]. periodont. Res. 1 : 1-13.

1966

Experimental gingivitis in man

II. A Longitudinal Clinical and Bacteriological Investigation

ELSE THEILADE, W . H . WRIGHT, S. BORGLUM JENSEN AND HARALD LOE •

The Departments of Microbiology, Oral Diagnosis, and Periodontology,
The Royal Dental College, Aarhus, Denmark

After 9-21 days without oral hygiene eleven experimental subjects with previously excellent oral
hygiene and healthy gingivae developed heavy accumulations of plaque and generalized mild gin-
givitis. The individual rate of development of gingivitis was closely correlated with the rate of
plaque accumulation. Characteristic bacteriological changes were revealed in the plaque along the
gingival margin during this experiment. Initially, i.e. when the teeth were clean and the gingiva
healthy, the extremely sparse plaque flora consisted almost exclusively of gram-positive cocci and
rods. The first phase of plaque development occurred during the first 2 days without oral hygiene
and consisted of a proliferation of the gram-positive cocci and rods and an addition of about 30
per cent gram-negative cocci and rods. During the second phase (after 1-4 days) fusobacteria and
filaments appeared and increased until they each made up about seven per cent of the flora.
During the third phase (after 4-9 days) the flora was supplemented with spirilla and spirochetes,
and at the end of the period without oral hygiene each of these two groups of organisms accoun-
ted for about two per cent of the plaque flora. In specific areas the gingival condition was corre-
lated with the composition of the plaque and it was found that mild gingivitis could be diagnosed
clinieally at approximately the same time as the complex flora was established. However, sub-
clinical inflammation started much earlier, probably as a reaction to the first phases of plaque
development. When oral hygiene was reinstituted, the plaque in most areas disappeared in 1-2
days and after 7-11 days the Plaque Index for each subject was as low as before the experiment.
Correspondingly, after 1-2 days most tooth surfaces only harbored the original sparse flora of
gram-positive cocci and rods. The gingival inflammation in an area usually disappeared one day
after the plaque had been removed.

