Original Paper
Caries Res 2000;34:502–508
Changes in the Cultivable Flora in Deep
Carious Lesions following a Stepwise
Excavation Procedure
L. Bjørndal T. Larsen
Departments of Cariology and Endodontics and Oral Microbiology, School of Dentistry, Faculty of
Health Sciences, University of Copenhagen, Denmark
Key Words
Microbiology · Deep carious lesions · Stepwise
excavation
Abstract
This study examined the cultivable microflora before
and after stepwise excavation procedures in deep cari-
ous lesions in 9 permanent teeth, categorized according
to degrees of proximal surface destruction. The final ex-
cavation was performed 4–6 months after the initial
treatment, which included peripheral dentine excava-
tion and removal of the central cariogenic biomass and
the superficial necrotic dentine. Dentine colour and con-
sistency were assessed by means of standardized scales
before the application of a Ca(OH)2 compound and tem-
porary sealing. Reassessments were performed before
and after the final excavation. Microbiological samples
of the central demineralized dentine were obtained with
a sterile bur before and after the first excavation, as well
as before and after the final excavation. After anaerobic
cultivation on enriched non-selective tryptic soy agar, 30
colonies from a representative area were identified by
standardized biochemical and physiological tests. Be-
fore temporary restoration, a yellowish and light brown
demineralized soft dentine was typically observed, and
gram-positive rods accounted for 70% and lactobacilli
for 50% of the total colony-forming units. Lactobacillus
casei subsp. rhamnosus and Actinomyces naeslundii
dominated the lactobacilli and the other gram-positive
 2000 S.Karger AG, Basel
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Received: November 3, 2000
Accepted after revision: April 4, 2000
rods, respectively. Gram-negative rods were the next
most frequent isolates, followed by streptococci, each
group accounting for about 20% of the colony-forming
units in positive samples. Before the final excavation,
which did not cause exposure of the pulp in any of the
cases, the retained demineralized dentine had changed
into a darker and harder tissue, and the total colony-
forming units, as well as the frequency and proportions
of lactobacilli were substantially reduced. Gram-nega-
tive rods also declined, and the flora was dominated by
A. naeslundii and various streptococci. In conclusion,
the cultivable flora detected following the treatment in-
terval had declined substantially, and the distribution of
bacterial species did not represent a typical cariogenic
microbiota of deep lesions, confirming the clinical find-
ings of arrested caries progression.
Copyright © 2000 S. Karger AG, Basel
In the Scandinavian countries a stepwise excavation pro-
cedure has traditionally been used on deep carious lesions,
particularly in the primary dentition [Magnusson and Sun-
dell, 1977], but also in permanent teeth [Leksell et al., 1996;
Bjørndal and Thylstrup, 1998], in order to reduce the risk of
causing exposure of the pulp. Previously, the first excava-
tion in a stepwise procedure was typically performed into
the residual level of carious dentine [Kerkhove et al., 1967],
implying a risk of iatrogenic exposure of the pulp. In order
to decrease pulp exposures, we employed a more gentle
procedure leaving relatively larger amounts of demineral-
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Lulea Tekniska Universitet
Lars Bjørndal
School of Dentistry, Department of Cariology and Endodontics
Faculty of Health Sciences, University of Copenhagen
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Nørre Allé 20, DK–2200 Copenhagen N (Denmark)
Tel. +45 35326700, Fax +45 35326505, E-Mail Lars.Bjoerndal@odont.ku.dk.
ized dentine behind after the first excavation of deep cari-
ous lesions [Bjørndal et al., 1997]. No clinical signs of ac-
tive caries progression, i.e. yellowish, soft and wet dentine,
could be observed following the treatment interval, and the
retained carious dentine changed to the clinical appearance
of arrested or slow-progressing lesions. Similar alterations
have been described for the activity of dentinal lesions
[Miller and Massler, 1962; Massler, 1967] as well as for
root surface lesions [Nyvad and Fejerskov, 1986; Schüp-
bach et al., 1990]. In accordance with studies on stepwise
excavation procedures applying a rigorous first excavation
step [Besic, 1943; Schouboe and Macdonald, 1962; King et
al., 1965; Aponte et al., 1966; Fisher, 1969], we also ob-
tained a marked reduction in bacterial growth following the
treatment interval [Bjørndal et al., 1997]. However, in some
lesions, a growth of streptococci and lactobacilli was still
detected.
