International Endodontic journal ( 1 9 9 6 ) 2 9 .
1 2 5 - 1 3 0
In-vitro antibacterial susceptibility of bacteria taken from
infected root dentine to a mixture of ciprofloxacin, metronidazole
and minocycline
E. HOSHINO*t, N. KURIHARA-ANDO**, I. SATO*?, H. UEMATSim,
M. SATO*t, K. KOTA** & M. IWAKU**
*Cariology Research Unit, fDepartment of Oral Microbiology and ^Department of Operative Dentistry and Endodontics.
Niicjata University School of Dentistry, Niigata, japan
Summary
The aim of this study was to clarify the antibacterial
effect of a mixture of ciprofloxacin, metronidazole and
minocycline, with and without the addition of
rifampicin, on bacteria taken from infected dentine of
root canal walls. The eflicacy was also determined
against bacteria of carious dentine and infected pulps
which may the precursory bacteria of infected root
dentine. This efficacy was estimated in vitro by
measuring bacterial recovery on BHI-blood agar
plates in the presence or absence of the drug combina-
tion. Bacteria ranging in number from 10^ to 10''
occurred in samples of infected root dentine (27 cases).
However, none was recovered from the samples in the
presence of the drug combination at concentrations of
25 |xg ml~^ each. The respective drug alone (10, 25, 50
and 75 (xg m l ' ) substantially decreased the bacterial
recovery, but could not kill all the bacteria. Bacteria
taken from carious dentine (25 cases) and infected pulps
(12 cases) were also sensitive to the drug combination.
These results may indicate that the bactericidal efficacy
of the drug combination is sufficiently potent to eradi-
cate bacteria from the infected dentine of root canals.
Keywords: antibacterial drugs, ciprofloxacin, endodon-
tics, metronidazole, minocycline
Introduction
One of the main causes of periapical pathosis is bacterial
infection of the periradicular tissues. Bacteria remaining
Correspondence: Dr Hoshino Etsuro. Cariology Research Unit.
Department of Oral Microbiology. Niigata University School of
Dentistry. Gakkocho-dori 2. Niigata 9 5 1 . Japan.
© 1996 Blackwell Science Ltd
in the root dentine or in the periradicular tissues should
be eliminated in order to improve the prognosis, and this
should be achieved without damaging the tissues. The
application of antibacterial drugs may represent one
method of eradicating bacteria in root canal treatment.
It has been found that obligate anaerobes from the
overwhelming majority of the isolates from carious
lesions (Hoshino 1985) and infected root dentine (Ando
& Hoshino 1990), and from non-exposed pulpal tissue
where bacteria have invaded through dentinal tubules
(Hoshino et al 1992). Metronidazole has a wide
spectrum of bactericidal action against oral obligate
anaerobes (Ingham et al 1975), and also against
isolates from infected necrotic pulps (Sundquvist 1976).
More than 99% of the bacteria isolated from carious
lesions of permanent teeth were not recovered in the
presence of 10 jxg ml-i of metronidazole (Hoshino et al.
1988). Similar bactericidal efficacy can be expected
against bacteria in pulpal and periradicular lesions, and
in infected root dentine. However, metronidazole, even
at concentrations of 100 [jug ml^i, could not kill all the
bacteria from carious lesions in vitro (Hoshino et al
1988), indictiting that some additional drugs may be
necessary to sterilize the lesions. It has been found that a
mixture of antibacterial drugs, i.e. ciprofloxacin.
metronidazole and minocycline (each at a concentration
of 100 )xg ml""i), can sterilize carious lesions, necrotic
pulps and infected root dentine of deciduous teeth (Sato
et al 1992). Only a small amount of the drug mixture
was required to sterilize lesions in situ (Sato et al 1992),
and such an amount did not cause any pathological
changes when injected into connective tissue of mice
and dogs (Hoshino et al, unpublished data) or when
placed on to human pulpal tissue (Ayukawa 1994).
The sensitivity of bacteria to drugs is usually deter-
mined as the minimum inhibitory concentration (MIC),
125
126 E. Hoshino et al.
but many species of bacteria exist in oral lesions and in
the normal oralfiora,so it is impossible to determine the
MIC values for every bacterial species. In order to deter-
mine whether a mixture of drugs can kill all the bacteria
in such populations, the MIC method may not he
suitable. Methods of estimating antibacterial efficacy
against mixed bacteria, such as oral fiorae, have not yet