In a previous study (Loe, Theilade and
Jensen 1965) it was shown that withdrawal
of toothbrushing in 12 healthy persons with
excellent oral hygiene and clinically normal
gingiva resulted in a rapid accumulation of
debris on the teeth. Marginal gingivitis
developed in all subjects in from 10 to 21
days without oral hygiene. Jn a subsequent
period, efficient oral hygiene was reinsta-
ted and in about a week, the plaque was
reduced to pre-experimental levels and
healthy gingival conditions were reestab-
lished. During the period of plaque accu-
mulation and development of gingivitis,
characteristic changes occurred in the
bacterial flora of the gingival margin which
seemed worthy of further investigation.
Therefore, the experiment was repeated in
an attempt to correlate the gingival condi-
tion of specific tooth surfaces with the
bacteriological findings from the same sur-
faces.
Material and Methods
Clinical Examination. Eleven male dental
students (aged 21-27) in the first clinical
year were the experimental subjects. After
the initial examinations, the participants
had their teeth scaled and polished and
were instructed to practice thorough oral
hygiene measures with toothbrush and
wood points. They were then examined
periodically until the individual Gingival
 EXPERIMENTAL G I N G I V I T I S IN MAN
Index scores (Loe and Silness 1963) and
Plaque Index scores (Silness and Loe 1964)
approached zero. At the start of the ex-
perimental period the Gingival Index scores
for the eleven participants ranged between
0.03 and 0.30 (average 0.15). The Plaque
Index scores ranged between 0.04 and 0.15
(average 0.08). The subjects were also
examined for periodontal disease and
pocket formation by means of the Perio-
dontal Index system (Russell 1956). The
Periodontal Index scores ranged between
0.07 and 0.39 (average 0.22). There were
four periodontal pockets present in the
experimental group.
At the start of the experimental period
the participants were instructed to cease all
oral hygiene measures. Clinical examina-
tions were performed daily for the first five
days, then every second day until the Gin-
gival Index score for each person exceeded
1.0 (mild gingivitis). At this point oral
hygiene measures were reinstituted and the
subjects were examined daily for three days
and then every second day until the indi-
vidual Gingival Index scores were the same
as the scores at the start of the experimental
period. At the end of the study the partici-
pants were again examined for periodontal
disease and pocket formation.
Intra-oral photographs were taken at the
beginning of the experimental period, when
gingival inflammation was manifest (GI =
1.0), and at the end of the experiment.
Bacteriological Examination. Bacteriological
samples were collected from the gingival
margin during the clinical examinations:
1) on the day before the participants
ceased to clean their teeth, 2) during the
»no oral hygiene« period, and 3) during
the subsequent period of oral hygiene. Two
methods of obtaining stained preparations
for microscopic examination were emplo-
yed.
1. The impression technique originally de-
veloped by Gins and Mattig (1941) was
used as described by Loe, Theilade and
Jensen (1965). In the impression prepara-
tions the gingival curvature can be followed
accurately and specific anatomic areas can
be identified during microscopic observa-
tion. The composition of the gingival flora
at the mesial papillae and at the buccal
marginal gingivae of the upper left bicu-
spids was assessed. The presence of cocci,
fusobacteria, leptotrichia, spirilla and spi-
rochetes was assessed using a 0 to + + +
score to designate the approximate number
in each observation.
2. Bacterial smears were made of plaque
collected from the gingival margin of the
lower left second bicuspid with a moist
wood point. Two samples were taken each
time, one from the mesial and one from the
buccal surface. In order to minimize inter-
ference with the plaque, very little material
was removed. The smears were prepared
directly from the wood point by mixing
with a loopful of sterile distilled water on
1 X 2 cm areas of the slides. Although
clumps were not entirely avoided, the
suspension made it possible to distinguish
the morphology and staining reaction of
the majority of the bacteria. Samples from
clean surfaces during the period of oral
hygiene contained so few bacteria that they
had to be spread on smaller areas ( 1 X 1
cm). The smears were heat-fixed and
stained with a gram staining procedure.
Differential counts were performed of the
following groups of bacteria according to
morphological and gram staining charac-
teristics: 1) gram-positive cocci and rods,
2) gram-negative cocci and small rods, 3)
gram-positive and gram-negative filaments,
4) fusobacteria (gram-negative, long, thin
rods with tapered ends), 5) spirilla (gram-
negative, curved rods i.e. vibrios and se-
lenomonas), 6) spirochetes. On the basis of
counts of 200 bacteria in representative
 THEILADE, WRIGHT, JENSEN AND L O E

fields, the percentage distribution of these
groups of bacteria in each sample was cal-
culated. Bibby (1938a) showed that a diffe-
rential count of morphological groups in
smears of plaque was reliable, provided
great care was taken during the gram
staining procedure and if at least 200 orga-
nisms were counted.
By these means it was possible to study
the changes in bacteriological composition
of the plaque on one and the same tooth
surface over a period of time and to corre-
late these changes with the gingival condi-
tion in the corresponding area.
Results
Clinical Results
The preliminary scaling and instruction in
oral hygiene techniques gave a group which
exhibited uniformly excellent oral hygiene
and gingival health. However, when active
oral hygiene procedures were withdrawn,
plaque accumulated rapidly. The Gingival

day of gingivitis
(GliUMJ)

15 10 5 2 3 4 5 6 7 8 9 10
.days of no oral hygiene days of oral h y g i » n » — •
group I (17-21 days)

group II (15 days)

group III (9-13 days)

day of g i n g i v i t i s
(GlilOO)

3 * 5 E 7 8 9 to
-days of no oral hygiene days of oral h y g i e n t — ^

Fig. 1. Changes in Gingival Index (top) and Plaque Index (bottom) during the period without oral
hygiene and the subsequent period of oral hygiene. Group I consisted of 3 subjects who developed
gingivitis in 17-21 days. Group II (5 subjects) developed gingivitis in 15 days. Group III (3 subjects)
developed gingivitis in 9-13 days.
 EXPERIMENTAL G I N G I V I T I S IN MAN

all surfaces interprox facial oral

INCISORS

BICUSPIDS

MOLARS

H plaque index
maxilla •""