The majority of micro-organisms in carious dentine, in-
cluding the microbiota in advanced root carious lesions
[Schüpbach et al., 1996], are Gram-positive cocci and rods,
and the predominant species are streptococci, lactobacilli
and Actinomyces [Edwardsson, 1974; Hoshino, 1985; Hahn
et al., 1991]. Focusing on these particular species, the aim
of the present study was to examine at a species level the
cultivable flora persisting in clinically excavated lesions
following a stepwise excavation procedure, and to examine
whether a cultivable flora after the final treatment was com-
parable to a specific composition of a microflora character-
istic of arrested lesion environments.
Materials and Methods
The material comprised 9 teeth (5 molars and 4 premolars) with
deep proximal carious lesions. Pretreatment radiographs were used to
classify lesion size and to exclude apical pathosis, and an electric pulp
tester confirmed pulp sensibility. None of the lesions had irreversible
signs of pulpitis (i.e. no information on spontaneous or provoked pul-
pal pain). All lesions were cavitated with exposure of the dentine, and
with proximal gingival inflammation. In 2/3 of the lesions the neigh-
bouring proximal surface was restored. Figure 1 illustrates progres-
sive clinical and radiographic signs of proximal surface destruction,
representing three different types of lesions. A closed lesion environ-
ment is shown in figure 1a,b where clinical signs of demineralization
of the cavity are not visible from the occlusal aspect. In figures 1c, d
the demineralization within the cavity is clinically visible as a change
in enamel translucency, indicating a more advanced coronal deminer-
alization along the enamel-dentine junction [Bjørndal et al., 1999] and
finally, in figures 1e, f a lesion environment with a total breakdown of
the proximal surface is noted. In table 1 the material is shown accord-
ing to the proximal surface status and lesion size, estimated on the ba-
sis of radiographs and expressed as depth of dentine demineralization
in relation to dentine thickness. The majority of teeth with lesion size
Microbiological Changes during Stepwise
Excavation
equal to 2/3 of the entire dentine thickness had a closed lesion envi-
ronment (a), whereas lesion sizes larger than 2/3 could also be associ-
ated with clinically visible changes of enamel translucency (b). Final-
ly, the deepest lesion examined (c3/4) was associated with a
breakdown of the proximal surface (c). A Scientific Ethical Commit-
tee approved the study and informed consent was given by the sub-
jects according to the Helsinki Declaration II.
Treatment Procedures and Clinical Recordings
The first step in the stepwise excavation procedure included (1)
removal of the central cariogenic biomasses and the superficial parts
of the necrotic and demineralized dentine, and (2) complete excava-
tion of the peripheral demineralized dentine. A calcium hydroxide-
containing base material and a temporary filling were applied. After a
treatment interval of 4–6 months, the temporary filling was removed.
The retained carious dentine was removed with a sharp excavator or a
slowly rotating bur (Komet Germany No. 1.204.025). The bottom of
the cavity was sealed with a calcium hydroxide-containing base ma-
terial, and the tooth was finally restored. Recordings of dentine alter-
ations during the treatment were made using standardized scales of
dentine colour and consistency [Bjørndal et al., 1997] as well as
recordings of moisture as previously described by Kidd et al. [1993a].
