Journal of Dental Research

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Study of Dental Plaque in Orthodontic Patients

J.W. Balenseifen and J.V. Madonia
J DENT RES 1970 49: 320
DOI: 10.1177/00220345700490022101

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Study of Dental Plaque in Orthodontic Patients
J. W. BALENSEIFEN and J. V. MADONIA
Departments of Oral Biology and Microbiology, Loyola University School of Dentistry
Maywood, Illinois 60153
Plaque samples were collected and studied
from orthodontic patients before and after
placement of orthodontic bands and arch
wires. The samples were assayed for changes
in pH, carbohydrate content, and microbial
populations of streptococci and lactobacilli.
Statistically significant increases of plaque
pH, carbohydrate content, and microbial
populations were noted.
Many factors can influence the physical,
chemical, and biologic characteristics of
dental plaque, thereby favoring the develop-
ment of dental caries or periodontal disease.
One environment change that alters the na-
ture of dental plaque is the placement of
orthodontic bands and arch wires. Bloom
and Brown' found an average increase of
90,000 lactobacilli/ml of saliva in ortho-
dontic patients after the placement of bands
and arch wires. Since lactobacilli require a
special plaque environment, this 3,500% in-
crease represents a striking change in the
plaque after banding procedures.
-The purpose of this investigation was to
evaluate changes in the dental plaque of or-
thodontic patients by studying pH, carbo-
hydrate content, and the microbial popula-
tions of streptococci and lactobacilli.
Materials and Methods
Twelve patients between 10 and 16 years
of age were selected for this study from the
orthodontic clinic at Loyola University
School of Dentistry. Plaque samples were
collected before the bands were placed and
one month after the bands and arch wires
were inserted.
The patients were required to brush their
teeth for five minutes using a modified Still-
man technic. Plaque samples and measure-
ments were collected one hour after the
Received for publication January 13, 1969.
320
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supervised brushing, and the patients were
not allowed to eat anything between brush-
ing and sample collection. To maintain good
oral hygiene, the patients were also in-
structed to brush their teeth for five minutes
after meals, using the modified Stillman
technic.
Plaque samples were collected and mea-
surements were recorded one week before
the teeth were banded. The pH of the plaque
was recorded in situ; and then the plaque
was collected for lactobacillus and strepto-
coccus counting. The last test of the plaque
sample was the quantitative microdetermi-
nation of carbohydrate in the plaque by use
of the modified anthrone test. The remain-
ing plaque sample was used for this purpose.
A second set of plaque samples was col-
lected four to five weeks after the bands and
arch wires had been attached to the teeth.
The same measurements were made as on
the first collection: pH, carbohydrate con-
tent, lactobacillus counts, and streptococcus
counts. Differences in the two sets of sam-
ples were compared statistically and eval-
uated for their importance.
PH MEASUREMENTS.-Plaque pH read-
ings were made with an antimony-silver-
exploring mercury electrode similar to the
one used by Stephan.2 The electrode was
constructed of an antimony-silver tip in a
glass tube that contained mercury as a con-
tact between the tip and the lead. A stan-
dard glass calomel electrode was used as a
reference electrode. The reference electrode
was placed in saturated potassium chloride
along with the patient's finger to complete
the electrical circuit. These electrodes were
used in conjunction with a Coleman Me-
trion IV pH meter.
The pH meter was standardized by using
a buffer of known pH before each test.
Comparative tests were made with the ex-
ploring (antimony-silver) electrode and the
Vol 49 No. 2
Coleman glass electrode in different known
pH solutions at 25 C. The maximum varia-
tion was less than 0.10 pH units in the range
of 4.0 to 7.0 pH. This variation is compa-
rable with that found by Parks and Beard.3
Before the antimony-silver electrode was
used in the mouth, it was standardized with
a buffer at pH 6.0, so that a reading could
be made directly on the pH scale of the
potentiometer-electrometer.
The pH readings were taken at various
sites on the teeth. The plaque was usually
found on the buccal aspect of the maxillary
molars, the lingual of the maxillary lateral
incisors, the lingual of the mandibular mo-
lars, and the lingual of the mandibular in-
cisors. A minimum of three readings were
recorded for each patient at a session.
COLLECTION AND WEIGHING OF PLAQUE
SAMPLES.-The plaque was removed from
the teeth with a no. 6 or 7 Garcy curet. The
plaque was placed in a no. 5 gelatin cap-
sule,* which had been shortened to reduce
the weight to approximately 16 mg. The gel-
atin capsules were preweighed and placed
in small vials bearing the patient's name.