_•=•- "-"
D
s
gingival index
start of the experiment
mandible m~^ g day of gingivitis

hs + g -^.g_i e end of the experiment

Fig. 2. Plaque Index scores and Gingival Index scores broken down into scores for various groups
of teeth and surfaces (average for 11 subjects).
 THEILADE, WRIGHT, JENSEN AND L O E
Index scores increased correspondingly until
the average score of each participant ex-
ceeded 1.0 (mild inflammation).
Although the time required to develop
gingivitis varied within the group the pat-
tern was essentially the same, and the devel-
opment of gingivitis correlated well with
plaque accumulation (Fig. 1). When the rate
of gingivitis development was evaluated,
the participants could be divided into three
groups. Three individuals developed gingi-
vitis in 9-13 days, five individuals in 15
days, and three in 17-21 days (Fig. 1, Top).
The group which required the longest period
of time to exhibit gingivitis accumulated
plaque at a slower rate than the group
which developed gingivitis more rapidly.
Regardless of the time required for the
Gingival Index to exceed 1.0 the total
plaque accumulation was essentially the
same (Fig. 1).
The total plaque accumulation did not
differ significantly between the maxilla and
the mandible. However, certain tendencies
were noted when specific areas or surfaces
were evaluated separately (Fig. 2). The
interproximal surfaces consistently exhi-
bited the highest Plaque Index scores
whereas the oral surfaces with the excep-
tion of those of the mandibular molars
had the lowest. The incisors exhibited the
greatest variation with the interproximal
areas accumulating heavy deposits and the
oral surfaces acquiring considerably less.
The Gingival Index scores did not ex-
hibit as wide a range of variation as the
plaque scores (Fig. 2). The interproximal
area scores were consistently the highest,
and those of the oral areas of the incisors
were the lowest. Also, the scores of the
facial and oral areas of the maxillary molars
were slightly lower than those of the corre-
sponding mandibular molars.
When active oral hygiene procedures
were reintroduced, the Plaque and Gingival
Index scores rapidly returned to pre-ex-
perimental levels (Fig. 1). Although some
individuals required a longer time to de-
velop gingivitis than others, the time re-
quired to return to clinically normal condi-
tions was essentially the same for all the
subjects (7-11 days). As evaluated by the
Periodontal Index system there did not
appear to be any residual effects of the
gingival changes which occurred during the
experimental period.
Bacteriological Results
Impression preparations: Microscopic exa-
minations of the impression preparations
revealed changes in the bacterial composi-
tion of the developing plaque identical to
the pattern observed in the first study (Loe,
Theilade and Jensen 1965). The change
from a predominantly coccal to a predomi-
nantly filamentous flora took place ap-
proximately two days (range 1-4 days)
after toothbrushing had stopped. Spirilla
and spirochetes were observed after about
seven days (range 4-9 days) without oral
hygiene. Manifestation of clinical gingivitis
at the local level coincided fairly well with
the establishment of this complex flora in
the area. However, 7-8 days usually elapsed
from the time when the total complex flora
was established in the area of the two upper
left bicuspids until the experimental sub-
jects had developed a generalized gingivitis
(Table I).
The change in the bacterial flora as re-
lated to Plaque Index and Gingival Index
of the area selected for bacteriological
examination is shown graphically for one
typical experimental subject in Fig. 3. Cli-
nical gingivitis in this area was detected on
the same day as the total complex flora had
been established. The Gingival Index score
for the individual reached 1.0 eight days
later.
When oral hygiene was reinstituted, the
gingival flora rapidly returned to pre-ex-
 EXPERIMENTAL G I N G I V I T I S IN MAN

Table I
Time of the Change in Local Bacterial Flora Related to the
Appearance of Local and Generalized Gingivitis during the Period
without Oral Hygiene

Bacteriological data *) Clinical data

Experimental First day observed
subject No.
Day of local Day of total
Fusobacteria Spirilla and GI > 1 **) GI > 1
and Filaments Spirochetes

8 4 9 7 9
2 1 7 4 11
7 2 5 7 13
11 2 9 5 13
4 1 7 7 15
9 2 7 9 15
10 2 7 5 15
1 2 7. 9 15
6 2 4 7 17
5 2 9 11 19
3 1 7 7 21

Average 1.9 7.1 7.1 14.8

Range 1-4 4-9 4-11 9-21

*) Based on impression preparations from the region of the upper left

bicuspids.
**) Average of two papillary and two marginal scores frcm the region of
the upper left bicuspids.