Microbiological Sampling
An initial cavity preparation was made and the tooth was isolated
with rubber dam, and washed with Savlon (5% savlon concentration,
95% ethanol), followed by a saline wash, and then dried with sterile
swabs. Samples to assess external contamination of the cavity were
obtained by a sterile swab, dampened in saline, which was transferred
to peptone yeast and incubated anaerobically for 3 days. If growth
was observed, dilution and plating was performed as described below,
and the resulting colonies compared to the samples of surface dentine
by colony morphology and Gram-staining. Samples of the dentine
were obtained with a sterile slow rotating bur [Kidd et al., 1993a;
Bjørndal et al., 1997] (Komet, Germany, No. 1.204 0.18) damped in
saline. Samples of the outer carious dentine (I) and the central dem-
ineralized dentine (II) were obtained before and after the first excava-
tion, respectively, and followed by the application of a temporary
restoration. After the treatment interval, the temporary restoration was
removed, the rubber dam procedure was applied and another two
samples of the central dentine were obtained before (III) and after the
final excavation (IV). Clinical photographs as well as impressions
were taken in order to identify the same sampling area following the
treatment interval.
Microbiological Procedures
The samples were transferred to reduced transport fluid [Syed and
Loesche, 1972], vortex-mixed for 30 s, and serially diluted 10-fold
before 0.1 ml was plated on tryptic soy agar (Difco), supplemented
with horse blood (5%), haemin (5 µg/ml) and menadione (0.5 µg/ml)
under anaerobic conditions (70% N2, 20% H2, 10% CO2). After anaer-
obic incubation at 37°C for 7 days, the total number of colony-form-
ing units (cfu) per millilitre was counted, and 30 colonies from a rep-
resentative area of an agar plate yielding 50–300 colonies were
Gram-stained and identified according to Cowan and Steel [Barrow
and Feltham, 1993], and as described below using the following cri-
teria. Gram-positive cocci were characterized by: colony morphology
on mitis salivarius agar (Difco), presence of catalase, fermentation of
acetylglucosamine, amygdalin, inulin, mannitol, melibiose, raffinose,
salicin and sorbitol, hydrolysis of arginin and esculin, production of
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Caries Res 2000;34:502–508 503
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 a b
c d
Fig. 1. Progressive clinical and radiograph-
ic signs of proximal surface destruction. a, b
Closed lesion environment in +7. c, d Clini-
cal signs of demineralization within the cavi-
ty in 7+. e, f Lesion environment with a to-
tal breakdown of the approximal surface in
another 7+. e f
acetoin and dextran, and presence of β-N-acetylglucosaminidase, al-
kaline phosphatase, α-L-fucosidase and β-glucosaminidase, using the
API ZYM system (Bio Mérieux SA) [Kilian, 1978; Kilian et al., 1989;
Beighton et al., 1991a,b]. Gram-positive rods growing on Rogosa SL
agar (Merck) were designated Lactobacillus and divided into species
according to Sharpe [1981], based on fermentation of amygdalin, cel-
lobiose, mannitol, melezitose, melibiose, raffinose, rhamnose and sor-
bitol, production of CO2 from glucose and hydrolysis of arginine and
esculin. Gram-positive rods not growing on Rogosa SL agar were
identified according to Kilian [1978] using the API ZYM system sup-
plemented by growth under aerobic conditions +7.5% CO2 and tests
for the presence of catalase and β-xylosidase [Kilian and Bülow,
1976]. Gram-negative organisms were tested for growth under aero-
bic conditions, and black-pigmenting species were characterized by
fluorescence in long-wave ultraviolet light and analysed by the API
ZYM system [Laughon et al., 1982; Slots and Reynolds, 1982].
Results
Clinical Observations
All temporary restorations were sufficient following the
treatment interval, and the final excavation did not cause
pulp exposure in any tooth. In all teeth with a lesion envi-
ronment defined as a (fig. 1a, b), a yellowish and light
brown demineralized soft dentine was observed to change
504 Caries Res 2000;34:502–508
into a dry, darker and harder demineralized dentine follow-
ing the treatment interval (table 1). In the lesions with a
depth of dentine demineralization larger than 2/3 of the en-
tire dentine thickness and with lesion status defined as b
(fig. 1c, d) or c (fig. 1e, f), respectively, the demineralized
dentine appeared darkly discoloured even before the treat-
ment, and no colour changes occurred following the treat-
ment interval, but the dentine became dry, and in 1 case al-
so harder (tooth A).
