INFECTION AND IMMUNITY, Sept. 1978, p. 821-829 0019-9567/78/0021-0821$02.00/0 Copyright i 1978 American Society for Microbiology
Vol. 21, No. 3
Printed in U.S.A.
Bacteriology of Human Experimental Gingivitis: Effect of Plaque Age S. A. SYED AND W. J. LOESCHE* Dental Research Institute, Department of Oral Biology, Department of Microbiology-Medical School, University of Michigan School of Dentistry, Ann Arbor, Michigan 48109
Received for publication 28 June 1978
Twenty-five subjects with previously excellent hygiene and healthy gingiva developed heavy plaque accumulations and bleeding or nonbleeding gingivitis about certain papilla after 21 days of no oral hygiene. Gingival marginal plaque about a single papilla was collected at 0, 1, 2, and 3 weeks of no oral hygiene.in each subject. The plaque was dispersed, serially diluted, and plated on MM10 sucrose agar in an oxygen-free atmosphere. From 50 to 100 colonies from a single high-dilution plate were characterized for each sample. Over 8,500 isolates were partially characterized and placed into one of 29 taxonomic species or groups. The flora was predominantly gram-positive at all time periods. Streptococcal species dominated in the 0- and 1-week-old plaques, i.e. 62 and 43% of the colonyforming units (CFU), but dropped to 26 to 32% of the CFU in the 2- and 3-weekold plaques. Actinomyces species dominated in the older plaques, i.e., 40 to 50% of the CFU. Actinomyces israelii was the most prominent species in the older plaques. Veillonella accounted for 15 to 20% of the CFU at all time periods. Although the other gram-negative species increased with time, collectively they averaged less than 5% of the CFU at week 3. The shift from a Streptococcusdominated plaque to an Actinomyces-dominated plaque was the most striking microbial change observed as the plaque aged.
Dental plaque has been associated with the human periodontal disease on the basis of epidemiological observations (28, 30, 34) and clinical observations (13, 17). However, it was not until 1965 that conclusive evidence was obtained which correlated plaque accumulation with gingivitis, the earliest stage of periodontal disease (17). Volunteers were brought into a state of excellent oral health by a supervised program of mechanical cleaning. Once optimal health was achieved, all mechanical procedures were withdrawn and plaque was allowed to accumulate. The amount of plaque and the degree of gingivitis were monitored during a 3-week period. When the plaque was removed at day 21, and oral hygiene procedures reinstituted, the gingivitis disappeared within 5 to 7 days. Bacteriological smears of the plaque made at different time intervals showed that gram-negative organisms increased in number and proportions at about the time that gingivitis occurred (17, 37). No cultural studies were performed, nor were the bacteriological smears associated with either plaque or gingivitis scores about a discrete site. In the present investigation the experimental gingivitis model was repeated, and extensive bacteriological analyses of plaque removed from discrete sites were performed at 0, 1, 2, and 3
weeks on the no-brushing regimen. The effect of plaque accumulation and degree of gingivitis upon the plaque flora will be presented in a separate report (22).
MATERIALS AND METHODS Subjects. Twenty-four male dental students and one female dental student suspended all oral hygiene procedures for a 3-week period. These students had been selected from a larger pool of candidates on the basis of having a minimal number of dental restorations in their mouths and having a generally high level of gingival health. Pilot studies had indicated that the comprehensive bacteriological studies could only be performed on about four students at a time. Accordingly the students were sorted into six groups of four or five students, and each group was consecutively brought through the no-hygiene experimental period. Sample sites. The students in each group were brought into a state of excellent oral hygiene by professionally administered mechanical cleanings and by vigorous brushing and flossing by the student. When this point was reached, a 0-time plaque sample was
removed from one or more of three separate interproximal sites, e.g., the mesial gingival interproximal surface of the upper left premolar, tooth no. 13; the mesial gingival interproximal surface of the lower left central incisor, tooth no. 24; and the mesial gingival interproximal surface of the lower right first molar, tooth no. 30. The 0-time sample was collected approx-
821
822
INFECT. IMMUN.