r 2.50

DAYS

and .spirochetes <>•"--•« plaque index

filaments gingival index
cocci

Fig. 3. Changes in the bacterial composition of the plaque in the region of the upper left bi-
cuspids in subject No. 4 as seen in impression preparations during 15 days without oral hygiene
and a subsequent period of oral hygiene.
shows the local Plaque Index scores and
the local Gingival Index scores for this region.
The complex flora is established at the day local gingivitis is diagnosed.
 THEILADE, WRIGHT, JENSEN AND LOE

Table II
Time of the Change in Local Bacterial Flora Related to the
Disappearance of Local and Generalized Gingivitis during the
Period with Oral Hygiene

Bacteriological data *) Clinical data

Experimental Last day observed
subject No. Day of local Day of total
Spirilla and Fusobacteria GI £ 0.2 **) GI ^ 0.2
Spirochetes and Filaments

8 0***) 0 9 11
2 0 0 7 9
7 5 5 7 7
11 0 3 5 7
4 0 1 3 9
9 0 1 5 9
10 0 0 4 7
1 0 1 3 7
6 2 5 5 7
5 0 0 3 7
3 0 7 7 7

Average 0.6 2.1 5.3 7.9

Range 0-5 0-7 3-9 7-11

*) Based on impression preparations from the region of the upper left

bicuspids.
*) Average of two papillary and two marginal scores from the region of
the upper left bicuspids.
*) 0 indicates that the organisms were observed on the final day without
oral hygiene and had disappeared on day 1 of the oral hygiene period.

perimental conditions. In nine out of eleven
subjects spirilla and spirochetes had dis-
appeared within 24 hours after toothbrush-
ing had started, and in seven out of eleven
subjects fusobacteria and leptotrichia could
not be detected after 48 hours (Table II).
In all cases but one, cocciform bacteria
were the only organisms observed at the
time when clinically healthy gingival con-
ditions were reestablished.
Smears, of plaque: The gram stained smears
of gingival plaque from the mesial and buc-
cal surfaces of the lower left second bicuspid
confirmed the results from the impression
preparations. At the start of the experiment
the Plaque Index and Gingival Index scores
were zero for all buccal surfaces. The pla-
que flora at that time was predominantly
gram-positive cocci and rods which com-
prised 77-100 per cent (mean 92 per cent)
of the bacteria in the samples (Table III).
The remaining bacteria were mainly gram-
negative cocci and rods. Very few filaments
and fusobacteria were observed, and spi-
rilla and spirochetes were absent.
The buccal gingiva of these teeth had
developed a mild gingivitis (GI = 1 ) after
a period of 7-^13 days without oral hygiene.
At that time, i.e. the day of local gingivitis,
the gram-positive cocci and rods comprised
35-79 per cent (mean 56 per cent) of the
total flora. The percentage of gram-nega-
tive cocci and rods as well as filaments and
fusobacteria had increased, and a few spi-
rilla, but no spirochetes were seen. No
further changes occurred during the re-
maining period of no oral hygiene except
that spirilla and spirochetes increased. Thus
the spirilla and spirochetes appeared later
 EXPERIMENTAL G I N G I V I T I S IN MAN

Table III
Percentage Distribution of Bacteria in Snnears fronn Buccal
Surfaces of the Lower Left Second Bicuspid in Eleven Subjects

start of day of local day of after one day end of oral

Bacteria experiment gingivitis *) generalized of oral hygiene
gingivitis **) hygiene period

gram-positive range 77-100 35-79 42-75 88-100 87-100

cocci and rods mean 92 56 54 97 97
gram-negative range 0-20 21-40 15-40 0-6 0-11
cocci and rods mean 7 30 28 2 2
range 0-3 0-17 1-17 0-3 0-3
filaments
mean 08 7 7 0.5 0.5
range 0-1 0-12 0-16 0-4 0
fusobacteria
mean 0.3 5 7 0.4 0
range 0 0-3 0-7 0 0
spirilla
mean 0 0.6 2 0 0
range 0 0 0-19 0 0
spirochetes
mean 0 0 2 0 0

