issue 4
dish was covered with a lid and
placed in the middle of the microwave
oven. After seeding the bacteria on
the specimens, 20 mL of hydrogen
peroxide was placed on the glass dish,
and the specimens were positioned on
the dish. The specimens were allowed
to sit in air for 1, 2, and 3 minutes and
were denoted H1, H2, and H3,
respectively. The specimens were then
microwaved for 1, 2, and 3 minutes
and were denoted M1, M2, and M3,
respectively. The specimens containing
hydrogen peroxide were treated in the
same manner described previously.
The specimens did not contact the
hydrogen peroxide. The specimens
microwaved for 1, 2, and 3 minutes were
denoted MH1, MH2, and MH3
(Table I), respectively. In all trials, fresh
hydrogen peroxide was used. Specimens
without any treatment were used as the
control group.
After each treatment, the specimens
were covered with a polyethylene film
and incubated for 24 hours at 37 C.
They were then transferred to a 24-well
plate, and 1 mL of culture medium
was inserted in each well. The bacteria
were suspended by sonication in an
ultrasonic cleaner (Ultrasonic cleaner
SH-2100; Saehan Ultrasonic) for 1
minute. After 100 mL of the bacteria
was seeded on a solid agar plate for 24
hours, the CFU of the bacteria were
counted.
The contact angles of the control and
microwaved specimens were measured.
Choi et al
October 2014 983
Table I. Test groups submitted to different conditions control and the experimental groups for
each test condition (Tables II and III).
Exposure In post hoc analysis, the Tukey honestly
Time/Disinfection Group H Group M Group MH significant difference test was used to
Groups (H2O2) (Microwave) (Microwave D H2O2) determine differences.

0 min (control groups) HC MC MHC RESULTS

1 min H1 M1 MH1
2 min H2 M2 MH2 A significant difference was found
3 min H3 M3 MH3 between the disinfection effect for each
group of S mutans and E coli specimens
(P<.05). The groups that were treated
with hydrogen peroxide, microwave
Table II. Two-way ANOVA analysis of Streptococcus mutans CFU count results for irradiation, or both microwave irradia-
different disinfection methods and duration of each method
tion and hydrogen peroxide showed
Source df Mean Square F P significant differences among them-
selves (P<.05).
Group 2 177.8 17.92 <.05 In terms of S mutans groups,
Time 2 285.8 28.81 <.05 compared with that of the control
Group  Time 4 3.66 0.37 .829 groups, the CFU count of the H, M,
Error 36 9.92 - - and MH groups decreased, demon-
strating significant differences (P<.05).
CFU, colony-forming units.
In the case of the experimental group
(comparing the 1-, 2-, and 3-minute
Table III. Two-way ANOVA analysis of Escherichia coli CFU count results for groups), only the CFU count of the
different disinfection methods and duration of each method 3-minute group showed a significant
difference (P<.05). Comparing the con-
Source df Mean Square F P trol and experimental groups revealed
that the entire set of experimental
Group 2 246.6 20.12 <.05
groups showed significant differences
Time 2 29.4 2.39 <.05
(P<.05). Only the 3-minute specimens
Group  Time 4 2.86 0.23 .917 in the MH groups showed no CFU.
Error 36 12.25 - - In terms of the E coli groups,
compared with that of the control
CFU, colony-forming units.
groups, the CFU count of the H, M,
and MH groups decreased, demon-
The contact angle was measured by mm were prepared, as instructed in ISO strating significant differences (P<.05).
using a contact angle analyzer (Phoenix 4823.18 In the experimental group (comparing
300; Surface Electro Optics Co Ltd). The To measure tear strength, specimens the 1-, 2-, and 3-minute groups),
contact angle of the specimens was of each material were made with a Type only the CFU of the 3-minute group
measured 30 seconds after dropping C mold of ASTM D624-0019 and showed a significant difference (P<.05).
10 mL of distilled water on the surface of loaded in tension until failure at a rate Moreover, the 3-minute specimens
each specimen. of 500 mm/min with a universal testing in the M and MH groups showed
The specimens were prepared as machine. The tear strength was calcu- no CFU.
instructed in ISO 4823.18 After the Type lated as tear strength ¼ ultimate tensile The specimens that were treated
3 gypsum was poured, all group speci- strength (N)/specimen thickness (mm). with microwave irradiation and hy-
mens were separated, and the replica- Software (PASW Statistics 18.0; IBM drogen peroxide (group MH) exhibited
tion of 20-mm, 50-mm, and 75-mm lines Inc) was used to evaluate significant a better disinfection effect than the
was evaluated with a 50 image differences in the disinfection effect H groups and M groups (Figs. 2, 3).
analyzer (Hirox KH-1000; Hirox), as for all possible combinations of spec- Additionally, the disinfection effect
instructed in ISO 4823.18 To conduct imen groups with 2-way ANOVA against E coli was better than that
strain-in-compression tests, cylindrical (a¼.05) for the contact angle, strain- against S mutans. The contact angle
specimens of the control and micro- in-compression, and tear strength. measurement results are shown in
wave treatment groups with a diameter Two-way ANOVA was used to deter- Table IV. No significant difference
of 10 mm and a maximum height of 20 mine significant differences between the was found between the control group
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984 Volume 112 Issue 4

