]. 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 gingivitis. 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 accounted for about two per cent of the plaque flora. In specific areas the gingival condition was correlated 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, subclinical 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 reinstated and in about a week, the plaque was reduced to pre-experimental levels and healthy gingival conditions were reestablished. During the period of plaque accumulation 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 condition of specific tooth surfaces with the bacteriological findings from the same surfaces.
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 experimental 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 Periodontal 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 examinations were performed daily for the first five days, then every second day until the Gingival 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 individual Gingival Index scores were the same as the scores at the start of the experimental period. At the end of the study the participants 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 employed. 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 preparations the gingival curvature can be followed accurately and specific anatomic areas can be identified during microscopic observation. The composition of the gingival flora at the mesial papillae and at the buccal marginal gingivae of the upper left bicuspids was assessed. The presence of cocci, fusobacteria, leptotrichia, spirilla and spirochetes 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 interference 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 characteristics: 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 (gramnegative, curved rods i.e. vibrios and selenomonas), 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 calculated. Bibby (1938a) showed that a differential 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 organisms 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 condition 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 group I group group (17-21 days)

10
.days of no oral

5
hygiene

4 5 6 7 8 9 days of oral h y g i n

10

II (15 days) III (9-13 days)

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

-days

of no oral

hygiene

3 * 5 E 7 8 9 days of oral h y g i e n t ^

to

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 maxilla

plaque index gingival index start of the experiment day of gingivitis experiment

_=-

"" "-"

D s g e

mandible

m~^

^.g_i hs + g -

end of the

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 exceeded 1.0 (mild inflammation). Although the time required to develop gingivitis varied within the group the pattern was essentially the same, and the development 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 gingivitis 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 exhibited the highest Plaque Index scores whereas the oral surfaces with the exception 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 exhibit 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 corresponding 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 develop gingivitis than others, the time required to return to clinically normal conditions 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 examinations of the impression preparations revealed changes in the bacterial composition 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 predominantly filamentous flora took place approximately 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 subjects had developed a generalized gingivitis (Table I). The change in the bacterial flora as related to Plaque Index and Gingival Index of the area selected for bacteriological examination is shown graphically for one typical experimental subject in Fig. 3. Clinical 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 *) Experimental subject No. First day observed Fusobacteria and Filaments
8 2 7 11 4 9 10 1 6 5 3 4 1 2 2 1 2 2 2 2

Clinical data Day of local GI > 1 **) 7 4 7 5 7 9 5 9 7 11 7 7.1 4-11 Day of total GI > 1 9 11 13 13 15 15 15 15 17 19 21 14.8 9-21

Spirilla and Spirochetes

9 7 5

9
7 7 7 7. 4 9 7
7.1 4-9

2
1
1.9 1-4

Average Range

*) 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 filaments cocci <>"-- plaque index

gingival index

Fig. 3. Changes in the bacterial composition of the plaque in the region of the upper left bicuspids 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 *) Experimental subject No. Last day observed Spirilla and Spirochetes 8 2 7 11 4 9 10 1 6
5

Clinical data Day of local GI 0.2 **) Day of total GI ^ 0.2

Fusobacteria and Filaments

0

0***) 0
5

0
0 0

0 5 3 1

9 7 7

11 9 7

1
0 1 5 0 7
2.1 0-7

0
0 2 0 0
0.6

5 3 5 4 3 5 3 7
5.3 3-9

7 9 9 7 7 7 7 7
7.9 7-11

Average Range

0-5

*) 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 disappeared within 24 hours after toothbrushing 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 conditions were reestablished. Smears, of plaque: The gram stained smears of gingival plaque from the mesial and buccal 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 plaque 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 gramnegative cocci and rods. Very few filaments and fusobacteria were observed, and spirilla 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-negative cocci and rods as well as filaments and fusobacteria had increased, and a few spirilla, but no spirochetes were seen. No further changes occurred during the remaining 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 Bacteria gram-positive cocci and rods gram-negative cocci and rods filaments fusobacteria spirilla spirochetes range mean range mean range mean range mean range mean range mean start of experiment 77-100 92 0-20 7 0-3 08 0-1 0.3 0 0 0 0 day of local gingivitis *) 35-79 56 21-40 30 0-17 7 0-12 5 0-3 0.6 0 0 day of generalized gingivitis **) 42-75 54 15-40 28 1-17 7 0-16 7 0-7 2 0-19 2 after one day of oral hygiene 88-100 97 0-6 2 0-3 0.5 0-4 0.4 0 0 0 0 end of oral hygiene period 87-100 97 0-11 2 0-3 0.5 0 0 0 0 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 preparations. One day after reinstitution of oral hygiene the percentage distribution of bacteria 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 composition for the mesial surfaces were essentially the same as those described for the buccal surfaces. At the start of the experiment some of the mesial surfaces showed plaque and a slightly more complex 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 bacterial flora on each tooth surface and to correlate these changes with plaque and gingival condition of the same surface, diagrams 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 increased 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

3020 10

per cent 100 90 80 ^ 70 60-1

50
AO-

30 20 10 0

gramt gram-

cocci and rods cocci and rods fusobacteria

filaments and mb 1 mb 2

subject no.