Removing the plaque from the teeth and
placing it in the gelatin capsule required
approximately five minutes. The total weight
of the filled capsule was then determined,
and the actual weight of the plaque was
calculated.
The plaque and gelatin capsule were in-
serted in a 5 ml Viritis microhomogenizer
cup. A recorded quantity of water was
added in the ratio of approximately 3 mg
of plaque to 1 ml of water. The total cap-
sule was homogenized in the water at 400
rrm for five minutes.
ENUMERATION OF THE MICROBIAL FLORA.
-The homogenized sample was diluted in
a test tube, 0.1 ml of sample with 0.9 ml
of sterile water. A serial dilution procedure
was then continued until the sample was
diluted to 10-15.
The plating of the diluted sample was
done on two different media. The selective
media for streptococci was Mitis-Salivarius
agar.t The selective media for lactobacilli
was lactobacillus Selective Agar.t All the
petri dishes were inverted and incubated for
48 hours at 37 C. The total streptococci,
Streptococcus mitis, Streptococcus salivar-
* Eli Lilly and Co., Indianapolis, Ind.
t Baltimore Biological Laboratories, Baltimore,
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DENTAL PLAQUE IN ORTHODONTIC PATIENTS 321
ius, and lactobacilli colonies were counted
and recorded after the incubation.
CARBOHYDRATE DETERMINATION.-Carbo-
hydrate determinations were done by a mod-
ification of the technic used by K. J. Weeks.4
The reagent used was a 0.2% solution of
purified anthrone in 98% (v/v) sulfuric
acid. This reagent had a three-day shelf
life. One ml of the carbohydrate solution
was overlaid on the surface of 5 ml of the
anthrone reagent in such a manner as to
prevent mixing of the layers. The layers
were mixed and placed in boiling water for
exactly five minutes, whereupon the solution
was removed and cooled rapidly to room
temperature. The optical density of the mix-
ture was determined at 620 micrometers
(axm) on a Coleman Model 6135, Junior II
Spectrophotometer. The optical-density read-
ings were converted to carbohydrate con-
centrations by comparing with standard glu-
cose solutions. As reported by Weeks, this
method gave results accurate to 0.5 tg.
STATISTICAL ANALYSIS.-The data from
the two groups of experiments were col-
lected and prepared for statistical analysis.
The data were obtained as two series of
measurements from the same groups of in-
dividuals, so that the measurements in each
series are not independent of, but in fact,
are correlated with each other. The statis-
tical analysis of the data was done by the
related-samples or paired-samples technic.
The ratio of the average difference to the
standard error of the average difference is
t = d/ SE - d. This ratio is distributed in
the t curve with N-1 degrees of freedom.
The t value was computed and checked at
the 1 and 5% level of significance.
Results
The results of these experiments are com-
pared in Table 1, and Table 2 shows the
statistical analysis of these comparisons. The
pH of the plaque dropped 0.4 pH unit after
banding procedures, falling from a mean
pH of 6.8 to 6.4. This drop of pH was
significant at the 1% level of significance.
The carbohydrate content of the plaque
went from a mean concentration of 0.0277
mg of carbohydrate/mg of plaque to a con-
centration of 0.0377 mg of carbohydrate/
mg of plaque. This represents a mean in-
crease of .01 mg of carbohydrate/mg of
plaque, and using the t test is significant at
Md. the 5% level. The lactobacilli count showed
322 BALENSEIFEN AND MADONIA J Dent Res March-April 1970
TABLE 1
MEAN PLAQUE MEASUREMENTS PER MG OF PLAQUE
Plaque Characteristics Before Banding After Banding Difference
pH 6.8 6.4 -0.4
Mg carbohydrate 0.0277 0.0377 +0.0100
Lactobacilli count 4.35 X 102 9.81 X 104 +9.80 X 104
S mitis count 2.28 X 107 1.51 x 1014 + 1.50 X 1014
Ssalivarius count 2.92 x 107 1.81 x 1010 +1.80 x 1010
Total streptococci 5.20 x 107 1.51 X 1014 +1.50 X 1014

TABLE 2
STATISTICAL ANALYSIS OF PLAQUE DIFFERENCES PER MG OF PLAQUE
Plaque Characteristics t d SEd P
pH 8.36 0.418 0.05 0.01
Mg carbohydrate 2.35 0.00915 0.0039 0.05
Lactobacilli count 6.33 9380.2 1481.5 0.01
Smitis count 7.89 151.6 X 1012 1.91 X 1013 0.01
S salivarius count 6.8 1.79 X 1010 2.63 X 109 0.01
Total Streptococci 7.22 1.50 X 1014 2.09 X 1013 0.01

a dramatic increase as was suggested by
other studies of saliva."5 The mean lacto-
bacilli count increased by 9.8 X 104 bac-
teria/mg of plaque. This increase was sig-
nificant at the 1% level. The streptococci,
the largest aerobic member of the plaque,
continued to be a large portion of the micro-
flora by increasing to 1.51 X 1014 bacteria
/ mg of plaque. The streptococci were
further broken down into a S mitis and S
salivarius count on the basis of colonial
morphology on Mitis-Salivarius agar. The
greatest increase was shown by S mitis with
a difference of 1.5 X 1014 bacteria/mg of
plaque. S salivarius showed an increase of
1.8 X 1010 bacteria/mg of plaque. The in-
crease in streptococci was statistically sig-
nificant at the 1% level.
