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JOURNAL OF ENDODONTICS
Copyright 9 1990 by The American Association of Endodontists

Printed in U.S.A.

VOL. 16, NO. 12, DECEMBER1990

In Vitro Bacterial Penetration of Coronally Unsealed

Endodontically Treated Teeth
Mahmoud Torabinejad, DMD, MS, Borasmy Ung, DDS, and James D. Kettering, PhD

In addition to dyes, radioisotopes have been used to study

microleakage in alloy, resins, temporary filling substances,
and root canal filling materials (4-9).
Although isotopes may be a good tool for comparing relative leakage, they cannot give a true picture of the leakage
which occurs clinically. This is because the ions used are
much smaller than dye molecules and they diffuse much
more rapidly than other small molecules (4).
Isotopes are indicators of ion exchange, diffusion, or metabolism within the tissues rather than indicators of true
leakage (10, 11).
Mortensen et al. (12) and Krakow et al. (13) have stated
that microorganism penetration might be more appropriate
than dye or isotope penetration for studying leakage in vivo.
Goldman et al. (14) have pointed out that bacteria performed
better than dye in testing for leakage of hydrophilic materials
and that dyes could give a false positive reading if their
molecules were small enough. Because air bubbles can prevent
dye leakage, the results of dye studies have been questioned
(15; Goldman et al., personal communication).
Because of inherent inadequacies in dye and radioisotope
studies, it appears that bacterial leakage studies can provide
more accurate information in clinical situations. We have
found no reports on the length of time that passes before the
entire obturated root canal is invaded by bacteria in obturated
root canals without coronal seals.
The purpose of this experiment was to determine the length
of time needed for bacteria to penetrate a standardized length
of obturated root canals which were intentionally exposed to
one of two species of microorganisms.

Forty-five root canals were cleaned, shaped, and

then obturated with gutta-percha and root canal
sealer, using a lateral condensation technique. The
coronal portions of the root filling materials were
placed in contact with Staphylococcus epidermidis
and Proteus vulgaris. The number of days required
for these bacteria to penetrate the entire root canals
was determined. Over 50% of the root canals were
completely contaminated after 19-day exposure to
S. epidermidis. Fifty percent of the root canals were
also totally contaminated when the coronal surfaces
of their fillings were exposed to P. vulgaris for 42
days.

Sealed root canals can be recontaminated under several circumstances: (a) if the patient has had endodontic treatment
but has delayed placement of permanent restorations; (b) if
the seal of the temporary filling material has broken down; or
(c) if filling materials and/or tooth structures have fractured
or been lost.
When these situations occur, the coronal portion of the
root canal system is exposed to oral flora. The question is
how quickly the entire root canal system becomes contaminated again, to the point that retreatment of the canal may
be necessary.
Swanson and Madison (1) evaluated the length of time that
the obturation material could be exposed to artificial saliva
before compromising the integrity of the seal. They exposed
the coronal portion of obturated root canals to artificial saliva
for various time periods, and then immersed them in Pelikan
ink for 48 h. They found that the dye penetrated from 79 to
85% of the root length in all exposed specimens. There was
no leakage in the control group which was not exposed to
artificial saliva, but was placed in contact with ink for 48 h.
The follow-up study by Madison et al. (2) showed that in
teeth exposed to artificial saliva for 7 days, the ink penetrated
between 33 and 80% of the root length, depending on the
type of sealer used.
The latest findings by Madison and Wilcox (3) evaluating
in vivo microleakage did not, however, confirm their in vitro
studies. They found that some of the positive controls (no
sealer) did not show microleakage, while some of the negative
controls (temporary not removed) showed dye penetration.

MATERIALS AND METHODS

To test bacterial penetration, a set-up similar to the one
used by Goldman et al. (14) and Williams and Goldman (16)
was used in this experiment.

Apparatus Set-up
By using a high-speed handpiece and a #2 round bur, a
small circular opening (about l mm in diameter) was made
through the cap of a 20-ml scintillation flask. A paper clip
was threaded through the opening and an alligator clip was
hung on the inside. The outside end was bent to stabilize the
alligator clip against the cap. The outside opening of the cap

566

Vol. 16, No. 12, December 1990

was resealed with acrylic material. The flasks, with the alligator clips attached to their caps, were steam sterilized.
After instrumentation and obturation of root canals, a 25m m length of latex tubing was placed over the coronal portion
of each tooth and the edges were sealed with epoxy resin
(Quick Gel, non-run super-glue; Loctite Corp., Cleveland,

OH).
The tubes, with the teeth attached, were sterilized in 5.25%
sodium hypochlorite for 15 rain (based on the results of a
pilot study) and then were rinsed with approximately 300 ml
of sterile water. The tubes were then fastened to the caps of
the previously sterilized scintillation vials.
Ten milliliters of sterile phenol red broth with 3% lactose
was added to the bottom of a flask, and the length of the
tubing was adjusted so that a m i n i m u m of 2 m m of the apical
part of each tooth was immersed in the solution (Fig. 1).