mb 3

mb

mb 5

mb 6

mb 7

mb 8

m b 9

m b 10

spirils and spirochPtes

m b 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

5 oral hygiene gingival index plaque index

Loo

Lno oral hygiene I I gram* cocci and rods

gram- cocci and rods 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 preexperimental 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 between the gingival condition and the occurrence of spirochetes in the smear. After 1-2 days of oral hygiene the bacterial 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 returned 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 impression preparation. Leukocytes were found to be virtually absent from all pre-

THEILADE, WRIGHT, JENSEN AND L O E

11

parations at the start of the experiment. However, after an average of four days without oral hygiene, leukocytes were present in almost all cases and gradually increased 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. Polymorphonuclear 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 leukocytes 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 accumulation has reached its maximum. Bacteriologically, 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-

rized by a proliferation of fusobacteria and filamentous bacteria in addition to the organisms already present. During the third phase (after 4-9 days) spirilla and spirochetes 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 originally 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 agreement between the results obtained with the two methods. However, spirilla and spirochetes 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, impression preparations are probably more accurate 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 individuals. 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 characterization of clinical oral conditions. Williams, 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 gingival 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 because of the variability in clinical scoring as well as the limitations of the bacteriological 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 increased 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 gingiva and subjects with gingivitis were observed 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 combination of microscopic and cultural counts, found no significant differences, except for an increased number of spirochetes, in gingival debris from individuals with periodontal disease as compared to debris from persons with healthy gingiva. They suggested that the increased amount of gingival debris present in individuals with periodontal disease, rather than a change in bacterial composition, was of etiologic significance. The findings of these authors may be explained 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 obtained 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 specific information regarding the role of the various types of organisms in the development 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 inflammation 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 reaction 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 possibly also fusobacteria and filaments. Certain 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 (SchultzHaudt and Scherp 1955 and 1956). Moreover, 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 grampositive 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 practically all gram-negative organisms indigenous to the oral cavity contain endotoxins (Mergenhagen, Hampp and Scherp 1961). Thus, some of the gram-positive and gramnegative cocci and rods of plaque have been shown to be potentially pathogenic. References Bibby, B. G. 1938a. Determination of percentage occurrence of various groups of mouth bacteria, J. dent. Res. 17: 265-272, Bibby, B. G. 1938b. A comparison of the bacterial flora of different mouths, J. dent. Res. 17: 423-429, Bibby, B. G. 1938c. The bacterial flora in different parts of the mouth. J. dent. Res. 17: 471-476. Carlsson, J. and J, Egelberg, 1965, Effect of diet on early plaque formation in man, Odont. Revy. 16: 112-125, Gibbons, R, J,, S. S, Socransky, S, Sawyer, B, Kapsimalis and J. B. MacDonald, 1963, The microbiota of the gingival crevice area of man-II, The predominant cultivable organisms. Arch, oral Biol. 8: 281-289, Gins, H. A, and G, Mattig. 1941, Ein Verfahren zur "topografischen" Mikroskopie des Zahnfleischsaumes, Zbl. Bakt. 1. Abt. Ref. 146: 189-194, Loe, H. and P. Holm-Pedersen, 1965. Absence and presence of fluid from normal and inflamed gingivae. Periodontics 3: 171-177,

13

Loe, H, and J. Silness, 1963. Periodontal disease in pregnancy. I. Prevalence and severity. Acta odont. scand. 21: 533-551. Loe, H,, Else Theilade and S. B. Jensen, 1965, Experimental gingivitis in man. J. Periodont. 36: 177-187. MacDonald, J, B,, S, S, Socransky and R. J, Gibbons, 1963, Aspects of the pathogenesis of mixed anaerobic infections of mucous membranes, /, dent. Res. 42: 529-544, Mergenhagen, S, E,, E. G, Hampp and H, W, Scherp, 1961, Preparation and biological activities of endotoxins from oral bacteria, J. infect. Dis. 108: 304-310, Russell, A, L, 1956, A system of classification and scoring for prevalence surveys of periodontal disease, J. dent. Res. 35: 350-359, Schultz-Haudt, S,, M, A. Bruce and B. G, Bibby. 1954, Bacterial factors in nonspecific gingivitis, /, dent. Res. 33: 454-458, Schultz-Haudt, S, D, and H. W. Scherp. 1955. Production of hyaluronidase and beta-glucuronidase by viridans streptococci isolated from gingival crevices, /, dent. Res. 34: 924-929. Schultz-Haudt, S, D, and H. W, Scherp, 1956. The production of chondrosulfatase by microorganisms isolated from human gingival crevices, J. dent. Res. 35: 299-307, Silness, J, and H, Loe, 1964. Periodontal disease in pregnancy, IL Correlation between oral hygiene and periodontal condition, Acta odont. scand. 11: 121-135. Socransky, S, S, and R, J, Gibbons, 1965, Required role of Bacteroides melaninogenicus in mixed anaerobic infections. /. infect. Dis. 115: 247-253, Socransky, S, S,, R, L Gibbons, A, C, Dale, L. Bortnick, E, Rosenthal and J, B, MacDonald, 1963, The microbiota of the gingival crevice area of man, I, Total microscopic and viable counts and counts of specific organisms. Arch, oral Biol. 8: 275-280, Williams, N, B., G. J, Parfitt and M, D, Richards, 1964. A preliminary study of microbial smears as an aid in diagnosis of gingival health, /, Periodont. 35: 197-201, 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.