P. Lingstr6ml*, F.O.J.
van Ruyven2,
J. van Houte2, and R. Kenf
'Department of Cariology, Faculty of Odontology, G6teborg
University, P.O. Box 450, SE 40530, Goteborg, Sweden,
2Department of Oral Microbiology, The Forsyth Institute, 140
The Fenway, Boston, MA 02115; 3Department of Clinical
Trials and Human Experimentation, The Forsyth Institute,
140 The Fenway, Boston, MA 02115; *corresponding author,
lingstromn@odontologi.gu.se
J Dent Res 79(2): 770-777, 2000
ABSTRACT
Dental caries appears to result from the action of
multiple, interrelated factors. A companion study
dealt with the plaque-flora/caries relationship (van
Ruyven et al., 2000). The plaque-pH/caries
relationship is the subject of this study. Since both
studies involve the same subjects, plaques, and tooth
surfaces, data on the examined factors have also been
integrated. In vivo plaque pH determinations
(microelectrode) were done on buccal sound (s) and
"white-spot" (ws) caries surfaces in a selected
dentition area in a low-caries (no ws) and higher-
caries subject group. The pH response to sugar was
evaluated before and after a sugar rinse, a local sugar
application, or sucking on a sugary lozenge. pH
profiles with sugar rinsing and normal or limited
salivary flow conditions, showed progressively
decreasing plaque pH values at various time points in
the order of: low-caries subjects (s sites), higher-
caries subjects (s sites), higher-caries subjects (s + ws
sites), and higher-caries subjects (ws sites). The
minimum pH values showed the same trend.
Analyses of all data indicated only a statistical
difference for minimum values for s sites in low-
caries subjects vs. ws sites in higher-caries subjects,
and for s and ws sites in the latter. Local sugar
application and sucking on a sugary lozenge induced
smaller pH drops than sugar rinsing; such suboptimal
sugar exposure caused a disappearance of the
difference between the minimum pH values for s and
ws sites observed with sugar rinsing in the higher-
caries subjects. Initial plaque pH values were similar
regardless of subject or tooth caries status. The
values were also not correlated with the plaque levels
of strongly iodophilic polysaccharide-storing
bacteria. Collectively, both studies indicate that
increasing subject caries status is characterized by
increasing plaque levels of highly-acid-tolerant,
acidogenic bacteria and an increasing plaque-pH-
lowering potential and support the dynamic
relationship between these parameters.
KEY WORDS: dental plaque, pH, mutans
streptococci, caries.
Received October 19, 1998; Last Revision March 23, 1999;
Accepted June 4, 1999
770
The pH of Dental Plaque in Its
Relation to Early Enamel Caries
and Dental Plaque Flora in Humans
INTRODUCTION
It appears increasingly that caries development is associated with a limited
number of major factors which are all dynamically interrelated (for review,
see van Houte, 1994). These are thought to include dietary carbohydrate
consumption, the microbial composition of dental plaque, the pH-lowering
ability of dental plaque, and the action of saliva. Although the above concept
is based on a considerable body of evidence, direct in-depth information from
studies with humans under natural in vivo conditions on the relationships
among some of the parameters is still lacking. Clearly, clarification of the true
relationship among these factors is of pivotal significance for our
understanding of caries etiology.
For this reason, we have recently focused on the factors plaque flora
composition and plaque-pH-lowering ability in relation to caries causation. A
pertinent basic observation is that both plaque factors as well as the
acidogenesis and acid tolerance of plaque organisms can vary widely (for
review, see van Houte, 1980, 1994). With respect to the plaque-flora/caries
relationship, the evidence indicates that increasing caries activity is associated
with an enrichment of plaque with organisms with a relatively high capacity
for acidogenesis and acid tolerance. Such organisms include the lactobacilli,
mutans streptococci (MS), and the so-called "low-pH" non-mutans
streptococci (non-MS), as well as, likely, other types of "low-pH" organisms
(for review, see van Houte, 1994; see also Handelman et al., 1968; Griffiths,
1979; Hayes et al., 1983; van Houte et al., 1996; van Ruyven et al., 2000).
Information on the plaque-pH/caries and, particularly, the plaque-
flora/plaque-pH relationship is more limited. The available in vitro evidence
suggests that, with few exceptions, caries development is accompanied by a
higher plaque-pH-lowering or acidogenic ability and that the above-cited
bacterial shifts (successions) within plaque contribute to this increased pH-
lowering ability (Miller et al., 1940; Manly et al., 1962; Minah and Loesche,
1977; Griffiths, 1979; Minah et al., 1981a,b; Vratsanos and Mandel, 1982;
Igarashi et al., 1987; van Houte et al., 199 1a,b; Margolis et al., 1993; Sansone
et al., 1993). Few in vivo studies of the plaque-pH/caries-activity relationship
exist. The classic study of the pH response of plaque to sugar (Stephan, 1944)
indicated a decreasing plaque pH profile with increasing subject caries status.
This finding is supported by some other studies (Englander et al., 1956;
Kleinberg and Jenkins, 1964; Rosen and Weisenstein, 1965; Turtola and
Luoma, 1972; Abelson and Mandel, 1981) but not by two recent studies, one
involving coronal (Fejerskov et al., 1992) and another involving root surfaces
(Aamdal-Scheie et al., 1996). Some studies have provided plaque pH data for
cavities (see Fejerskov et al., 1992), but none exists for initial "white spot"
caries lesions. Information about such caries lesions is critical for the
evaluation of the role of plaque pH in the transition from a caries-inactive to a
caries-active state. Of the few studies dealing with the plaque-flora/plaque-pH
relationship, most support the association between the earlier-noted bacterial
shifts and increased plaque acidogenesis (Minah and Loesche, 1977; Minah et
al., 1981a,b; van Houte et al., 1991a,b; Sansone et al., 1993), whereas one
J Dent Res 79(2) 2000 Plaque pH, Microflora, and Early Enamel Caries
does not (Scheie et al., 1984).