Discussion
These experiments show that the presence
of orthodontic appliances in the mouths of
patients stimulates an environmental change.
This change has been characterized by
showing a drop in pH and an increase of
carbohydrate, streptococci, and lactobacilli
(Table 1). These effects do not merely rep-
resent an increase of total plaque, bacteria,
or carbohydrate, but are an indication of the
concentration of these substances in one unit
of mass, such as a mg of plaque. Obviously,
in the orthodontic patient, there is an in-
crease in plaque; but there also is a greater
concentration of bacteria and carbohydrate
in each mg of plaque. This makes this type
of plaque more cariogenic, since there is a
greater concentration of acid-producing bac-
teria on the surface of the teeth. The plaque,
therefore, has a greater potential to lower
the pH to a range that will decalcify the
enamel surface. This drop of pH was clearly
shown in our results.
The amount of S mitis in the plaque of
the orthodontic patients with bands and
arch wires seems high. These data, however,
appear to be accurate, since the same meth-
ods and technics in the same patients before
orthodontic treatment showed plaque bac-
terial counts similar to those found by other
investigators.6-8 The experimental error and
standard deviation were the same in both
measurements.
A number of changes in the plaque flora
can account for this high concentration of
bacteria. A shift in the bacterial flora to
bacteria of a smaller weight and size, eg, S
mitis, could account for these increases and
allow a greater number of bacteria per unit
of weight. The ratio of nonmicrobial sub-
stances to bacteria may also shift, thus in-
creasing the amount of bacteria per unit of
weight. These are just a few of the altera-
tions in plaque suggested by the results of
this experiment.
The pH of plaque was 0.4 of a pH unit
lower in the patients wearing bands and
arch wires (Table 1). This is a significant
difference at the 1% level. It should be kept

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Vol 49 No. 2 DENTAL PLAQUE IN ORTHODONTIC PATIENTS
in mind that this pH reading represents the
acidity of the plaque at rest. The plaque
might have shown a more dramatic drop in
pH if the measurements had been taken
after stimulation with glucose or some other
carbohydrate. The drop in pH of the plaque
at rest does not necessarily represent the
acidogenic capability of the plaque. The
drop in pH would be magnified by making
carbohydrate substrate available to the bac-
teria of the plaque.
The increased concentration of carbohy-
drate is not just an increase of food and
debris but may be an increase of bacterial
products. This increased carbohydrate makes
the plaque more tenacious and prevents the
neutralization of acids and the washing of
the tooth surface by saliva. The observed
increase in carbohydrate represents an in-
crease of both extracellular and intracellular
polysaccharide. The role of extracellular car-
bohydrates in the form of dextrans has been
well established by many investigations.9-12
These dextrans make the plaque tenacious,
resistant to bacterial breakdown, and insol-
uble in saliva. Extracellular levans, such as
those produced by S salivarius, are undoubt-
edly also a part of the plaque carbohydrate.