been estabfished. In previous papers (Hoshino et al,
1988, Sato et al 1992), stepwise concentrations of the
drug combination were tested, and it was found that no
bacteria were recovered at a drug concentration of
100 |xg ml^i each. Anaerobic conditions should he used
to estimate the efficacy against obligate anaerobes.
Bearing these limitations in mind, the present study
the in vitro antibacterial efficacy of the drug combination
(i.e. ciprofioxacin, metronidazole, minocycline and
rifampicin) was determined against the hacteria of
infected root dentine, infected pulps and periapical
lesions under strictly anaerobic conditions.
Materials and methods
Samples
Infected dentine from root canal walls was obtained
from a total of 27 freshly extracted human teeth
diagnosed clinically as 'hopeless' teeth because of their
advanced caries. Immediately after extraction, the root
apex and the open carious coronal cavity were sealed
with blue inlay wax under a gentle stream of N2
containing 5% CO2. The tooth surfaces were wiped for a
few seconds with gauze which had been immersed in
70% ethanol solution. After being notched with a sterile
diamond disc along the long axis of the tooth to facilitate
splitting, the tooth was immediately transferred into an
anaerobic glove box (Model AZ-Hard, Hirasawa, Tokyo,
Japan) containing 80% N2,10% H2 and 10% CO2. While
in the box, the teeth were split with sterile forceps.
Samples were taken from lesions on the split surfaces
using sterile excavators or smafi round burs at low speed
(< 120 rpm; Edwardsson 1974).
Carious dentine was obtained directly from 25 patients.
The occlusal or proximal carious lesions did not extend to
the pulp. Samples for in vitro experiments (25 samples)
and in vivo experiments (10 samples), respectively, were
taken as described previously (Hoshino et ah 1988,
1989a).
Fresh pulpal samples were obtained from 14 human
teeth that had been diagnosed and extirpated clinically
as acute pulpitis (4 cases), chronic ulcerative pulpitis
(6 cases) and chronic pulpitis (2 cases). Intact pulps
(2 cases), extirpated for prosthodontic treatment, were
used as controls. Each of the pulps was removed with a
sterile barbed broach, transported in a tightly capped
vialfifiedwith anaerohic gases (80% N2, 10% CO2 and
10% H2) and transferred to the anaerobic glove box
within a few minutes. While in the box, the samples
were suspended in sterile pre-reduced 40niM potassium
phosphate (pH 7.0).
Infected periapical tissue was obtained from
amputated tooth roots at the time of apicoectomy, as
described previously (Kiryu et al, 1994).
Bacterial recovery
Each of the samples was weighed, suspended in sterile
pre-reduced 40 mM potassium phosphate (pH 7.0) and
dispersed with a motor-driven homogenizer (Tissue-
Tearor, Biospec Products, Bartlesvifie, OK, USA) and/or
a glass homogenizer in the buffer solution. The dispersed
samples were inoculated with an automatic spiral plater
(Model D, Spiral System Instruments, Inc., Bethesda,
MD, USA) on to the surfaces of brain heart infusion-
yeast extract-blood (sheep) agar plates (BHI-blood agar;
Holdemann et al 1977). Aliquots of 0.1 ml of each
sample were also spread over BHI-hlood agar plates
containing either a mixture of ciprofioxacin, metronida-
zole, minocycline (3Mix) and rifampicin (4Mix), or one
of the respective drugs. The plates were incuhated in the
anaerobic glove box at 37°C for 10 days. Colony-
forming units were counted, and to ensure aseptic proce-
dures, sterile buffer was also treated in the homogenizer,
diluted, and spread over the blood agar plates and
cultured. Plates, media and bulTer solution were kept in
the anaerobic glove box for at least 24 h prior to use.
Statistieal analysis
The data were analysed using the paired Student's t-test.
Results
More than 10^ bacteria mg^i sample (range, lO^-lO^;
median, 10*) were recovered from samples of infected
root dentine (Tables 1-3). However, the addition of
10 (xg nil"i metronidazole to BHI-hlood agar plates
significantly decreased (paired t-test; P<0.0001) the
hacterial recovery (Table 1), indicating that most of the
bacteria were sensitive to metronidazole. The bacterial
recovery was also substantially reduced when the
samples were cultured with concentrated metronidazole
(100 |jLg ml-i, data not shown), or one of the other
> i996BlackweIlScienceLtd,/ nflJ. 29, 125-130
 Mixed drugs for dentine sterilization 127