In Vitro Bacterial Penetration

567

Monitoring of the Samples

The results from a pilot study showed that viable P. vulgaris
were present 3 wk after incubation, while the S. epidermidis
organisms were viable for only 1 wk. Fresh overnight cultures
of organisms and sterile artificial saliva were added to the
tubes at 5-day intervals for S. epidermidis and at 5- to 10-day
intervals for P. vulgaris. When the bacterial culture was
replenished, the old culture was plated to confirm continued
viability o f the microorganisms.
The samples were monitored daily until the red indicator
solution at the bottom of the flask turned yellow. After color
change, a sample of the yellow medium was plated on blood
agar to assure that it contained the same type o f bacteria as
that placed in the tubing.

Preparation of Teeth
Bacterial Preparations
Two species of bacteria were used as contaminants for this
experiment; Proteus vulgaris, which is highly motile, and
Staphylococcus epidermidis, which is nonmotile. These were
grown overnight in 30 ml of trypticase soy broth (approximately 4.7 x l08 per ml o f / ' . vulgaris and 7.5 x 1 0 6 per ml
of S. epidermidis).
Two milliliters of the bacterial suspension and 0.7 ml of
sterile artificial saliva as specified by Swanson and Madison
(1) (1 mM CaC12, 3 mM NAH2PO4, 20 mM NaHCO3) were
placed into the tubing. Since both organisms are acid formers,
we expected the phenol red indicator solution to change to a
yellow color when the bacteria reached it (16, 17).

Forty-five maxillary incisors and cuspids with straight canals were used in this study. The teeth had been stored
previously in 10% formalin and were kept moist at all times
throughout the experiment. After initial radiographs, standard
access cavities were prepared, and the coronal portions of the
canals were enlarged with #2 to #4 Gates Glidden drills.
In order to obtain a standardized diameter, the apical
foramina of the teeth were enlarged and kept patent to a #40
file, using a step-back filing technique. Approximately 2 ml
of 5.25% NaOC1 were used between each file size, to remove
debris.
The prepared teeth were divided into experimental and
control groups.

Experimental Groups
The root canals of 33 teeth were obturated with guttapercha and Roth's sealer (Roth Drug Co., Chicago, IL) using
the lateral condensation technique. To obtain a standardized
length of filling, the coronal portion of the gutta-percha was
removed with hot pluggers until only l0 m m of the filling
material remained in the canal. To prevent bacteria from
penetrating the root surfaces, two layers of fingernail polish
were applied to the outside of the root except for 1 m m at the
apex.
GROUP 1
The coronal portions of the root canals of 16 teeth in this
group were placed in contact with 2 ml of P. vulgaris in
trypticase soy broth and 0.7 ml of sterile artificial saliva as
described above. The tubing was then suspended over the
phenol broth so that approximately 2 m m of the apex of the
tooth was immersed in it.
GROUP 2

FiG 1. Diagram of a tooth attached to a tube and suspended in the

flask by an alligator clip.

The coronal portions of the root canals of the rest of the

teeth in the experimental group (17 teeth) were exposed to 2
ml ofS. epidermidis and 0.7 ml of artificial saliva. The apices
of these teeth were also immersed in phenol red broth.

568

Torabinejad et al.

Joumal of Endodontics

Control Groups

TABLE 2. Rate of total recontamination of obturated root canals

exposed to S. epidermidis

To test the reliability of our results, the rest of the prepared

teeth were divided into two control groups.
GROUP 3
The root canal in each of the eight teeth that were used as
positive controls was filled with a single gutta-percha cone
without sealer, simulating poorly obturated root canals. Each
of the two species of bacteria were placed separately in the
tubing to contaminate four root canals (per microorganism),
as described in the experimental group.
GROUP 4
To verify that no contamination developed during the
experiments (negative controls), four instrumented root canals were filled with gutta-percha and sealer. After removing
the coronal portion of the filling material and leaving 10 m m
of it in the root canal, the coronal portions of the filling were
exposed to sterile artificial saliva applied as described above.