2 Results obtained for Streptococcus mutans test group. A, Control (nontreated). B, H3 (hydrogen treatment for 3 minutes).
C, M3 (microwave treatment for 3 minutes). D, MH3 (microwave þ hydrogen treatment for 3 minutes).

and the group treated with microwave
irradiation (P>.05). Both the control
group and microwave-treated groups
reproduced the 20-mm, 50-mm, and
75-mm line. The results for the strain-
in-compression test are shown in
Table IV. No significant difference was
found between the control group and
the microwave-treated groups (P>.05),
which showed a minimum value of
2.86% and a maximum of 3.26%.
The tear strength results are shown in
Table IV. Again, no significant differ-
ence was found between the control
group and the microwave-treated
groups (P>.05).
DISCUSSION
The results demonstrated a statisti-
cal difference between experimental
group and control group dental
impression material, in terms of disin-
fection effect. Therefore, the null hy-
pothesis was rejected.
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In dental practice, disinfection is
essential before and after all clinical
procedures. In particular, different
dental materials need different disin-
fection, depending on their materials
standard level.20-22 Chemical disinfec-
tion is generally used in dentistry;
however, chemicals should be used with
caution.8,9
Microwave ovens are widely used for
heating or defrosting frozen food items
by creating electromagnetic waves with
a frequency of 2.45 GHz. In a previous
study on disinfection with a microwave
oven, microwaves not only changed the
biologic state of bacteria and killed
them but also influenced the charge
distribution of the cell membrane
because of the electric field generated
and thus damaged the semi-
permeability of the cell membranes.
This effect influenced the function of
the Na-K pump and disabled the
cell membrane. Microwave irradiation
either alters or destroys the normal
metabolism of cells and therefore sup-
presses and prevents the growth of
bacteria, which has proven excellent for
disinfection.23-25
Although bacterial death is crucial
in disinfecting dental materials, the
properties of the material being steril-
ized should not change. Several studies
on the effect of microwaves in dentistry
have been performed, especially on
dentures, because patients can easily
use such a device in their own homes.
Such studies have indicated that sig-
nificant shrinkage occurs when liner
denture resin is dipped in sodium
perborate and treated with microwaves.
Significant shrinkage is also observed
when acrylic resin is dipped in distilled
water and treated with microwaves.26,27
Likewise, when dental materials are
subjected to microwave treatment, the
materials may be modified. However,
because these studies involved speci-
mens immersed in water during testing,
a rise in temperature is inevitable.
Choi et al
October 2014 985

3 Results obtained for Escherichia coli test group. A, Control (nontreated). B, H3 (hydrogen treatment for 3 minutes). C, M3
(microwave treatment for 3 minutes). D, MH3 (microwave þ hydrogen treatment for 3 minutes).