In view of the above, we initiated a study to examine further
the relationships among the pH response of coronal plaque to
sugar, its composition with respect to certain selected groups of
organisms, and the caries status of the tooth surface beneath this
plaque. The bacteriological aspects of this study have been dealt
with in the companion study (van Ruyven et al., 2000), whereas
the present report focuses on the plaque pH response.
MATERIALS & METHODS
Test Subjects
The test subjects were the same as used in the companion study
(van Ruyven et al., 2000). Informed consent was obtained from all
subjects. All procedures had received prior approval by the
Institutional Review Board of The Forsyth Institute. Low-caries
subjects (eight) were without "white-spot" caries (ws) in a selected
test dentition area, and higher-caries subjects (17) had ws in this
area (van Ruyven et al., 2000; Table 1). Subject AK, with 12 ws in
this area (coronal DMFS of 34), was added to the latter group. The
low-caries subjects had a mean (median) coronal DMFS of 5.8 (1).
The higher-caries group was divided into groups of eight and 10
subjects. The first group of eight (subjects CG, DC, FJ, KC, MJ,
PB, SL, and TK; van Ruyven et al., 2000; Table 1) had a mean
(median) coronal DMFS (including the ws) of 13.9 (6.5) and a
mean (median) of 3.6 (3.5) buccal ws sites in the selected dentition
area. For the second group, with 10 subjects, these figures were
16.1 (10.5) and 5.3 (5.0), respectively. The low-caries and eight
higher-caries subjects were used for plaque pH tests involving a
rinse with sugar solution; the 10 higher-caries subjects were used
for tests involving topical application of sugar solution as well as
sucking on a sugary lozenge.
Plaque pH Measurement
All subjects refrained from oral hygiene for 3 days to allow for
sufficient plaque formation; they also refrained from eating or
drinking prior to the tests, either since the night before (if they
arrived early in the moming) or for at least 2 hrs (if they arrived
later in the day), to prevent undue influence on the test pH results.
The tooth-surface test sites and plaques were the same as used for
bacteriological plaque analyses (van Ruyven et al., 2000), which
permits direct comparison among the data for the plaque pH
response to sugar exposure, the composition of the plaque flora,
and the caries status of the tooth-surface sites. Preliminary
measurements of plaque pH at one-week intervals with a few
subjects indicated that quite reproducible readings were obtained if
the plaque mass exceeded 0.5 to 0.75 mg wet weight per site. With
all subjects, this could be accomplished after discontinuation of
Table 1. Examples of Variability of the Initial Plaque pH Observed with Some of the Test Subjects
Caries Initial pH Values for the 12 Dentition Sites
Subject Group Test 16 15 14 13
TK higher 1 5.85 5.68 5.63 5.55
caries 2 6.47 6.56 6.74 6.75
JJ low 1 5.92 5.37 5.35 5.41
caries 2 6.53 6.17 6.12 6.25
771
oral hygiene for 3 days; with few exceptions, the range of plaque
wet weight for each site was 1.0 to 2.0 mg or, frequently, even
higher. In view of other preliminary results (Table 1), the
important issue of food intake as related to the pH measurements
was regularly discussed with the test subjects throughout the study.
Plaque pH profiles were obtained during different regimens.
Preliminary tests with some subjects with different-strength
glucose solutions and various exposure times indicated that an
optimal pH drop to about 4.0 (i.e., close to the theoretical
minimum) could be obtained after subjects rinsed their mouths for
3 min with 15 mL of a 20% glucose solution; the use of 10%
glucose solution did sometimes yield somewhat higher pH values.
Hence, the first and main test regimen involved subjects rinsing
with glucose solution (20%) followed by either normal salivary
exposure or limited access of saliva to the test dentition sites
during a 30-minute test period. In the latter case, cotton rolls were
placed in the buccal fold immediately after the three-minute rinse.
In some subjects, these rolls had to be replaced during the test
period so that the plaque would not be exposed to saliva. The
attempted additional use of Dry Angle, a triangular pad which was
placed over Stensen's duct during some tests, yielded no superior
results and was discontinued. Plaque pH measurements suggested
that salivary access to the plaque was probably not always totally
prevented, especially during the last 10 min of the test period.
Tests with each subject, involving either normal or limited salivary
exposure, were done within 1 or 2 wks.
In addition to sugar rinsing, test designs consisted of a local
application of 50 FL of a 40% glucose solution per tooth-surface
site on top of the plaque, while salivary exposure was limited by
prior placement of cotton rolls, during a 20-minute test period. For
this purpose, the 6 teeth on both sides of the dentition area were
studied separately. The glucose solution was applied to each tooth
site with a micropipet, starting with the first molar and ending with
the central incisor. This was then followed by plaque pH
determinations. This procedure was repeated for the left side.
Another design consisted of subjects sucking on a sugary lozenge
tablet (Lifesaver, 1.7 grams of sugar) for 5 min with normal
salivary access during a 30-minute test period. Both types of tests
with each subject were done likewise within 1 or 2 wks.