*) The day when gingivitis was first diagnosed (Gingival Index = 1) on the particular buccal surface.
**) At the end of the no oral hygiene period i.e. when the Gingival Index for the individual reached a value
of one or more.

in the smears than in the impression prepa-
rations. One day after reinstitution of oral
hygiene the percentage distribution of bac-
teria was identical to that at the start of
the experiment. This distribution remained
unchanged throughout the oral hygiene
period (Table III).
The changes in bacterial plaque compo-
sition for the mesial surfaces were essen-
tially the same as those described for the
buccal surfaces. At the start of the ex-
periment some of the mesial surfaces
showed plaque and a slightly more com-
plex flora than that of the buccal surfaces
(Fig. 4). However, this did not seem to
influence the final composition of the flora.
To test the possibility that the frequent
sampling had interfered with the plaque
flora, control samples were taken on the
mesial and buccal surfaces of the lower
right second biscuspid after seven and after
thirteen days without oral hygiene, at the
end of the period without oral hygiene, and
at the end of the cleansing period. These
samples showed a proportional distribution
of bacteria similar to that observed on
corresponding days on the teeth which were
continuously examined.
To demonstrate the changes in the bac-
terial flora on each tooth surface and to
correlate these changes with plaque and
gingival condition of the same surface, dia-
grams were made for each of the mesial
and buccal surfaces examined. A typical
example of the findings is shown in Fig. 5.
At the start of the experiment, the Plaque
Index and Gingival Index were zero and
the smear showed 98 per cent gram-positive
cocci and rods and 2 per cent gram-negative
cocci and rods. During the period without
oral hygiene the quantity of plaque in-
creased as seen by the Plaque Index rising
to 1 after one day and to 2 after seven days.
The earliest change was an increase in
numbers of gram-negative cocci and rods
during the first two days. After two or
three days an increase in filaments and
fusobacteria occurred. These two types of
organisms were usually found together in
about equal numbers and are combined in
 THEILADE, WRIGHT, JENSEN AND L O E

per cent
100

90 •

80 •
70 •

60 •
50
40 •
30-
20 •

10 •

0
I

per cent
100

90

80 ^

70

60-1

50

AO-

30 gramt cocci and rods

20 • gram- cocci and rods

10 - filaments and fusobacteria

0
mb mb mb mb M
mb mb mb mb m b m b
m
m b
spirils and spirochPtes

subject no. 1 2 3 5 6 7 8 9 10 11
Fig. 4. Percentage distribution of bacteria in smears of piaque from the mesiai (m) and the buccal
(b) surfaces of the iower left second biscuspid in each of 11 subjects.
A) At the start of the experiment,
B) At the end of the period without oral hygiene,
C) After one day of oral hygiene.
10 EXPERIMENTAL G I N G I V I T I S IN MAN

30

3
a.

•1-0

Ol
c

Loo
0 1 2 3 A 5
Lno oral hygiene oral hygiene

I I gram* cocci and rods gingival index

gram- cocci and rods plaque index

filaments and fusobacteria

m^l spirils and spirochetes

Fig. 5. Changes in the percentage distribution of bacteria in smears of piaque from the mesial
surface of the lower left second bicuspid in subject No. 10 during 15 days without oral hygiene
followed by 7 days of oral hygiene.
shows the local Plaque Index scores and
the local Gingival Index scores for this surface.