No. of
Samples

% of
Total

Cumulative
%

1
1
5
2
2
4
1
1

6
6
29
12
12
23
6
6

6
12
41
53
65
88
94
100

Total 17

100%

No. of Days
15
16
17
19
20
30
45
51

Average: 24.1 days

0.4 mm/day

t test of independent samples shows that the difference is

statistically significant (t = 4.68), with p < 0.01.
Except for one sample in the positive control group (group
3), the rest of the samples (seven of eight) caused a color
change in the phenol red medium after 1 to 4 days. The
culture medium did not change color in teeth whose coronal
segments were in contact with only sterile saliva (group 4)
throughout the experiment (over 90 days).

RESULTS
DISCUSSION
In group 1, contaminated with P. vulgaris, two of the
samples became positive after 2 days. One of these may have
been a sample from the positive control, whereas the other
one was found to have leakage through the latex tubing. These
two samples were discarded.
Table 1 shows the time it took P. vulgaris to reach the apex
through 10 m m of the filling material. It varied from 10 to
73 days. The average length of time for leakage was 48.6 days.
The time periods required for S. epidermidis to reach the
apex in group 2 are shown in Table 2. Compared with those
obtained in group 1, the results were more consistent, i.e.
most apical leakage occurred between 15 and 30 days, with a
range between 15 and 51 days. The average length of time for
total penetration was 24.1 days. A statistical analysis using a

TABLE 1. Rate of total recontamination of obturated root canals

exposed to P, vulgaris

No. of
Samples
2 discarded
1
1
1
2
2
2
1
1
1
1
1
Total 14

% of
Total
7
7
7
14.3
14.3
14.3
7
7
7
7
7

99.9%

Cumulative
%
7
14
21
36
50
64
71
79
86
93
100

No. of Days

10
29
31
39
42
57
63
64
66
68
73
Average: 48.6 days
0.2 mm/day

Most of the samples in the positive control group with

poorly filled canals showed leakage by 1 to 4 days, except for
one which did not show a change until the 22nd day. There
are two possible technical errors which could have caused the
latter; one is that this tooth may have been switched by
mistake with one of the experimental samples, the other is
that the space prepared may have been circular enough to
provide a very tight seal.
The results of the positive control group are a confirmation
of studies by Marshall and Massler (4), Evans and Simon (5),
and Skinner and Himel (17) who showed that sealers are
needed to improve the apical seal.
As none of the negative controls led to a color change in
the phenol red medium, it appears that our set-up did provide
a contamination-free chamber.
Over 85% of the teeth inoculated with P. vulgaris became
completely penetrated in 66 days, whereas most (88%) of
those inoculated with S. epidermidis were totally infected in
30 days, suggesting that motility may not be a factor in rate
of penetration to the apices.
We found a significant variability in the time it took the
bacteria to penetrate the entire root canal system. Similar
results were reported by Swanson and Madison (1) and Madison et al. (2) when they studied dye penetration in obturated
root canals. This might be due to the shape of the prepared
canal, type of sealer used, or the nature of the solution to
which the coronal portions of the root canal were exposed.
Goldman et al. (14) studied bacterial penetration in root
canals filled with poly-HEMA, a hydrophilic, plastic polymer.
They found no bacterial penetration after 42 days. The main
reasons for lack of bacterial penetration in their results could
be that poly-HEMA does not support bacterial growth as
reported by Kronman et al., (18) because the pores are substantially smaller than the bacteria, or that the polymers have

Vol. 16, No. 12, December 1990

the same adhesive properties as the acrylic nail polish which

was used to cover the external surfaces of the teeth.
Compared with clinical conditions, the model used in our
study was static, its media for bacterial growth was not totally
similar to saliva and for ease of management of experimental
conditions and interpretations of the data, its bacterial contents were purposely limited to only two species. Because of
these limitations and their possible effects on the results, in
vitro models simulating clinical conditions are needed to
investigate the rate of leakage in unsealed, obturated root
canals.

We gratefully acknowledge the assistance of Dr. Junichi Ryu and William

Keeler with the bacterial culture aspects of the experiment.
Dr. Torabinejad is director, Postgraduate Endodontics, Dr. Ung is a former
graduate student in endodontics, and Dr. Kettering is professor of microbiology,
Loma Linda University School of Medicine, Loma Linda, CA.

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In Vitro Bacterial Penetration

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