Following Lingstrom et al. (1993), measurements of plaque pH
in all tests were done at time 0 min (initial pH) immediately prior to
sugar rinsing, the local application of sugar solution, or the sucking
of the tablet, and at 5, 10, 15, 20, and (for 2 of the regimens) 30 min
thereafter. The pH measurements were done with a touch
microelectrode (Beetrode MEPH-1, WPI, New Haven, CT, USA)
connected with an Orion 901 pH/ISP meter (Orion Research, Inc.,
Cambridge, MA, USA) in combination with a glass reference
electrode (Beetrode, MERE-1). A reference salt bridge was
established by each test subject dipping one finger into a 3 mol/L
12 11 21 22 23 24 25 26
5.82 5.76 5.52 5.85 5.68 6.22 5.99 5.74
6.65 7.16 7.00 6.22 6.67 7.19 6.99 6.98
5.58 5.58 5.74 5.77 5.65 5.83 5.88 6.20
6.30 6.52 6.48 6.77 6.42 5.74 5.98 7.07
772 Lingstr6m et al. J Dent Res 79(2) 2000
Table 2. Plaque pH Profiles Obtained for Low-caries and the Group of Eight Higher-caries Subjects subject was based on the readings for all
and Normal and Limited Salivary Access after a Sugar Rinse buccal surfaces regardless of their caries
status. The same type of calculation was
Caries Salivary Time (min) after Start of Rinsing with Glucose Solution made with respect to the pH profiles for s
Group n Accessa 0 5 10 15 20 30 and ws tooth-surface sites (Table 3), the
minimum pH values for subjects (Table 4)
Low caries 7 N 6.756 5.38 5.20 5.19 5.37 5.39 and for s and ws tooth-surface sites
6.69c 5.09 4.99 5.00 5.08 5.13 (Tables 5 and 6), and the initial pH values
for subjects and s and ws sites (Table 7).
Low caries 7 L 6.74 5.15 5.12 5.13 5.20 5.24 For converted pH values, the value for
6.60 4.96 4.78 4.74 4.94 4.91 each of the 10 to 12 plaques in each
subject was first converted to free H+
Higher caries 7 N 6.76 5.20 4.91 5.03 4.96 5.06 concentration. This yielded a mean of
6.44 4.76 4.61 4.74 4.71 4.67 these antilogs for each subject which, in
turn, was used to calculate a mean of the
Higher caries 8 L 6.62 4.97 5.03 4.81 5.02 5.31 antilogs for groups. Finally, the latter
6.53 4.73 4.88 4.65 4.82 5.00 mean was reconverted to pH, yielding an
average pH for groups.
a Normal (N) and limited (l) access.
b Mean of the means of all selected tooth-surface sites, sound as well as ws, in each subject. Plaque Flora and Plaque pH
Actual, unconverted pH values.
c As under "b", but converted pH values. Determination of the relationship between
the plaque flora and the plaque pH for the
same plaque sample (Table 8) involved
Table 3. Plaque pH Profiles Obtained for Sound and ws Tooth-surface Sites in the Group of Eight the following information. The data for
Higher-caries Subjects under Conditions of Normal and Limited Salivary Access after a Sugar Rinse the plaque flora were those reported in the
companion study (van Ruyven et al.,
Tooth-surface Subjects Salivary Time (min) after Start of Rinsing with Glucose Solution 2000; first sampling series) and involved
Sites n Accessa 0 5 10 15 20 30 3 groups of organisms: the MS, the total
undifferentiated "low-pH" bacterial flora
s 7 N 6.75b 5.30 5.00 5.12 5.01 5.09 as enumerated on blood agar, and the
6.48c 4.79 4.63 4.77 4.70 4.68
strongly iodophilic polysaccharide-storing
organisms. These data formed the basis
ws 7 N 6.63 5.10 4.82 4.90 4.88 4.92 for the results shown in Tables 2 through
6.37 4.75 4.59 4.67 4.75 4.65 5 and Table 7 of the companion study,
and the levels of each bacterial group
s 8 L 6.64 5.06 5.09 4.95 5.18 5.48 were expressed as a percentage of the
6.54 4.76 4.93 4.70 4.87 5.11 total flora. Further, the data for the MS
and the iodophilic polysaccharide-storing
ws 8 L 6.64 4.82 4.91 4.75 4.96 5.08 organisms were derived from the samples
6.51 4.67 4.80 4.56 4.71 4.79 from all the buccal surfaces (s and ws
a See Table 2. sites) in each subject, whereas those for
b Mean of the means of sound or ws tooth-surface sites in each subject. Actual unconverted the total "low pH" bacteria included only
pH values. plaque samples from 2 s sites in the low-
c As under "b", but converted pH values. caries subjects and from 2 s and 2 ws sites
in the higher-caries subjects. The data for
KC1 solution into which the glass reference electrode was inserted. plaque pH were obtained from tests with low-caries and higher-
The microelectrode was calibrated against standard pH buffers (pH caries subjects under conditions of normal and limited salivary flow
5.0 and 7.0; Coming, Inc., NY 14831, USA) prior to and after each and rinsing with sugar solution only. They were comprised of data
test as well as during tests if necessary. on the minimum pH in this study (Table 4) and the pH at 10 min
For data presentation, pH values have been used in unconverted after rinsing (Table 2). The latter data were considered to reflect
form as well as after conversion to free H+ concentration (antilog). most closely the pH drop rate (bacterial acidogenic rate). Evaluation
This permits our data to be compared with those from some of the relationships between the plaque percentages of the 3 groups
pertinent studies on plaque pH and caries in which only of organisms and the plaque pH values in this study (Table 8) was
unconverted pH values were used (Stephan, 1944; Fejerskov et al., based on a pairing of both parameters for each sample from the
1992) and, simultaneously, the use of, in certain situations, more specific sites in each subject. It should be noted that the
precise data by pH conversion. The data in Tables 2 through 7 for bacteriological data used pertain to only one plaque sampling
groups of low-caries and higher-caries subjects or sound (s) and ws occasion in the case of each subject (first sampling series). For
tooth-surface sites are based on single tests with each subject and some of the subjects, then, bacterial data were available from the
have been obtained as follows: The mean pH value for each subject test with normal and for other subjects from the test with limited
group at each time point of the pH profiles (Table 2) was calculated salivary access. Hence, the bacteriological data used for either of
from the mean of the readings for each subject; the mean for each both types of categories of salivary access in Table 8 (N or L)
J Dent Res 79(2) 2000 Plaque pH, Microflora, arnd Early Enamel Caries
Table 4. Minimum pH Values Obtained for Low-caries and the Group of
Eight Higher-caries Subjects under Conditions of Normal and Limited
Salivary Access after a Sugar Rinse
Caries Salivary
Group n Accessa Minimum pHb Minimum pHc
Low caries 7 N 5.06 (4.59-5.66) 4.87 (4.53-5.56)
Low caries 7 L 4.86 (4.15-5.14) 4.55 (4.07-5.06)
Higher caries 8 N 4.75 (4.07-5.33) 4.52 (3.98-5.29)
Higher caries 8 L 4.66 (4.25-5.09) 4.45 (4.05-5.00)
a See Table 2.