the diagram. In the last phase the spirilla
and spirochetes appeared among the other
components of the flora. In the example
illustrated, spirilla were found in all samples
taken on or after the ninth day, whereas
spirochetes were not seen until the fifteenth
day without oral hygiene. The Gingival
Index for this surface rose to a score of 1
(mild inflammation) on day nine. When
tooth cleansing was resumed, the Plaque
Index fell from 2 to zero within one day
and the bacterial flora returned to pre-
experimental composition in one or two
days. The Gingival Index returned to zero
within two days.
The other surfaces examined in this way
followed the same general pattern. During
the no-cleansing period, spirilla appeared
after 4-11 days and were seen regularly in
later samples. However, spirochetes were
seen in only some of the smears after the
ninth day. In all cases gingivitis developed
within 4-9 days on the mesial and within
7-13 days on the buccal aspects. Thus, there
seemed to be no specific correlation be-
tween the gingival condition and the occur-
rence of spirochetes in the smear.
After 1-2 days of oral hygiene the bac-
terial flora again consisted predominantly
of gram-positive cocci and rods. The other
groups of bacteria persisted longer only on
those surfaces which were insufficiently
cleaned. Generally, the Gingival Index re-
turned to zero one day after the bacterial
plaque had been removed.
Leukocyte Accumulation: The presence or
absence of leukocytes and the approximate
size of any leukocyte accumulation at the
gingival margin were assessed in each im-
pression preparation. Leukocytes were
found to be virtually absent from all pre-
 THEILADE, WRIGHT, JENSEN AND L O E
parations at the start of the experiment.
However, after an average of four days
without oral hygiene, leukocytes were pre-
sent in almost all cases and gradually in-
creased in number during the no-cleansing
period. Immediately after oral hygiene was
reinstituted the number of leukocytes fell
rapidly, and at the end of the experimental
period leukocytes were absent, or present
only in very small numbers. Differential
counts showed that 95-100 per cent of the
cells were polymorphonuclear leukocytes.
Small lymphocytes and larger mononuclear
cells were occasionally observed. Polymor-
phonuclear leukocytes were sometimes seen
to have engulfed bacteria and in certain
areas practically all leukocytes contained
one or more bacteria. In other areas,
however, no signs of phagocytosis could be
seen, and the overall impression was that
only few of the enormous number of leuko-
cytes had participated in phagocytic activity.
Discussion
Changes in Bacterial Composition with
Increasing Age of the Plaque
The present bacteriological results confirm
and elaborate the trends previously reported
(Loe, Theilade and Jensen 1965). Teeth
which are clinically free from plaque harbor
a sparse flora consisting almost exclusively
of gram-positive cocci and rods. During
plaque accumulation definite changes in the
bacterial composition of the plaque take
place. These changes are apparently related
to the age of the plaque more than to its
quantity, since some occur after plaque ac-
cumulation has reached its maximum. Bac-
teriologically, it is possible to distinguish
three phases in the development of plaque.
The first phase consists of proliferation
of resident gram-positive cocci and rods
and the appearance of gram-negative cocci
and rods during the first 2 days. The second
phase occurs after 2-4 days and is characte-
11
rized by a proliferation of fusobacteria and
filamentous bacteria in addition to the or-
ganisms already present. During the third
phase (after 4-9 days) spirilla and spiroche-
tes are added so that a complex flora is
formed. After about 7 days, the various
groups have proliferated to the extent that
the gram-positive cocci and rods which origi-
nally predominated now only constitute
about 50 per cent of the flora. This is in
agreement with the findings of Carlsson
and Egelberg (1965). During the remaining
period without oral hygiene (lasting up to
21 days in some of the subjects) no further
changes occurred.
This pattern of bacterial proliferation was
observed both in impression preparations
and in smears, and there was good agree-
ment between the results obtained with the
two methods. However, spirilla and spiro-
chetes tended to appear earlier and more
constantly in the impressions, where these
organisms were often found in clumps
simulating pure colonies. With the smear
technique these first few isolated colonies
could easily be missed during sampling or
overlooked in the smear. Therefore, impres-
sion preparations are probably more ac-
curate in determining the first appearance
of the various groups of organisms.
Relationship between Plaque Composition
and Gingival Health
Bibby (1938 b and c) found a great variation
in the percentage distribution of bacteria in
smears of plaque from different parts of
the oral cavity and from different indivi-
duals. He failed to establish any relationship
between plaque composition and oral health
and suggested that this might in part be due
to the lack of a definite and detailed charac-
terization of clinical oral conditions. Wil-
liams, Parfitt and Richards (1964) also failed
to find a correlation between gingival health