Microbiological Observations
The microbiological analysis was based on 32 samples
each containing 30 isolates. The remaining 4 samples were
excluded owing to external contamination. Table 2 presents
the median number and range of colony-forming units per
millilitre in the samples. In the 2 samples taken before and af-
ter the first excavation, the composition of the microflora was
rather similar, though the median number of total colony-
forming units per millilitre was reduced from 5.1E105 to
1.6E104 (97%) as a result of the initial excavation removing
the outer demineralized dentine. The majority of micro-or-
ganisms isolated before filling were Gram-positive rods ac-
counting for 70% of the total colony-forming units.
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Table 1. The material in relation to clinical signs of proximal surface destruction (a, b, c), lesion size and changes in consistency, colour and
moisture of demineralized dentine during the treatment interval

Tooth Proximal surface Lesion size a After first excavation Before final excavation
identification status

F ab = 2/3 yellow-light brown/medium hard/wet dark brown/medium hard/dry

H a = 2/3 yellow-light brown/medium hard/wet dark brown/medium hard/dry
J a = 2/3 yellow-light brown/medium hard/wet dark brown/medium hard/dry
K a c2/3 yellow-light brown/medium hard/wet dark brown/medium hard/dry
C b d2/3 yellow/medium hard/wet light brown/medium hard/dry
B b = 2/3 yellow-light brown/medium hard/wet dark brown/medium hard/dry
D b c2/3 dark brown/medium hard/wet dark brown/medium hard/dry
E b c2/3 light brown/medium hard/wet light brown/medium hard/dry
A c c3/4 dark brown/medium hard/wet dark brown/medium hard/dry

a Lesion size estimated on the basis of radiographs and expressed as depth of dentine demineralization in relation to dentine thickness.
b a = The cavity is not visible from the occlusal aspect; b = the cavity is clinically visible from the occlusal aspect as a change in enamel
translucency; c = total breakdown of the proximal surface.

Table 2. Number of colony-forming units

per millilitre in each sample before (I) and I II III VI
after (II) first excavation and before (III) and
after (IV) final excavation n 9 9 6 8
Median cfu 5.1 E105 1.6E 104 2.2E 103 4.3E102
Range 2.9 E104–4.4 E106 1.6E 103–5.3E105 0–3.9E103 0–9.0E104
Samples without growth 0 0 1 2

Lactobacillus was the dominating genus, constituting a
median of 57 and 45% of the total colony-forming units in
samples I and II, respectively. The most frequently isolated
and predominant species was Lactobacillus casei subsp.
rhamnosus, followed by Lactobacillus brevis, Lactobacil-
lus plantarum, Lactobacillus acidophilus and Lactobacillus
casei subsp. casei (table 3). Among the other Gram-positive
rods Actinomyces naeslundii (including species formerly
classified as A. viscosus) and Actinomyces israelii predom-
inated, while Arachnia and other species were occasionally
isolated. Mutans streptococci (S. mutans and S. sobrinus)
were only isolated from 1 tooth that had the least advanced
of all lesions among the 9 teeth (tooth C). Other streptococ-
ci, including S. mitis, S. oralis, S. anginosus and S. sanguis,
were isolated from several teeth, although generally in
rather low proportions. Gram-negative cocci, primarily
anaerobic, were isolated from 3 teeth, while both facultative
and obligate anaerobic Gram-negative rods were found in 7
teeth, constituting about 20% of the total colony-forming
units per millilitre. The latter included 3 teeth positive for
black-pigmented species (Prevotella intermedia, Prevotella
melaninogenica and Porphyromonas endodontalis, table 3).