b Mean (range) of the means of all selected tooth-surface sites,
sound as well as ws, in each subject. Actual, unconverted pH
values.
c As under "b", but converted pH values.
comprise a mixture obtained from plaques studied in both types of
tests.
Another type of evaluation involved the relationship between
the initial plaque levels of strongly iodophilic polysaccharide-storing
bacteria and the initial plaque pH at time 0 min prior to sugar
exposure (Table 9). The bacterial data were derived from the first
sampling series in the companion study (van Ruyven et al., 2000);
the pH data originated from the same plaques used in tests which
involved sugar rinsing as well as topical sugar application. Utilizing,
again, pairing of the bacterial and pH data for each plaque site, we
placed the plaque pH values for all sites into one of 3 categories of
percentage of strongly iodophilic polysaccharide-storing bacteria for
each subject. This permitted the mean pH for the sites in each
bacterial category to be calculated for each subject. These means
were then used for calculation of the mean pH for each bacterial
category for the low-caries and higher-caries subject groups.
Statistical Evaluation
The data obtained were analyzed for statistical significance by
appropriate tests. These include the two-tailed t test, analysis of
variance (ANOVA), and the Spearman rank correlation test. A p
value < 0.05 was considered to indicate statistical significance.
RESULTS
During preliminary tests with the low-caries and higher-caries
test subjects to establish suitable experimental regimens,
multiple plaque pH measurements at different times yielded
generally initial (time 0) pH values of about 6.5 or higher.
However, with some of the subjects, such pH values were found
to be much lower (Table 1, test 1). In these cases, we suspected
unwanted carbohydrate intake shortly before the tests. Support
for this view was obtained when the issue of food intake was re-
discussed with these subjects, and subsequent pH
determinations yielded higher pH values (Table 1, test 2).
Tables 2 through 5 show data from 2 series of pH readings,
one during normal and another during limited salivary access,
which were obtained with the low-caries and eight higher-
caries-group subjects under conditions of a three-minute rinse
with 20% glucose solution. The readings for individual tooth-
surface sites in each subject varied considerably. They also
fluctuated with time, i.e., a reading at 10 min for a specific site
could be higher or lower than that at 5 min. Nevertheless, in
773
Table 5. Minimum pH Values Obtained for Sound and ws Tooth-surface
Sites in the Group of Eight Higher-caries Subjects under Conditions of
Normal and Limited Salivary Access after a Sugar Rinse
Tooth-surface Subjects Salivary
Sites n Accessa Minimum pHb Minimum pHc
s 8 N 4.88 (4.04-5.41) 4.56 (3.95-5.36)
ws 8 N 4.65 (4.16-5.18) 4.43 (4.00-5.17)
s 8 L 4.78 (4.24-5.15) 4.50 (4.04-5.14)
ws 8 L 4.64 (4.29-4.98) 4.35 (3.95-4.88)
a See Table 2.
b Mean (range) of the means of sound or ws tooth-surface sites in
each subject. Actual, unconverted pH values.
c As under "b", but converted pH values.
keeping with the classic "Stephan pH profile", an initially rapid
pH drop to a minimum occurred generally within 5 to 10 min;
this was followed by a discemible gradual return to, in some
instances, the initial ("resting") pH (Tables 2 and 3).
A general trend was observed for lower pH values
(unconverted and converted type) in the higher-caries subjects (s
+ ws) than in the low-caries subjects (s) (Table 2) and on ws
than on s sites in the higher-caries subjects (Table 3) at various
time points under both conditions of salivary access. Only the
pH values (both types) for ws sites during limited salivary
access at all time points except 0 min (Table 3) were statistically
significantly different from those for s sites (t test). Comparison
of normal and limited salivary flow indicated no such trend for
subjects (Table 2) or tooth-surface sites (Table 3).
A same general trend was observed for the minimum pH
values (unconverted and converted), i.e., they were lower in
higher-caries subjects (s + ws) (Table 4) and on ws sites (Table
5) under both conditions of salivary access. It is noteworthy that
the lowest pH values at 10 or 15 min for subjects (Table 2) and
tooth-surface sites (Table 3) differed from the minimum pH
values (Tables 4 and 5), because the latter were not always
reached at the same time at different buccal sites in each subject.
For subjects (Table 4), none of the differences between both
groups was statistically significant (t test, two-way ANOVA).
For tooth-surface sites (Table 5), however, the pH values for ws
sites were significantly lower for unconverted pH values (t test
and two-way ANOVA, p = 0.05) and particularly for converted
values (t test, p < 0.01) under both conditions of salivary access.
For neither subjects (Table 4) nor tooth-surface sites (Table 5)
were the lower pH values (both types) during normal salivary
access statistically different from the values obtained during
limited salivary access (t test).
Comparison of the minimum, unconverted, and converted
pH values for s sites in low-caries subjects (Table 4) with either
the s or the ws sites in higher-caries subjects (Table 5) showed
only a statistical difference between the s sites in low-caries
subjects and ws sites during normal salivary access only (t test).