Table 1 Bactericidal eflicacy of metronidazole (MN) against bacteria of Table 3 Bactericidal efiicacy of the drug comhinations against
infected root dentine bacteria of infected root dentine

Bacterial recovery on BHI-hlood agar plates, Bacterial recovery on BHI-blood agar.

expressed as log (colony-forming units m g ' expressed as log (colony-forming units mg" ^')

Sample No drugs WithMN(10ixgml- 3Mix(^JLgml~') ml-1)

4.34 1.80 Sample No drugs 50 100 50 100

3.56 1.15
1.79 9 3.32 0* 0 0 0
3.93
10 7.36 0 0 0 0
3.08 1.04
11 7.61 0 0 0 0
5.78 2.88 5.88 0 0 0 0
12
5.76 2.93 13 3.28 0 0 0 0
5.23 2.87 14 5.38 0 0 0 0
6.30 3.08 15 4.15 0 0 0 0
16 4.97 0 0 0 0
17 5.72 0 0
18 3.41 0 0
drugs, i.e. ciprofloxacin, minocycline or rifampicin
3Mix(fji,gi nl-')
(25, 50 or 75 [xg m l " ' ; Table 2), but all of the bacteria
were not always killed by the single drug alone (Tables 1 Sample No drugs 1 10 25 50
&2). 19 2.00 1.00 0 0 0
On the other hand, bacterial recovery was 20 5.04 >3 2.78 0 0
significantly decreased (paired t-test; P<0,0001) to zero 21 2.28 1.00 0 0 0
22 >6 >3 0 0 0
in the presence of the mixture of ciprofloxacin, metron- 23 2.00 0 0 0 0
idazole, minocycline and rifampicin (4Mix) at a concen- 24 >5 >4 >3 0 0
25 4.04 0 0 0
tration of 50 or 100 jjLg ml^i each (Table 3). Bacterial 26 4.00 0 0 0
recovery was also significantly decreased (paired £-test; 27 3.87 1.48 0 0
P < 0,0001) to zero in the presence of the mixture of
3Mix, ciprofloxacin, metronidazole and minocycline; 4Mix, 3Mix +
ciprofloxacin, metronidazole and minocycline (3Mix) at rifampicin.
a concentration of 25 or 50 |JLg mh^ each (Table 3), *No bacteria were recovered.
indicating that the bactericidal eflicacy was sufficient
with 3 Mix at the concentration of 25 ml~i each
(Table 3). These results may indicate that infected root in the presence of 3Mix or 4Mix (50 or 100 jxg ml~i
dentine can be sterilized using this drug combination. each; Tables 4 & 6), These results may indicate that
Similarly, more than lO-^ bacteria were present in bacteria in carious and pulpal lesions can also be kflled
samples taken from carious dentine (Tables 4 & 5), by the cotnbination of drugs. In fact, the bactericidal
infected pulps and periapicai lesions (Table 6), respec- efficacy of the drug combination against bacteria in
tively. However, bacterial recovery was also carious lesions was clearly demonstrated in situ
significantly decreased (paired t-test; P<0.0001) to zero (Table 5).

Table 2 Bactericidal efficacy of minocycline (MIMO), rifampicin (RFP) and ciprolloxacin (CPl'X) against bacteria of infected root dentine
Bacterial recovery on BHI-blood agar plates, expressed as log (colony-forming units mg-1)

+ MINO + RFP + CPFX

(fxgml"M

Sample 25 50 75 25 50 75 25 50 75
No drugs

9 0* 0 0 0 0 0 0 0 0
3 32
10 7 36 >4 0 0 >5 4.91 6.63 4.78 4.72 4.62
11 7 61 4.57 3.63 2.26 4.46 2.77 0 >5 3.45 2.26
12 5 88 4.72 3.20 0 2.30 1.23 1.23 4.57 4.48 4.26
13 3 28 0 0 0 2.08 1.78 2.08 0 0 0
14 5 38 2.36 0.63 0.63 4.48 4.00 4.00 2.99 2.84 1.98
15 4.15 0 0 0 1.48 1.70 1.60 1.60 0 0

*No bacteria were recovered.