A lesion-specific comparison of the microbiological da-
ta showed some variation, however. In the 3 cases illustrat-
ed in figure 1 we found a homogeneous lactobacilli mi-
croflora in the closed lesion environments (a, tooth F),
whereas in b and c we saw a heterogeneous microflora with
more than 10 recovered species before the treatment inter-
val. Furthermore, the open lesion (c, tooth A) was the only
one positive for Staphylococcus aureus and Staphylococcus
epidermidis.
Table 4 presents the occurrence of the different bacterial
groups after the first excavation (II) and just before the final
excavation (III). The frequency and proportions of lacto-
bacilli were substantially reduced. In addition, while 3–6
different Lactobacillus species were represented in each
tooth before temporary restoration, only one (in 1 case two)
species were isolated from each positive sample following
the treatment interval. A. israelii was not detected before
the final excavation, and A. naeslundii predominated among
the other Gram-positive rods (table 3). In all 5 cases where
a facultative Gram-negative flora was recovered before the
treatment interval, no growth of these bacteria was found
following the treatment interval. Moreover, black-pigment-
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Microbiological Changes during Stepwise Caries Res 2000;34:502–508 505

Excavation
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Table 3. Number of samples yielding growth of the speciated bacte- ed Gram-negative anaerobic rods, i.e. Prevotella and Por-
ria (and median proportions in the positive samples) at the four sam- phyromonas, were not recovered following the treatment in-
pling sites (I, II, II, IV)
terval (table 3), and in those lesions where Gram-negative
Bacterial species I II III IV anaerobic rods accounted for a relatively large proportion
(20–43.3%) of the total colony-forming units per millilitre
Gram-positive cocci 4 (23.3) 6 (18.4) 3 (23.3) 6 (18.4) before the placement of a temporary restoration, the fre-
S. mutans 1 (3.3) 3 (6.7) quency and proportions of these species were either sub-
S. sobrinus 1 (13.3) 1 (3.3) 1 (26.7)
stantially reduced, or not detected at all.
S. sanguis 1 (16.7) 1 (3.3)
S. gordonii 1 (20.0) In the final sample (IV), the median total colony-form-
S. mitis/oralis 3 (3.3) 2 (8.4) 1 (20.0) 3 (16.7) ing units per millilitre was reduced by a factor E102 (table
S. anginosus 1 (3.3) 2 (6.7) 1 (6.7) 1 (10.0) 2), and no growth was detected in 2 of 8 samples. The pre-
Streptococcus sp. 1 (63.3) 2 (17.4) 1 (23.3) 2 (15.0) dominant organisms isolated were various streptococcal
species and A. naeslundii.
Gram-positive rods 9 (70.0) 9 (73.3) 5 (26.7) 5 (50.0)
L. brevis 5 (20.0) 3 (6.7)
L. plantarum 4 (5.0) 4 (18.4)
L. salivarius 1 (30.0) 2 (18.4) Discussion
L. acidophilus 5 (3.3) 3 (13.3)
L. casei, casei 2 (8.4) 3 (6.7) 1 (6.7)
The stepwise excavation approach has proved to be a
L. casei, rhamnosus 8 (30.0) 6 (23.3) 1 (33.3)
Lactobacillus sp. 7 (20.0) 4 (3.3) 1 (33.3) suitable method to manage deep carious lesions without
A. viscosus/naeslundii 3 (10.0) 5 (10.0) 3 (6.7) 5 (16.7) symptoms of irreversible pulpitis. Long-term prognostic da-
A. isrealii 3 (6.7) 2 (5.0) ta based on the stepwise excavation procedure performed
Arachnia 1 (10.0) 2 (23.4) by pedodontists [Leksel et al., 1996], as well as by general
Rothia 1 (6.7)
Bifidobacterium 1 (10.0)
practitioners [Bjørndal, 1999], show high success rates of
Other gram-positive rods 92% following 3.5–4.5 years of follow-up. In this respect
(non-lactobacilli) 3 (6.7) 4 (5.0) 4 (10.0) 2 (36.7) data have indicated that the first excavation step during a
step-by-step procedure need not be as rigorous as tradition-
Gram-negative cocci 3 (16.7) 3 (10.0) 4 (6.7) 2 (18.4) ally performed [Bjørndal et al., 1997]. Consequently, it has
Gram-negative rods 6 (18.4) 7 (20.0) 3 (6.7) 4 (8.4)
become possible to evaluate clinically as well as microbio-
P. intermedia 2 (4.4) 2 (3.9) logically a relatively larger amount of demineralized den-