The tests with topical sugar application and sucking of a
sugary lozenge with the other nine or 10 higher-caries subjects
showed generally higher minimum pH values for the s and ws
sites, particularly in the case of the tests involving the sugary
lozenge, than observed with sugar rinsing (Table 6, see also
Table 5). The differences between the pH values (unconverted
774 Lingstr6m et al.
Table 6. Minimum pH Values Obtained for Sound and ws Tooth-surface
Sites in the Group of 10 Higher-caries Subjects after Topical
Application and Sucking on a Sugary Lozenge
Tooth-
surface Subjects Mode of Sugar
Sites n Administration Minimum pHa Minimum pH6
s 10 topical 4.95 (4.55-5.40) 4.77 (4.50-5.35)
ws 10 topical 4.88 (4.45-5.39) 4.75 (4.51-5.26)
s 9 lozenge 5.22 (4.71-5.66) 4.92 (4.34-5.59)
ws 9 lozenge 5.15 (4.54-5.70) 4.90 (4.55-5.63)
a Mean (range) of the means of sound or ws tooth-surface sites in
each subject. Actual, unconverted pH values.
b As under 'a', but converted pH values.
and converted) for s and ws sites were smaller, and none was
statistically significant (t test).
Table 7 shows data on the initial pH values for subjects and
tooth-surface sites. In view of the narrower range of the pH data,
only unconverted pH values are shown. The results are based on
the combined data from the tests with sugar rinsing and normal or
limited salivary access; the data for 16 of the 18 WS+ subjects (s
+ ws and s and ws sites separately) have been supplemented with
data from the test involving subjects sucking a sugary lozenge. As
can be seen, the initial pH values were closely comparable
regardless of subject or tooth-site caries status (t test).
Table 8 shows results for the relationship between plaque pH
and 2 of the major bacterial groups that were studied
simultaneously (van Ruyven et al., 2000) in relation to tooth-
surface caries status. The data shown are based on a pairing of the
bacterial and pH data for each plaque sample (see "Materials &
methods"). For the Spearnan test, only unconverted pH values
were used. This test evaluated whether lower pH values were
correlated with higher plaque levels of the different types of
bacteria. Under conditions of normal salivary access, this
correlation was highly statistically significant for both bacterial
groups for either the pH at time point 10 min or the minimum pH
reached. However, under conditions of limited salivary access,
this correlation did not reach statistical significance for either of
the bacterial groups. The relationship between MS and plaque pH
(data not shown) was not significant for any of the 4 parameters.
This was also true for the relationships between the total "low-
pH" flora and plaque pH in the tests with either topical sugar
application or sucking of a sugary lozenge (data not shown).
Data on the relationship between the plaque levels of the
strongly iodophilic polysaccharide-storing bacteria and the
initial pH are shown in Table 9. The sample sources are given in
"Materials & methods". No different initial pH values were
observed for the different levels of strongly iodophilic
polysaccharide-storing bacteria in either the low- or higher-
caries subjects.
DISCUSSION
Plaque pH and Caries
It is of interest to compare our pH profiles (unconverted values)
for the tests with sugar rinsing and normal salivary access with
those from the similarly-conducted studies of Stephan (1944)
J Dent Res 79(2) 2000
Table 7. Initial pH Values Obtained for Sound and ws Tooth-surface
Sites in Low-caries and Higher-caries Group Subjects
Caries Group n Site Initial pH
Low caries 8 s 6.75 (6.05-7.22)b
Higher caries 16 s + ws 6.69 (6.02-7.33)
(combined)
Higher caries 6a s + ws 6.77 (6.47-7.26)
Higher caries 16 s 6.78 (6.28-7.43)
(combined)
Higher caries 16 ws 6.69 (5.84-7.46)
(combined)
a The six higher-caries subjects with the highest DMFS indices.
b Mean (range) of the means of one or both types of tooth-surface
sites in each subject. Actual, unconverted pH values.
and Fejerskov et al. (1992). Stephan's study involved 5 subject
groups (I-V) with increasing caries activity (caries-free to
rampant caries) and test plaques on upper and lower labial
tooth surfaces which were sound for groups I, II, and III but a
mixture of sound and carious ("white spot" caries or cavities)
for group V and, to some extent, group IV. The classic
presentation of Stephan's work depicts pH profiles which, with
regard to initial pH and lowest pH reached within 10 min, are
situated in progressively lower pH ranges with increasing
caries status. Leaving aside the issue of the caries-reducing
effect of fluoride (Stephan's study was done in the pre-fluoride
era), our low-caries subjects would fall in Stephan's groups I
and II (caries-free and caries-inactive but evidence of past
caries activity), with the higher-caries subjects in group III
(slightly caries-active) and some in group IV (rapid caries
development and progression). Hence, our data for initial pH
and lowest pH values reached within 5 to 10 min in our caries
groups, also taking into account their range for the subjects,
agree with Stephan's data for groups I, II, and III and perhaps
also IV (upper teeth) not only in terms of trend but also in
terms of actual pH values attained.
The study by Fejerskov et al. involved caries-inactive (C-)
and -active (C+) subject groups and test plaques on only sound
upper and lower interproximal sites. They considered their C+
group similar to Stephan's groups II and III. Regarding initial
pH values, our data agree with those of Fejerskov et al., i.e., the
values for our low-caries and higher-caries subjects are
statistically similar to those of the C- and C+ subjects (upper
teeth). The initial pH values reported by Fejerskov et al., being
lower than ours or Stephan's, could reflect unwanted
carbohydrate consumption shortly before the tests (see Table 1);
in fact, the effect of carbohydrate intake on initial pH can still be
shown 3.5 hr thereafter (Kleinberg and Jenkins, 1964). The C-
and C+ subjects of Fejerskov et al. were also statistically similar
with respect to the lowest pH profile values reached. This agrees
well with the statistically similar lowest pH profile values for s
sites in our higher-caries subjects and in the low-caries subjects.