Table IV. Means and SDs of contact angle, strain-in-compression, and tear bacterial death, which is why hydrogen
strength peroxide is applied to improve the
Contact Strain-in-Compression Tear disinfection effect.28,29
Gram-negative and gram-positive
Group Angle (Degrees) (%) Strength (N/mm)
bacteria respond differently to various
C 57.36 1.12 3.08 0.13 4.25 0.36 disinfectants. Therefore, to represent
these 2 types of bacteria, S mutans and E
H1 58.16 2.58 2.98 0.24 4.27 0.22
coli were used in this study. S mutans, a
H2 57.59 1.23 3.21 0.31 4.24 0.56
gram-negative bacillus, has a thick
H3 58.70 2.34 2.99 0.24 3.88 0.42 peptidoglycan layer that protects it
M1 59.16 2.88 3.01 0.14 3.93 0.33 from changes in the external environ-
M2 57.59 2.48 3.07 0.17 3.55 0.42 ment and also contains teichoic acid.
M3 57.70 2.94 3.16 0.11 3.86 0.35 The covalent bond between teichoic
MH1 57.58 2.36 2.98 0.14 4.46 0.42
acid and the peptidoglycan layer en-
hances the physical strength of the
MH2 57.86 2.51 3.12 0.25 3.47 0.17
bacteria’s cell membrane.30-32 E coli, a
MH3 56.55 2.49 2.92 0.06 3.80 0.29 gram-negative bacillus, has lipoprotein
C, nontreated; H, treated with hydrogen peroxide; M, treated with microwave; MH, treated with and a membrane around its peptido-
microwave þ hydrogen peroxide. glycan layer, and the cell membrane
contains a layer composed of lipo-
In contrast, in this study, micro- destroy the cell membranes of bacteria, polysaccharide. The external membrane
waves were applied to impression ma- simultaneously producing oxygen free of the cell has a structure similar to that
terials in the dry condition. Microwaves radicals. In other words, oxygen free of other biologic membranes. The
also split hydrogen peroxide into ion radicals destroy the RNA and DNA peptidoglycan layer is thin (approxi-
particles to form hydroxides (OH
) and within the cell and thereby induce mately a tenth of that of other
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986
gram-negative species), rarely has pep-
tide bonds, and does not contain tei-
choic acid. Therefore, the cell membrane
of the gram-negative species is weak.33-
35
In this study, the treatment
completely eradicated the E coli bacteria.
No significant difference was ob-
served in the contact angle of the
control group and microwave-treated
groups. A decrease in the contact
angle after microwave treatment would
have been a better outcome clinically.
However, the fact that the contact
angle did not increase indicates that
the microwave treatment would not
cause any problem when applied clini-
cally. All of the experimental groups,
as well as the control group, were
able to replicate the 20-mm, 50-mm,
and 75-mm lines, therefore demon-
strating that the microwave treatment
does not influence compatibility with
gypsum.
Strain-in-compression is an index
that evaluates the physical properties of
impression materials. In ISO 4823, the
strain-in-compression of Type 3 im-
pression materials is regulated to be
between 2% and 20%. In this study, the
strain-in-compression of the control
group, 3.08%, satisfied the ISO 4823
standard,18 and the groups that were
treated with microwaves met the stan-
dard as well. In addition, no significant
difference was found between the con-
trol group and the experimental groups
(P>.05).
Polyvinyl siloxane should resist
tearing when tensile stresses are applied
during impression removal and cast
separation from the set impression. In
this study, no significant difference was
found in tear strength between the
control group and the experimental
groups (P>.05).
This study focused on the disin-
fection of dry dental impressions
composed of polyvinyl siloxane by mi-
crowave radiation. The physical prop-
erties of polyvinyl siloxane impressions
were investigated. Unlike in this study,
previous authors have performed
similar experiments with specimens
immersed in distilled water during mi-
crowave treating, and thereby inducing
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a change in the physical properties of
the impression material.36
The authors identified no previous
study on the disinfection of dental
polyvinyl siloxane impressions with mi-
crowaves. In this study, microwave
treatment was performed under dry
conditions for 1, 2, and 3 minutes to
achieve effective disinfection without
the influence of heat. When microwave
treatment was applied, a significant
reduction occurred in the number of
bacteria, and no adverse effect was
noted on the physical properties of
the impression material. Moreover, the
disinfection effect against bacteria was
much stronger when hydrogen peroxide
was used in the microwave treatment.
However, further studies on the appli-
cation of microwave treatment to a
variety of bacteria and the use of a tray
model will be useful.
CONCLUSION
When polyvinyl siloxane impressions
were subjected to microwave treatment,
a disinfection effect was observed, with
no modification of the physical prop-
erties of the impressions. Therefore,
a microwave oven can serve as an
effective disinfection tool for dental
impression materials.
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Corresponding author:
Dr Kwang-Mahn Kim
Dental Materials and Bioengineering
Yonsei University College of Dentistry
50 Yonsei-ro, Seoul
REPUBLIC OF KOREA
E-mail: kmkim@yuhs.ac
Copyright ª 2014 by the Editorial Council for
The Journal of Prosthetic Dentistry.