© 1996 Blaciswell Science Ltd. International Endodonticlournal, 2 9 . 125-130

128 Jl J-Joshino et al

Table 4 Antibacterial efficacy of the drug combinations in vitro Table 6 Antibacterial efficacy of the drug combination in vitro against
against bacteria of carious lesions bacteria of pulpal and periapicai lesions

Bactcricil recovery on BHf-blood agar plates, Bacterial recovery on BHI-blood agar.

expressed as log (colony-forming units mg"') expressed as log (colony-forming units mg' 1)

Sample No drugs 3Mix 4Mix 3Mix(|xgml-i) 4Mi.'<(|xg m|-i)

(100|a.gmI~l)
Sample No drugs 50 100 50 100
1 4.08 0 0
2 2.26 0 1 6.53 0*
3 3.75 0 2 3.15 0 0
0 3 6.54 0 0 0 0
4 4.]1
0 4 2.48 0 0 0 0
5 5.72
6 4.88 0 5 3.41 0 0 0 0
0 6 3.t)8 0 t) 0 0
7 7.32
7 3.79 0 0 0 0
8 6.58 a
>5 0 8 3.78 0 0 0 0
9
3.85 0 9 4.36 0 0 0 0
10
11 4.04 0 10 6.08 0 0 0 0
12 >5 t) 11 2.32 0 0 0 0
0 12 3.79 0 0 0 0
13 4.69
14 3.30 0 13 0
15 2.91 t) 14 0
16 5.68 0 15 4.11 0 0
17 5.26 0 16 4.30 0 0
18 3.26 0
19 5.28 0 3Mix, ciprolloxacin. metronidazole and minocycline; 4Mix, 3Mix +
20 3.08 0 rifampicin.
21 4.79 0 Samples: 1-4, acute pulpitis with spontaneous pain; 5-10. chronic
22 >3 0 ulcerative pulpitis with cold pain and exposure of the pulp; 11-12.
23 4.79 0 ascending pulpitis; 13-14, intact pulp (intentional pulpcctomy);
24 >5 0 15-16, periapicai lesions.
25 5.00 0 *No bacteria were recovered.

3Mix, ciprofloxacin, metronidazole and minocycline; 4Mix, 3Mix +

rifampicin.
*No bacteria were recovered. Discussion
As a matter of principle, bacteria in root canal systems
Table 5 13act:ericidal eflicacy ol the drug combinations in vivo against should be eliminated during treatment, for a successful
bacteria of carious lesions outcome. It has been reported that sterilization of the
Bacterial recovery on BHI-blood agar plates, root canal and periradicular region results in good
expressed as log (colony-forming units m g " ' ) healing of periapicai diseases in adults (Bystrom et al,
After
1987). The application of antibacterial drugs to
endodontic lesions is one of the clinical procedures that
Sample Baseline 1 day 1 month 1 year
may be used to sterilize lesions (Grossman 1972), In
With 3Mix (MN. MINO and CPFX; 100 |xg m | - i each) order to sterflize such lesions, a single antibacterial drug
1 5.68 0* —" may not be eflective (Molander et al 1990; Tables 1 and
2 5.26 0 —
2), even if it has a broad antibacterial spectrum, because
With 3Mix (MINO, CPFX and RIP; 100 (xg ml~i each) the bacterial composition of the infected root canals is
3 3.26 0 0
5.28 — 0
complex. Bacteria may invade root canals from other
4
5 3.08 0 0 oral sites, e.g. dental plaque and saliva and, in carious
6 ' 4.79 — dentine, may also smear the root canal during
7 >3 0 endodontic treatment. All such bacteria may be targeted
Without drugs (sterile saline) by antibacterial drugs. The selection of antibacterial
8 4.79 4.99 5.28 — drugs should be based on the most up-to-date bacterio-
9 >5 >5 4.79 — logical knowledge.
10 5.00 4.96 5.28 —
It was found with strict anaerobic procedures that
CPFX, ciprofloxacin; MfNO, minocycline; MN, metronidazole; RIP.
obligate anaerobes represented the overwhelming
rifampicin.
*No bacteria were recovered.
majority of the isolates from infected root dentine of
**Not tested. adults (Ando & Hoshino 1990) and children (Sato et al