The plaque changes after the placement
of orthodontic bands and arch wires are the
result of inherent irregularities of fixed or-
thodontic appliances; it is quite conceivable
that these appliances provide additional sur-
face for the collection and retention of food
and debris. This increased supply of sub-
strate for bacteria permits the luxuriant
growth of bacteria, which accounts for the
increased concentration of bacteria in the
plaque. The appliances also protect the
plaque from the actions of brushing, masti-
cation, and salivary flow. They are acidic
and have a low oxygen tension. These char-
acteristics favor lactobacilli growth and ac-
count for the extremely high lactobacilli
levels in orthodontic patients. Omisi and
Kondo13 demonstrated a similar effect by
placing a plastic plate on the palate of pa-
tients and showed an increased number of
lactobacilli due to this type of food trap.
A comparison was made between the lac-
tobacilli and streptococci content of the
plaque and the pH of the plaque. There was
an increase in lactobacilli and streptococci
with an increase in acidity. There was no
direct correlation between the increase in
bacteria and the pH of the total experi-
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323
mental group, but the results of individual
subjects were related positively. The fact
that the experimental group did not show
a linear relationship was probably because
of the tremendous variation in increase of
bacterial content with a small change in pH.
Conclusions
Some chemical and biologic changes in
dental plaque after the placement of ortho-
dontic bands and arch wires were deter-
mined and evaluated. Plaque samples of 12
patients from the orthodontic clinic were
collected before and after the placement of
orthodontic bands and arch wires. They
were assayed for changes in pH, carbohy-
drate content, and the microbial populations
of streptococci and lactobacilli. After the
placement of the orthodontic appliances, the
plaque showed a mean pH drop of 0.4 units,
an increase of 0.01 mg of carbohydrate/mg
of plaque, an increase of 9.8 X 104 lacto-
bacilli/mg of plaque, and an increase of 1.5
X 1014 streptococci/mg of plaque. The strep-
tococci increase was divided into a 1.8 X
1010 increase of S salivarius types and a 1.5
X 1014 increase of S mitis. These observa-
tions were compared statistically and an-
alyzed for their implications concerning oral
health.
References
1. BLOOM, R.H., and BROWN, L.R.: A Study
of the Effects of Orthodontic Appliances
on the Oral Microbial Flora, Oral Surg,
17:758-767, 1964.
2. STEPHAN, R.M.: Changes in Hydrogen-Ion
Concentration on Tooth Surfaces and in
Carious Lesions, JADA 27:718-723, 1940.
3. PARKS, L.R., and BEARD, H.C.: J Amer
Chem Soc 54:856, 1932.
4. WEEKS, K.J.: The Microestimation of Car-
bohydrate by Anthrone, Soc Chem Ind
Victoria 54:1185-1187, 1954.
5. OWEN, O.W.: A Study of Bacterial Counts
(Lactobacilli) in Saliva Related to Ortho-
dontic Appliances, Amer J Orthodont 35:
672-678, 1949.
6. RITZ, H.L.: Microbial Population Shifts in
Developing Human Dental Plaque, Arch
Oral Biol 12:1561-1568, 1967.
7. POOLE, A.E.; GILMOUR, M.N.; and MILLS,
J.R.: The Microbial Content of Natural
and Membrane Plaques, Caries Res 1:239-
246, 1967.
8. STRALFORS, A.: Investigations into the Bac-
terial Chemistry of Dental Plaque, Odont T
58:290-297, 1950.
324 BALENSEIFEN AND MADONIA
9. WOOD, J.M.: Polysaccharide Synthesis and
Utilization by Dental Plaque, J Dent Res
43:955, 1964.
10. WOOD, J.M., and CRITCHLEY, P.: The Ex-
tracellular Polysaccharide Produced from
Sucrose by a Cariogenic Streptococcus,
Arch Oral Biol 11:1039, 1966.
11. GIBBONS, R.J.; BERMAN, K.S.; KNOETTE,
P.; and KAPSIMALIS, B.: Dental Caries and
Alveolar Bone Loss in Gnotobiotic Rats
Infected with Capsule Forming Strepto-
Downloaded from jdr.sagepub.com at BROCK UNIV on September 30, 2014 For personal use only. No other uses without permission.
J Dent Res March-April 1970
cocci of Human Origin, Arch Oral Biol
11:549-560, 1966.
12. GIBBONS, R.J., and BANGHART, S.B.: Syn-
thesis of Extracellular Dextran by Cario-
genic Bacteria and Its Presence in Human
Dental Plaque, Arch Oral Biol 12:11-24,
1967.
13. ONISI, M., and KONDO, W.: Establishing
an Environment for Growth of Acidogenic
Bacteria in the Oral Cavity, J Dent Res
35:596, 1965.