SYED AND LOESCHE
imately 4 to 5 h after the volunteers' morning brushing and prior to the noon meal. The participants then began their 3-week period of no oral hygiene. For each participant, a 0-, 1-, 2-, or 3-week plaque sample was obtained from one of the test teeth. The tooth sites were rotated among the participants so as to give an approximately similar number of 0-, 1-, 2-, and 3-weekold plaque samples from each tooth. These particular teeth were chosen because they represent different anatomical locations within the dentition and are included among the teeth examined in the widely used clinical Periodontal Index (27). At the 3-week sampling period, additional plaques were collected from maxillary premolars and anterior teeth (teeth numbered 4 to 12) that exhibited obvious plaque and/or gingivitis. There were no significant differences between these new sites and the original test sites in regard to the clinical and bacteriological parameters under investigation, as determined by an analysis of variance and the Kruskal-Wallis test. Bacteriological procedures. Efforts were made to remove all the visible plaque present at the gingival margin of the test site. The plaque was collected with the tip of one or more Morse scalers held with a hemostat. The tips plus plaque were dropped into 10 ml of reduced transport fluid (35) and immediately brought to the laboratory. This collection procedure was used to minimize the plaque loss that might occur in the transfer of plaque from a scaler (curette) to a transport fluid in those instances in which the plaque is simply shaken off the dental instrument. In particular, this method reduced the plaque loss which could occur in the 0-time samples in which the plaque amount was so minimal as not to be visible. The plaque was dispersed for 5 s by sonic disruption under a stream of oxygen-free gas, i.e. 85% N2, 10% H2, and 5% CO2. The samples were brought into an anaerobic chamber and serially diluted in reduced transport fluid, and 0.05-ml samples of suitable dilutions were plated in duplicate on MM10 sucrose agar (21, 36) and Schaedler agar within 30 to 60 min after collection. The plates were incubated for 5 to 7 days anaerobically or for 2 to 5 days aerobically. One MM10 sucrose plate from each plaque sample with about 50 to 100 colonies was retained within the chamber. Each colony was subcultured to a basal esculin nitrate broth, the composition of which is shown in Table 1. Growth in this broth was inoculated onto MM10 sucrose plates for aerobic incubation and was used for Gram staining and for measurement of esculin hydrolysis, nitrate reduction, and indole production. Samples of this broth were passed into suitable media for measurement of catalase production, gelatin liquefaction, and glucose and mannitol fermentations. Gas-liquid chromatographic analysis of volatile and nonvolatile acid end products (6) was performed when necessary either to confirm the identity of representative isolates within a grouping shown in Table 2, or to assist in the identification of an isolate which did not fall into any of these groupings. These particular tests were chosen because they could be formulated into a simple taxonomic scheme that would permit the recognition of 29 species or groups of organisms in the plaque. This scheme, given in Table 2, was compiled from several references (2, 6,
TABLE 1. Composition of basal esculin nitrate broth Constituents"
Tryptone Yeast extract (Difco) Potassium nitrate Esculin (Difco) Glucose Sodium chloride Mineral salt solution no. 1' Mineral salt solution no 2' Distilled water
Amt 10 g 5g Ig 0.5 g 0.5 g 5g 75 ml 75 ml 800 ml
I ml Hemin (1-mg/ml stock solution)' 1 ml Menadione (0.5-mg/ml stock solution)Y 0.2 g Dithiothreitol (Sigma) 0.4 g Sodium carbonate (anhydrous) 50 ml Distilled water " The first nine constituents formed a medium which was autoclaved at 121'C for 15 min. A solution of the last five compounds was sterilized by means of a membrane filter (0.22-tLm pore size) and added to the sterile medium, cooled to 50'C. The solution should be freshly prepared each time the medium is made. bContained 1.2% NaCi, 1.2% (NH4)2SO4, 0.6% KH2PO4, and 0.25% MgSO4 in distilled water. Contained 0.6% K2HPO4 in distilled water. d Prepared by dissolving 0.2 g of hemin (Eastman Kodak Co., Rochester, N.Y.) in 200 ml of 0 iN KOH solution in 50% ethanol. The solution was stored in a screw-cap bottle in the refrigerator. e Prepared in 50% ethanol in distilled water, filter sterilized, and stored in the refrigerator. Commercial source of menadione: Nutritional Biochemical Corp., Cleveland, Ohio. 