P. melaninogenica 1 (3.3) tine. Even though the literature has identified predominant
P. endodontalis 1 (10.0) species in deep carious pathology it is a typical feature that
Other gram-negative rods 5 (13.3) 7 (20.0) 3 (6.7) 4 (8.4) variation is always obtained. Therefore, in the present study
Unidentified 1 (13.3) 4 (10.0) 3 (70.0) 2 (84.0)
we have examined a limited number of well-defined clinical
lesions and particularly focused on the intra-lesion changes
over time. In addition, we applied a sufficient number of le-
sions that made it possible to categorize the deep lesions in-
to stages of progressing signs of caries. In short, we intro-
Table 4. Frequency and proportions of the different bacterial groups duced the stepwise excavation as a model to gain more
before (II) and immediately after (III) temporary restoration
information on ecological changes in the oral cavity
II III [Schüpbach et al., 1996], with respect to the interaction be-
tween the pulpdentinal reaction and the changes in the com-
n 9 6 position of the microbial flora in the carious lesion over
Mutans streptococci 1 (6.6) a 0 time. Variation of results due to inaccurate localization of
Other streptococci 6 (8.4) 3 (26.7)
Lactobacillus sp. 8 (45.0) 3 (20.0)
the sampling site after the treatment interval was precluded
Actinomyces sp. 5 (16.7) 3 (6.7) using clinical photographs as well as impressions of the in-
Other gram-positive rods 5 (6.7) 4 (10.0) dividual cavities.
Gram-negative cocci 3 (10.0) 4 (6.7) In the present study the microbiota recovered in the first
Gram-negative rods 7 (20.0) 3 (13.3) two samplings before the treatment interval was similar to
a Median percentage only for samples showing positive growth.
the microbiota normally harboured in deep lesions [Ed-
wardsson, 1974; Hoshino, 1985; Hahn et al., 1991]. In par-
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506 Caries Res 2000;34:502–508 Bjørndal/Larsen

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ticular, Gram-positive rods predominated, accounting for
70% of the total colony-forming units per millilitre. Among
these, Lactobacillus species were the principal isolates, and
L. casei subsp. rhamnosus was the most frequently isolated,
followed by A. naeslundii and A. israelii. Also, facultative
and obligate anaerobic Gram-negative cocci as well as rods
were recovered in the majority of samples, making up about
20% of the total colony-forming units in positive samples,
and with the occasional recovery of black-pigmented Pre-
votella and Porphyromonas. In accordance with the litera-
ture [Loesche and Syed, 1973], relatively high levels of S.
mutans and S. sobrinus were found in the less advanced le-
sion examined. As previously found in a limited number of
cases [Schüpbach et al., 1996], the composition of each mi-
crobial flora investigated disclosed variation. In the present
study we have performed a detailed clinical examination of
each lesion environment prior to treatment in order to ex-
plain the microbial variation as a possible consequence of
clinical differences in lesion types. A remarkably homoge-
neous lactobacilli microflora was found in tooth F, which
happened to be the only closed lesion environment without
a neighbouring proximal restoration (fig. 1a). Operative
procedures during such proximal restorative work are
known to cause potential damage to neighbouring teeth
[Qvist et al., 1992], and the quality of the restoration per se
influences the health status of the periodontal tissue [Lang
et al., 1988]. These factors, taken together, could possibly
imply ecological alterations. In lesions with various signs of
proximal surface destruction a more heterogeneous mi-
croflora was observed, including usual representatives of
gingival plaque along inflamed gingiva, and even normally
non-oral species like staphylococci were recovered in the
most open lesion environment (fig. 1e). Thus, we suggest
that the composition of the microflora in each lesion is a re-
flection of the local environment, including the clinical
characteristics of that particular lesion.