O 1996 Btackwell Science Ltd. IntenmtioiKifKridodontic Joimifl!. 2 9 . 1 2 5 - 1 3 0


1993b), as well as from dental plaque (Hoshino et al,
I989h), carious lesions (Hoshino 1985), non-exposed
pulpal tissue where bacteria invaded through dentinal
tubules (Hoshino et ai, 1992), cementum lesions (Kiryu
et al. 1994), periodontal pockets (Uematsu & Hoshino
1992) and saliva (Sato et al 1993) and denture plaque
of edentulous patients (Hoshino & Sato 1988). In these
studies, obligate anaerobes were strictly defined as
bacteria which grew only in the anaerobic glove box,
but not in air containing 30% CO2, and which were
demonstrated at least three times to conform with the
definition. Various anaerohic species have been isolated
from necrotic pulps (Bergenholtz 1974, Wittgow &
Sabiston 1975, Sundqvist 1976, Zavistoski et al 1980)
and from root canals with periapical pathosis (Yoshida
<^t al 1987). In these studies, obligate anaerobes were
again defined as bacteria which grew only in the anaer-
obic glove box hut not in air with 30% CO2, and it was
confirmed at least three times that there was no growth
in air containing 30% CO2. These findings suggest that
the bacteria in endodontic lesions and the related areas
Were predominantly anaerobes.
The bactericidal efficacy of a mixture of ciprofioxacin,
metronidazole and minocycfine, with and without the
addition of rifampicin (4Mix and 3Mix, respectively),
against bacteria in carious lesions and infected root
dentine has been clearly demonstrated in vitro (Tables 3.
4 & 6), although each of the respective drugs alone was
not capable of sterilizing the lesions (Tables 1 & 2). The
combination of drugs has been shown to penetrate
efficiently through dentine from prepared root canals,
especially from ultrasonically irrigated root canals (Sato
etal 199?).
These results suggest that topical application of the
drug combination may be potent in sterilizing lesions in
root canal treatment. In addition, the drug combination
is bactericidal against hacteria of carious dentine
(Table 4), dental plaque taken from adults (Hoshino
fit al, unpubfished data) and children (Sato et al 1992),
cementum lesions (Kiryu et al 1994) and periodontal
pockets (Hoshino et al, unpublished data), which may
possibly be associated with infection in endodontic
regions. Furthermore, it has been suggested that
softened dentine of carious lesions of permanent teeth
can be left after being disinfected in vivo, and the disin-
fected lesions can be expected to re-ctilcify (Hoshino et al
1989a, Hoshino 1990). If this is the case, softened
dentine of canal walls might also be left, after steriliza-
tion, to avoid too much enlargement of root canals and
unnecessary irritation of periapical tissues
The antibacterial drugs in this study have been used
® 1996 Blackwell Science Ltd, Internationai Endodontic Jownai 29. 125-1 30
Mixed drugs for dentine sterilization 129
clinically. Although side-effects of ciprofioxacin have
been reported, the drug has been shown to be cfinicafiy
safe when applied in the recommended doses (Black et al
1990). For topical application of the drug combination,
a very low dose is required, and thus any adverse
systemic side-efi'ects should be minimized. Even so, one
should consider that unnecessary, inadequate and
lengthy application of antibacterial drugs may induce
drug-resistant bacteria.
Since the red colour of rifampicin is not always
aesthetically appropriate for topical application in the
oral cavity, a combination of drugs was made without
rifampicin (3Mix). Minocycline sometimes causes
pigmentation, especially in calcifying teeth, so the bacte-
ricidal efficacies of the mixture of ciprofioxacin and
metronidazole plus amoxicifiin (Mixed-drug I; Sato et al.
1993a). cefaclor (Mixed-drug 11), cefroxadine (Mixed-
drug III), fosfomycin (Mixed-drug IV) or rokitamycin
(Mixed-drug V) have been compared. It was found that
the new drug combinations (100 |xg ml-' each) were
able to sterilize carious lesions and infected necrotic
pulps of deciduous teeth (Sato et al. 1993a).
Acknowledgements
This investigation was supported in part by the Japanese
Ministry of Education, Science, Sport and Culture under
Grants-in-aid for Scientific Research (60440088,
61440076, 01790543 and 03404055).
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© 1996 Biackweii Science Ltd. lntcrnationai Endodontic Journal, 29, 1 2 5 - 1 3 0