19) and, in the case of the actinomycetes, drew upon the numerical taxonomic study of Holmberg and Hallander (7) which separated Actinomyces viscosus and Actinomyces naeslundii by the single criterion of being catalase positive or negative. All other anaerobic gram-positive rods with branching Gram-stain morphology were categorized as Actinomyces israelii, with the exception of a group which was unable to hydrolyze esculin. These esculin-negative isolates are listed as unidentified actinomyces in the tables and would include those strains of Actinomyces odontolyticus that are esculin negative (32). Approximately 200 to 400 isolates were characterized each week, and during the entire study approximately 8,500 isolates were characterized. Plaque and gingivitis scores. The amount of plaque and gingivitis about each site was estimated by modifications of the plaque and gingivitis indexes (15). In the described plaque index, the amount of plaque is visually estimated on the tooth surface at the gingival margin and given a score. We used this score to assign an initial maximal value to the plaque. Because we physically removed all the plaque from the site, we were able to use the amount of plaque on the scaler to assign the final score, i.e., 0.5 or 1, 1.5 or 2, 2.5 or 3.0. Two examiners concurred on each score. The ability of this scoring system to reflect plaque wet weight had
VOL. 21, 1978
EFFECT OF TIME ON PLAQUE BACTERIA
823
TABLE 2. Identification to species ofgingivitis isolates Taxonomic criteria"
Group Gram stain
Gram-positive cocci Micrococci Streptococci S. mutans S. sanguis S. salivarius S. mitis (mitior) Enterococci
Colony type
Aero- Terminal bic pH in Catalase growth glucose
Nitrate Esculin reduc- hydroltion ysis
GLC acids"
Other'
+C + +
5.5 5.5
+
-
-
Heaped Adherent Mucoid
MOH+ MOH-
MOH+,
+
Gel V Anaerobic cocci Unidentified
Gram-negative cocci Neisseria Veillonella Gram-positive rods Actinomyces Viscous naeslundii israelii Unidentified Bacillus
Clostridium Corynebacterium Rothia
-C +
-
5.5 6.5
+ -,+
5-6 5-6 5-6 5-6 5-6 5-7
+
AP
+
+R
Branching
ALS + +
+
Spores Spores
_
+
Nocardia Bacterionema
Propionibacterium
-
+ +
+ +
-
V
+
+
+
-
V V
V V
+
+
_
+ + + +
+ + + +
+ + + +
GelGelGelGelGel+ Gel V Gel+ Gel+ GelGel-
Unidentified
Gram-negative rods
-R
Bacteroides Black
melaninogenicus Other F. nucleatum C. sputorum Unidentified
Speckled Motile
-
_ -
5-7 5.5 6-7 6.5
-
ABS
Indole V
ABL None
Indole V
+, Positive; -, negative; V, variable. "GLC, Gas-liquid chromatography. A, Acetic acid; P, propionic acid; L, lactic acid; S, succinic acid; B, butyric acid. ' MOH, Mannitol; gel, gelatin hydrolysis.
"
been shown in a previous study, where the correlation coefficient for plaque wet weight and plaque score was r = 0.84 (20). The majority of the plaque samples removed in the present study were too small to weigh reliably. However, some estimate of plaque mass was obtained by measuring the plaque DNA content with ethidium bromide using a fluorometric procedure (3). Sufficient numbers of sample sites had a degree of gingivitis that was intermediate to the described criteria. Accordingly, if a site was not perfectly healthy, i.e. gingivitis score = 0, but failed to show the edema and redness associated with a gingivitis score of 1.0, a value of 0.5 was assigned. If a site bled by spotting and did not flow along the gingival margin, a score of 1.5 was given. A score of 2.0 was given when the blood flowed along the gingival margin. These intermediate values increased the sensitivity of our clinical disease measurements and, since they represented the concurrence of two examiners, were thought to be more
accurate than scoring systems involving only one examiner. Statistical analysis. The various clinical and bacteriological data were obtained for each visit and recorded for statistical analysis using the MIDAS statistical package contained in the Michigan Terminal System. The compiling of the bacteriological data was independent of the clinical data. Analyses of variance were performed on the data, stratified according to age of plaque, plaque score, and gingivitis score. Whenever significance was found, comparisons between entries were performed using the Scheffe test. The Scheffe test is a method for testing the significance of one or more comparisons of mean values arising in an analysis of variance where the entire experimental error rate is taken into consideration (29). The same data set, again stratified according to age of plaque, plaque score, and gingivitis score, was analyzed using the nonparametric Kruskal-Walhis ranking test (31).
INFECT. IMMUN.