values for the entire mouth and the results
of microscopic examination of tooth debris.
 EXPERIMENTAL G I N G I V I T I S IN MAN
They did, however, find some correlation
between the gingival health rating for each
tooth and the result of the smear from that
tooth. Their data further indicated that
microbial activity is already well advanced
in cases of suspicious or early gingival
changes.
In the present study it was possible to
establish a correlation between the state of
the gingiva and the bacterial composition
of the plaque by using a detailed system of
scoring the earliest clinical changes in gin-
gival condition for individual tooth surfaces
(Loe and Silness 1963).
In areas with healthy gingiva the tooth
surface is virtually free from plaque and
shows a sparse flora of gram-positive cocci
and rods. Appearance of clinical gingivitis
in a certain area coincides fairly well with
the time when the total complex flora is
established. However, the timing of the
events cannot be determined exactly be-
cause of the variability in clinical scoring
as well as the limitations of the bacterio-
logical techniques. During the period with
oral hygiene it was found that the variations
in time required for individual gingival
areas to return to normal were correlated
with variations in the time necessary to
remove the plaque and reduce the bacterial
flora to its original simple composition. With
efficient oral hygiene this is achieved in 1-2
days for most surfaces. The gingiva in an
area becomes healthy one day after the
bacterial plaque has been removed so that
the tooth surface only harbors a sparse flora
of gram-positive cocci and rods.
Thus gingivitis is associated with an in-
creased number of bacteria (presence of
plaque), and a change in the flora towards
a richer representation of gram-negative
organisms. Similar differences between the
bacterial flora in subjects with healthy gin-
giva and subjects with gingivitis were ob-
served by Schultz-Haudt, Bruce and Bibby
(1954). They found a somewhat higher per-
centage of gram-negative bacteria than was
observed in the present study. This may
indicate that further minor changes would
have taken place if our experimental period
had been extended.
On the other hand. Gibbons et al. (1963)
and Socransky et al. (1963) using a com-
bination of microscopic and cultural counts,
found no significant differences, except for
an increased number of spirochetes, in gin-
gival debris from individuals with periodon-
tal disease as compared to debris from per-
sons with healthy gingiva. They suggested
that the increased amount of gingival
debris present in individuals with periodon-
tal disease, rather than a change in bacterial
composition, was of etiologic significance.
The findings of these authors may be ex-
plained by the fact that even the mouths
deemed periodontally healthy probably had
a few areas with plaque accumulation and
mild gingivitis. By using a pooled sample of
debris from all teeth they may have obtain-
ed a sample consisting partly of debris from
areas of gingivitis. In this way the complex
flora would be present also in samples from
"periodontally healthy mouths".
The present study does not provide spe-
cific information regarding the role of the
various types of organisms in the develop-
ment of gingivitis. However, certain leads
may be found concerning the initiation of
gingival inflammation. Although clinical
gingivitis is manifest about the time when
the total complex flora is established, this
may be a mere coincidence. Subclinical in-
flammation starts much earlier as indicated
by the presence and increasing number of
leukocytes in the impression preparations
already after the first few days. Flow of
gingival fluid (exudate) also starts before
clinical gingivitis can be diagnosed (Loe and
Holm-Pedersen 1965). This suggests that the
initiation of gingivitis may occur as a reac-
tion to the early changes in the bacterial
flora, i. e. the proliferation of gram-positive
 THEILADE, WRIGHT, JENSEN AND L O E
and gram-negative cocci and rods and pos-
sibly also fusobacteria and filaments. Cer-
tain streptococci and diphtheroids are known
to produce enzymes such as hyaluronidase,
beta-glucuronidase, and chondrosulfatase,
all of which have been thought to play a
role in the initiation of gingivitis (Schultz-
Haudt and Scherp 1955 and 1956). More-
over, animal experiments have shown that
transmissible subcutaneous infections can be
produced with the mixed flora of plaque
and that similar infections can be produced
with mixtures of a few strains of gram-
positive and gram-negative rods provided
Bacteroides melaninogenicus is included in
the mixture (MacDonald, Socransky and
Gibbons 1963, Socransky and Gibbons
1965). Finally, it has been found that prac-
tically all gram-negative organisms indige-
nous to the oral cavity contain endotoxins
(Mergenhagen, Hampp and Scherp 1961).
Thus, some of the gram-positive and gram-
negative cocci and rods of plaque have been
shown to be potentially pathogenic.
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Addresses:
The Royal Dental College.
Vennelyst Boulevard,
Aarhus C, Denmark.
Dr, W, H, Wright's present address:
Department of Periodontology,
School of Dental Medicine,
University of Pennsylvania,
Philadelphia, Pa. 19104, U.S.A.