The microbial alterations during the treatment showed a
substantial reduction in the frequency and proportions of
lactobacilli, reflecting a change towards a non-acidic envi-
ronment. As many as six different Lactobacillus species
were represented in the lesion environment before tempo-
rary restoration, decreasing to typically one species in posi-
tive samples following the treatment interval. Taking other
Gram-positive rods into account, A. israelii was never de-
tected after the treatment interval, nor was Rothia or Bifi-
dobacterium.
The frequency and proportions of Gram-negative rods
were reduced following the treatment interval, and black-
pigmented species were eliminated. It has previously been
speculated that the recovery of black-pigmented species
Microbiological Changes during Stepwise
Excavation
may account for the darkening of demineralized dentine
[Lynch and Beighton, 1994]. Again, taking the small mate-
rial into account, we could not confirm this hypothesis, be-
cause the development of dark demineralized dentine dur-
ing the treatment interval appeared independently of the
recovery of Prevotella and Porphyromonas. The lesions
with dark demineralized dentine before the treatment inter-
val did not harbour black-pigmenting species and, further-
more, these species were eliminated from all positive le-
sions following the treatment interval, where all lesions
turned dark. In this respect studies examining the associa-
tion between micro-organisms in carious dentine and the
cellular response have shown that Gram-negative rods, in
particular Prevotella, produce an extensive mononuclear in-
flammatory infiltration within the pulp [Massey et al.,
1993]. Therefore, it was an important finding that although
they appeared in positive samples before temporary restora-
tion, Prevotella and Porphyromonas were not detected fol-
lowing the treatment interval. Apart from the overall eco-
logical change induced by the first excavation procedure,
this might be explained by a previous in vitro finding that
Prevotella hardly appears to migrate into dentinal tubules
[Perez et al., 1993].
Following the final excavation two samples were with-
out growth, whereas the median total colony-forming units
per millilitre was reduced by a factor E102. Compared to
the results from studies applying the same sampling method
the acceptable level of growth after excavation has been
claimed to be 102 cfu/ml or less [Kidd et al., 1993b]. The
predominant organisms isolated were various streptococcal
species and A. naeslundii, which has previously been recov-
ered from arrested root surface lesions, being good indica-
tors of a change in the cariogenic environment. Even though
a different ecosystem is harboured at a root surface lesion
compared to the environment following a stepwise excava-
tion procedure, we have previously indicated the clinical
similarity between the retained carious dentine following a
treatment interval and the arrested root carious lesion in
terms of a dark brown discolouration and a harder consis-
tency of the surface [Bjørndal et al., 1997]. A microbial
comparison between findings in arrested lesions [Schüp-
bach et al., 1995; Brailsford at al., 1998] and our results dur-
ing stepwise excavation show similar features. After the
treatment interval, the total colony-forming units as well as
the numbers of lactobacilli and Gram-negative species were
reduced. These findings are in accordance with the changes
previously found in arrested root surface lesions [Schüp-
bach et al., 1995; Brailsford et al., 1998]. In conclusion, the
cultivable flora detected following the treatment interval
had declined substantially, and the distribution of bacterial
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Caries Res 2000;34:502–508 507
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species did not represent a typical cariogenic microbiota of
deep lesions, confirming the clinical findings of arrested
caries progression. Studies are needed to obtain more infor-
mation on the changes in the specific composition of a mi-
croflora over time in relation to the nature of tertiary
dentinogenesis [Bjørndal and Darvann, 1999].
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