SYED AND LOESCHE
824
RESULTS No subject withdrew from the experiment, nor complained of any inconveniences caused by the period of no oral hygiene. No systemic antibiotics were taken by the subjects for medical reasons during the experimental period. There was an obvious clinical increase in plaque about all the teeth in the subjects' mouths, but the number of gingival sites at 3 weeks which exhibited bleeding were few. This was particularly so among the sample sites, where only 6 of 24 sites had a gingivitis that bled with a flow upon probing (Table 3). Additional gingival sites were sampled at 3 weeks from maxillary anterior teeth so as to increase the number of plaques associated with gingival bleeding (Table 3). The correlation between plaque and gingivitis scores with each other and with the total count, DNA content, and plaque age was determined in those plaques with a measurable amount of DNA (Table 4). The plaque score correlated significantly with plaque age, gingivitis score, total count, and DNA content. The gingivitis score correlated significantly with plaque age and with the plaque score, but showed no relationship with DNA content and total bacterial count (Table 4). At 0 time, the plaque score averaged 0.35 and the gingivitis score averaged 0.25 (Table 5). Approximately 3 x 106 organisms could be cultured from the tooth surface sites, even though half the sites had a plaque score of 0.0 (Table 3). After 1 week of no brushing, the plaque significantly increased to about 70 to 100% of its 3-week-old value as judged by the increase in plaque scores, DNA content in miTABLE 3. Frequency distribution of plaque and gingivitis scores as a function of time Time Score 0
Plaque 0.0
week
1
week 2 weeks
3 weeks"
score
0.5 1.0 1.5 2.0 2.5
13 9 3 0 0 0
1 0 8 7 8 0
0 0 2 9 13 0
17 4 4 0 0
2
1 2 14 6 1
0 0 0 9 11 4
(3) (2) (13) (6)
Gingivitis score
0.0 0.5 1.0 1.5 2.0
8 13 1 0
Figures in parentheses sites sampled at 3 weeks.
are
0 3 (3)
12 (5) 3 (4) 6 (12)
number of additional
TABLE 4. Correlation coefficient of plaque and gingivitis scores with various plaque parameters' Plaque score Parameter
Age of plaque Plaque score Gingivitis score DNA content (jig) Total bacterial
Gingivitis score
r coeffi- Signifi- r coeffi- Significient
cance
cient
cance
0.66
Vol. 21, No. 3
Printed in U.S.A.
Bacteriology of Human Experimental Gingivitis: Effect of Plaque Age S. A. SYED AND W. J. LOESCHE* Dental Research Institute, Department of Oral Biology, Department of Microbiology-Medical School, University of Michigan School of Dentistry, Ann Arbor, Michigan 48109
Received for publication 28 June 1978
Twenty-five subjects with previously excellent hygiene and healthy gingiva developed heavy plaque accumulations and bleeding or nonbleeding gingivitis about certain papilla after 21 days of no oral hygiene. Gingival marginal plaque about a single papilla was collected at 0, 1, 2, and 3 weeks of no oral hygiene.in each subject. The plaque was dispersed, serially diluted, and plated on MM10 sucrose agar in an oxygen-free atmosphere. From 50 to 100 colonies from a single high-dilution plate were characterized for each sample. Over 8,500 isolates were partially characterized and placed into one of 29 taxonomic species or groups. The flora was predominantly gram-positive at all time periods. Streptococcal species dominated in the 0- and 1-week-old plaques, i.e. 62 and 43% of the colonyforming units (CFU), but dropped to 26 to 32% of the CFU in the 2- and 3-weekold plaques. Actinomyces species dominated in the older plaques, i.e., 40 to 50% of the CFU. Actinomyces israelii was the most prominent species in the older plaques. Veillonella accounted for 15 to 20% of the CFU at all time periods. Although the other gram-negative species increased with time, collectively they averaged less than 5% of the CFU at week 3. The shift from a Streptococcusdominated plaque to an Actinomyces-dominated plaque was the most striking microbial change observed as the plaque aged.