Based on their scrutiny of Stephan's data, Fejerskov et al.
concluded that the pH values among Stephan's groups I, II, and
III overlapped considerably and seemed essentially similar.
Hence, they suggested that the lower pH values for the truly
different groups V and, perhaps, IV were caused by caries
lesions on part of the test surfaces. In this connection, they had
J Dent Res 79(2) 2000 Plaque pH, Microflora, and Early Enamel Caries 775
found very low initial and minimum pH values in their C+ Table 8. Relationships among the Total 'Low pH" Flora, Strongly
subjects for deep, active occlusal caries lesions; these were lodophilic Polysaccharide-storing Bacteria, and Plaque pH during
much lower than such pH values for sound occlusal surfaces or Normal and Limited Salivary Access
inactive occlusal caries lesions. Fejerskov et al. proposed,
therefore, that plaque pH differences between sound surfaces in Bacterial Salivary Spearman Rank Test
caries-free (or inactive) and caries-positive (or active) subjects Group Accessa pH Correlation (r) Significance
do not generally exist; true differences exist only between sound
surfaces and active caries lesions. "Low-pH" N 10 min -0.41 p = 0.005
The critical issue not addressed by Fejerskov et al. is the "Low-pH" N minimum -0.39 p = 0.007
transition from a caries-inactive to a caries-active tooth-surface "Low-pH" L 10 min -0.10 p = 0.51
state from the standpoint of plaque pH. First, the classic picture "Low-pH" L minimum 0.21 p = 0.16
of Stephan's pH profiles for subject groups with various levels Polysacch. storers N 10min -0.37 p = 0.01
of caries activity should be re-examined by in situ pH Polysacch. storers N minimum -0.42 p = 0.004
measurements of plaque overlying clinically sound buccal Polysacch. storers L 10 min -0.11 p = 0.47
surfaces in Stephan's groups IV and V-type subjects. Second, Polysacch. storers L minimum 0.23 p = 0.15
earlier studies report pH profiles with much lower pH a See Table 2.
minimums and somewhat lower initial pH values for plaques
on sound tooth surfaces in caries-active subjects than for
plaques on sound surfaces in caries-free subjects (Clement et sites was only detectable in tests with sugar rinsing but not
al., 1956; Englander et al., 1956; Moore et al., 1956). Other when sugar exposure was suboptimal. Insight into the plaque-
information from Stephan's study also indicates a progressive pH/caries relationship would therefore benefit from tests
increase of the percentage of plaque pH determinations at involving optimal as well as suboptimal sugar exposure of
which the minimum pH on sound test surfaces was at or below plaques, so that misleading data can be avoided. For example,
5.0 or 5.5 in groups I, II, and III. during our repetition of tests with a few subjects which
Our present data on plaque pH for coronal s and ws sites, involved repeated rather than single topical applications of
the only ones so far available, also support the idea that a sugar solution, differences between s and ws sites which were
decrease of the minimum plaque pH may occur during the hardly discernible after the single sugar application became
transition from a sound to a carious enamel tooth surface. By clearly evident after a second application. It is possible,
contrast, in a recent study, sound and carious (past the early therefore, that the interpretation of findings from some reported
caries stage) root surfaces in the same subjects yielded studies of plaque pH might have been different if the test
undistinguishable plaque pH profiles (Aamdal-Scheie et al., plaques had been exposed to a suboptimal as well as an optimal
1996). However, neither our nor this latter study has yielded sugar concentration (e.g., Abelson and Mandel, 1981).
the most desirable information, i.e., sequential pH data for a The plaque pH response in our study was evaluated under
given tooth-surface area during its transition from a caries- conditions of normal and restricted salivary access, in imitation
inactive to a caries-active state. Thus, surfaces in our higher- of the natural situation. The plaque-pH-elevating effect of
caries subjects considered to be sound are probably a mixture saliva has been documented in various studies focusing
of surfaces without and those with ongoing caries activity. This specifically on this issue (e.g., Englander et al., 1959; Abelson
is supported by the fact that the plaque pH values for sound and Mandel, 1981). In our sugar-rinsing tests, saliva's effect
surfaces, observed in both studies, showed a wide variation. was often only slightly discernible. As indicated by the work by
Further, the development of ws sites or actual cavities may Lindfors and Lagerl6f (1988), this is probably due to the
modify the local milieu and, hence, the pH response to sugar continued exposure of plaque to salivary glucose after the rinse
(Fejerskov et al., 1992). Also, caries development is a time- during conditions of normal but not of restricted salivary
related process, whereas both studies are cross-sectional rather access. Tests in which saliva was restricted after sugar
than longitudinal. Finally, dental plaque pH may not be the exposure indicated that differences between the plaque pH
single decisive factor in caries causation (Margolis et al., values for s and ws sites, observed during normal salivary
1985). Clearly, our understanding of the plaque-pH/caries access, persisted during salivary restriction (Tables 3, 5). This
relationship is still very incomplete, and much more systematic suggests that differences between the pH responses of different
study of this critical issue appears warranted.
Some other methodological aspects of our study Table 9. Relationship of Initial Plaque pH to the Plaque Levels of Strongly lodophilic
also deserve consideration. Under natural acchharide-storing Bacteria
conditions, the intensity of plaque's exposure to Polysacd
carbohydrate will vary widely, from virtually zero % of lodophilic Polysaccharide-storing Bacteria
to a level which appears to permit full expression of Caries Grroup n 0-15 16-30 > 30
plaque's pH-lowering potential. In our study, only
during sugar rinsing but not during tests involving Low cane.s 8 6.74 (5.63-7.22)0 6.96 (6.58-7.46)b
topical sugar application or sucking of the sugary iries 16 6.67 (5.82-7.35) 6.60 (5.94-7.60) 6.63 (5.62-7.51)
lozenge, plaque pH values of around 4.0 were Higherncc'd)
obtained, i.e., close to the presumed theoretical (combine
minimum, as dictated by the acid tolerance of 0 Me(!an (range) of pH values of all selected tooth-surface sites.
plaque bacteria. Analysis of our plaque pH data b Cattegories 16 to 30 and > 30% are combined because of the low sample
indicates that a pH difference between s and ws nurnnber in each category.
776 Lingstr6m et a!.
plaques in the dentition to sugar are caused not only by the
differences in the local action of saliva but also by variations in
the composition of the plaque flora.
Plaque pH, Plaque Flora, and Caries
The plaque pH response on clinically sound tooth surfaces in
vivo is not a constant but varies from negligible to none in
people with little or no oral exposure to carbohydrate, such as
among the Kalahari Bushmen or in stomach-tube-fed
individuals, to very significant (for review, see van Houte,
1980). Earlier-cited evidence indicates that the same is true for
plaque acidogenesis (or pH) in vitro. The literature also
suggests that this variation of the plaque pH response reflects
variations of the intensity of carbohydrate exposure to the teeth
over time (for review, see van Houte, 1980, 1994). This
combined evidence suggests that a gradual increase of
carbohydrate consumption induces a gradual increase of
plaque's pH-lowering potential.
The literature also indicates that increasing caries activity is
accompanied by significant changes in the composition of the
plaque flora toward organisms with a relatively high
acidogenicity and acid tolerance (for review, see van Houte,
1994). This trend is also amply supported by the findings of our
companion study (van Ruyven et al., 2000). Comparison of the
low-caries and higher-caries subjects in this companion study
showed a statistically significant increase of the plaque levels of
4 groups of organisms, the MS (only borderline significance), the
undifferentiated "low-pH" segment of the total plaque flora, the
"low-pH" non-MS, and the strongly iodophilic polysaccharide-
storing bacteria in the higher-caries subjects. The
undifferentiated "low-pH" segment of the total plaque flora,
which includes the MS, lactobacilli, the "low-pH" non-MS, and
many of the iodophilic polysaccharide-storing bacteria, increased
from about 20% for the low-caries subjects to about 60% for the
higher-caries subjects with the highest caries experience. This
indicates bacterial succession of a major magnitude.
Comparison of the pH and bacterial trends for the order of s
sites (low-caries subjects) to s sites (higher-caries subjects) to
ws sites (higher-caries subjects) showed, in both cases, the
greatest difference between the low-caries subjects and the ws
sites in the higher-caries subjects. However, the downward trend
for the factor pH was much more gradual than the upward trend
for the bacterial factor. Thus, the pH difference between the s
sites in the low-caries and higher-caries subjects was relatively
small, whereas the bacterial differences here were very large;
further, the pH difference between s and ws sites in higher-
caries subjects was statistically significant, whereas this
difference for the bacterial groups was only small and not
significant. Overall, our findings support a trend for a positive
correlation between a plaque flora with a higher acid-tolerance
potential and lower plaque pH values. This trend is supported
further by the results of an analysis which is based on a direct
pairing of bacterial and pH factors for each plaque sample under
normal conditions of salivary access (Table 8). Collectively,
these correlations support further the earlier-proposed
hypothesis (van Houte, 1994) according to which a gradual
increase of carbohydrate intake induces specific bacterial
successions which cause a gradual intensification of the plaque
acidity after sugar exposure. Plaque acidity may eventually
reach or exceed a critical level and, as such, contribute to the
undersaturation of the plaque milieu with respect to tooth
J Dent Res 79(2) 2000
minerals and, hence, the induction of mineral loss from the tooth
surfaces (Margolis et al., 1985).
Finally, in view of the progressively decreasing initial pH
values for subjects with increasing cares activity as observed by
Stephan (1944), Gibbons and Kapsimalis (1963) suggested that
iodophilic polysaccharide storage by plaque bacteria could be
responsible for this trend. However, our results (Table 9),
indicating similar initial pH values for plaques with a wide
variety of proportions of strongly iodophilic polysaccharide-
storing bacteria, do not support this suggestion. Although
iodophilic polysaccharide, synthesized by bacteria during
carbohydrate exposure, can prolong bacterial acidogenesis in the
absence of environmental carbohydrate in vitro (Gibbons and
Kapsimalis, 1963), such an effect was not demonstrable in our
study under natural conditions characterized by no carbohydrate
consumption for at least 2 hr prior to the initial pH
determinations.
ACKNOWLEDGMENTS
This work was supported by USPHS Research Grants DE-
07493 and 07009 from the National Institute of Dental and
Craniofacial Research, National Institutes of Health, Bethesda,
MD 20892, USA, and by Grants from Henning och Johan
Throne-Holst Stiftelse, Sweden. We thank Ms. Yi Fan for her
excellent assistance in the analysis of our data.
REFERENCES
Aamdal-Scheie A, Luan WM, Dahlen G, Fejerskov 0 (1996). Plaque
pH and microflora of dental plaque on sound and carious root
surfaces. JDent Res 75:1901-1908.
Abelson DC, Mandel ID (1981). The effect of saliva on plaque pH in
vivo. JDent Res 60:1634-1638.
Clement AJ, Plotkin R, Fosdick LS (1956). The formation of lactic
acid in dental plaques. II. Oral conditions of primitive Bushmen of
the western Kalahari desert. JDent Res 35:786-791.
Englander HR, Carter WMJ, Fosdick LS (1956). The formation of
lactic acid in dental plaques. III. Caries-immune individuals. J
Dent Res 35:792-799.
Englander HR, Shklair IL, Fosdick LS (1959). The effects of saliva on
the pH and lactate concentration in dental plaques. I. Caries-
rampant individuals. JDent Res 38:848-853.
Fejerskov 0, Scheie AA, Manji F (1992). The effect of sucrose on
plaque pH in the primary and permanent dentition of caries-
inactive and -active Kenyan children. J Dent Res 71:25-31.
Gibbons RJ, Kapsimalis B (1963). Synthesis of intracellular iodophilic
polysaccharide by Streptococcus mitis. Arch Oral Biol 8:319-329.
Griffiths SJ (1979). The acidogenic potential of plaque from caries-
free and caries-prone subjects and the effects of nonanoate-glucose
mouthrinses. BrDentJ 147:329-33 1.
Handelman SL, Mills JR, Meggo L (1968). A medium for
differentiating acidogenic bacteria. Arch Oral Biol 13:1187-1196.
Hayes ML, Carter EC, Griffiths SJ (1983). The acidogenic microbial
composition of dental plaque from caries-free and caries-prone
people. Arch Oral Biol 28:381-386.
Igarashi K, Hamada Y, Nishimaki H, Sakurai S, Kamiyama K (1987).
The acidogenic potential of plaque from sound enamel, white spot
lesions, and cavities in children. Pediatr Dent 9:212-215.
Kleinberg I, Jenkins GN (1964). The pH of dental plaques in the
different areas of the mouth before and after meals and their
relationship to the pH and rate of flow of resting saliva. Arch Oral
J Dent Res 79(2) 2000 Plaque pH, Microflora, and Early Enamel Caries
Biol 9:493-516.
Lindfors B, Lagerl6f F (1988). Effect of sucrose concentration in
saliva after a sucrose rinse on the hydronium ion concentration in
dental plaque. Caries Res 22:7-10.
Lingstrom P, Imfeld T, Birkhed D (1993). Comparison of three different
methods for measurement of plaque-pH in humans after
consumption of soft bread and potato chips. JDent Res 72:865-870.
Manly RS, Shiere FR, O'Brien A, Harrington D (1962). Glycolysis in
films of oral samples from persons with different caries rates. J
Dent Res 41:1461-1474.
Margolis HC, Murphy BJ, Moreno EC (1985). Development of
carious-like lesions in partially saturated lactate buffers. Caries
Res 19:36-45.
Margolis HC, Zhang YP, van Houte J, Moreno EC (1993). Effect of
sucrose concentration on the cariogenic potential of pooled plaque
fluid from caries-free and caries-positive individuals. Caries Res
27:467-473.
Miller BF, Muntz JA, Bradel S (1940). Decomposition of carbohydrate
substrates by dental plaque material. JDent Res 19:473-478.
Minah GE, Loesche WJ (1977). Sucrose metabolism in resting-cell
suspensions of caries associated and non-caries-associated dental
plaque. Infect Immun 17:43-54.
Minah GE, Lovekin GB, Finney JP (1981a). Sucrose-induced
ecological response of experimental dental plaques from caries-free
and caries-susceptible human volunteers. Infect Immun 34:662-675.
Minah GE. Matheus M, Finney JP (1981b). The effect of pH on
sucrose metabolism in vitro of sucrose- and saline-exposed
experimental dental plaques in man. Arch Oral Biol 26:153-157.
Moore BW, Carter WJ, Dunn JK, Fosdick LS (1956). The formation of
lactic acid in dental plaques. I. Caries-active individuals. J Dent
Res 35:778-785.
Rosen S, Weisenstein PR (1965). The effect of sugar solutions on pH
of dental plaques from caries-susceptible and caries-free
individuals. J Dent Res 44:845-849.
777
Sansone C, Van Houte J, Joshipura K, Kent R, Margolis HC (1993).
The association of mutans streptococci and non-mutans
streptococci capable of acidogenesis at a low pH with dental caries
on enamel and root surfaces. JDent Res 72:508-516.
Scheie AA, Arneberg P, Orstavik D, Afseth J (1984). Microbial
composition, pH-depressing capacity and acidogenicity of 3-week
smooth surface plaque developed on sucrose-regulated diets in
man. Caries Res 18:74-86.
Stephan RM (1944). Intra-oral hydrogen-ion concentration associated
with dental caries activity. J Dent Res 23:257-266.
Turtola LO, Luoma H (1972). Plaque pH in caries-active and inactive
subjects modified by sucrose and fluoride, with and without
bicarbonate-phosphate. Scand JDent Res 80:334-343.
van Houte J (1980). Bacterial specificity in the etiology of dental
caries. Int Dent J 30:305-326.
van Houte J (1994). Role of micro-organisms in caries etiology. J Dent
Res 73:672-681.
van Houte J, Sansone C, Joshipura K, Kent R (199la). In vitro
acidogenic potential and mutans streptococci of human smooth-
surface plaque associated with initial caries lesions and sound
enamel. J Dent Res 70: 1497-1502.
van Houte J, Sansone C, Joshipura K, Kent R (1991b). Mutans
streptococci and non-mutans streptococci acidogenic at low pH,
and in vitro acidogenic potential of dental plaque in two different
areas of the human dentition. JDent Res 70:1503-1507.
van Houte J, Lopman J, Kent R (1996). The final pH of bacteria
comprising the predominant flora on sound and carious human
root and enamel surfaces. JDent Res 75:1008-1014.
van Ruyven FOJ, Lingstrom P, van Houte J, Kent R (2000).
Relationship among mutans streptococci, "low-pH" bacteria, and
iodophilic polysaccharide-producing bacteria in dental plaque and
early enamel caries in humans. J Dent Res (this issue).
Vratsanos SM, Mandel ID (1982). Comparative plaque acidogenesis of
caries-resistant vs. caries-susceptible adults. J Dent Res 61:465-468.