Dental plaque has been associated with the human periodontal disease on the basis of epidemiological observations (28, 30, 34) and clinical observations (13, 17). However, it was not until 1965 that conclusive evidence was obtained which correlated plaque accumulation with gingivitis, the earliest stage of periodontal disease (17). Volunteers were brought into a state of excellent oral health by a supervised program of mechanical cleaning. Once optimal health was achieved, all mechanical procedures were withdrawn and plaque was allowed to accumulate. The amount of plaque and the degree of gingivitis were monitored during a 3-week period. When the plaque was removed at day 21, and oral hygiene procedures reinstituted, the gingivitis disappeared within 5 to 7 days. Bacteriological smears of the plaque made at different time intervals showed that gram-negative organisms increased in number and proportions at about the time that gingivitis occurred (17, 37). No cultural studies were performed, nor were the bacteriological smears associated with either plaque or gingivitis scores about a discrete site. In the present investigation the experimental gingivitis model was repeated, and extensive bacteriological analyses of plaque removed from discrete sites were performed at 0, 1, 2, and 3
weeks on the no-brushing regimen. The effect of plaque accumulation and degree of gingivitis upon the plaque flora will be presented in a separate report (22).
MATERIALS AND METHODS Subjects. Twenty-four male dental students and one female dental student suspended all oral hygiene procedures for a 3-week period. These students had been selected from a larger pool of candidates on the basis of having a minimal number of dental restorations in their mouths and having a generally high level of gingival health. Pilot studies had indicated that the comprehensive bacteriological studies could only be performed on about four students at a time. Accordingly the students were sorted into six groups of four or five students, and each group was consecutively brought through the no-hygiene experimental period. Sample sites. The students in each group were brought into a state of excellent oral hygiene by professionally administered mechanical cleanings and by vigorous brushing and flossing by the student. When this point was reached, a 0-time plaque sample was
removed from one or more of three separate interproximal sites, e.g., the mesial gingival interproximal surface of the upper left premolar, tooth no. 13; the mesial gingival interproximal surface of the lower left central incisor, tooth no. 24; and the mesial gingival interproximal surface of the lower right first molar, tooth no. 30. The 0-time sample was collected approx-
821
822
INFECT. IMMUN.
SYED AND LOESCHE
imately 4 to 5 h after the volunteers' morning brushing and prior to the noon meal. The participants then began their 3-week period of no oral hygiene. For each participant, a 0-, 1-, 2-, or 3-week plaque sample was obtained from one of the test teeth. The tooth sites were rotated among the participants so as to give an approximately similar number of 0-, 1-, 2-, and 3-weekold plaque samples from each tooth. These particular teeth were chosen because they represent different anatomical locations within the dentition and are included among the teeth examined in the widely used clinical Periodontal Index (27). At the 3-week sampling period, additional plaques were collected from maxillary premolars and anterior teeth (teeth numbered 4 to 12) that exhibited obvious plaque and/or gingivitis. There were no significant differences between these new sites and the original test sites in regard to the clinical and bacteriological parameters under investigation, as determined by an analysis of variance and the Kruskal-Wallis test. Bacteriological procedures. Efforts were made to remove all the visible plaque present at the gingival margin of the test site. The plaque was collected with the tip of one or more Morse scalers held with a hemostat. The tips plus plaque were dropped into 10 ml of reduced transport fluid (35) and immediately brought to the laboratory. This collection procedure was used to minimize the plaque loss that might occur in the transfer of plaque from a scaler (curette) to a transport fluid in those instances in which the plaque is simply shaken off the dental instrument. In particular, this method reduced the plaque loss which could occur in the 0-time samples in which the plaque amount was so minimal as not to be visible. The plaque was dispersed for 5 s by sonic disruption under a stream of oxygen-free gas, i.e. 85% N2, 10% H2, and 5% CO2. The samples were brought into an anaerobic chamber and serially diluted in reduced transport fluid, and 0.05-ml samples of suitable dilutions were plated in duplicate on MM10 sucrose agar (21, 36) and Schaedler agar within 30 to 60 min after collection. The plates were incubated for 5 to 7 days anaerobically or for 2 to 5 days aerobically. One MM10 sucrose plate from each plaque sample with about 50 to 100 colonies was retained within the chamber. Each colony was subcultured to a basal esculin nitrate broth, the composition of which is shown in Table 1. Growth in this broth was inoculated onto MM10 sucrose plates for aerobic incubation and was used for Gram staining and for measurement of esculin hydrolysis, nitrate reduction, and indole production. Samples of this broth were passed into suitable media for measurement of catalase production, gelatin liquefaction, and glucose and mannitol fermentations. Gas-liquid chromatographic analysis of volatile and nonvolatile acid end products (6) was performed when necessary either to confirm the identity of representative isolates within a grouping shown in Table 2, or to assist in the identification of an isolate which did not fall into any of these groupings. These particular tests were chosen because they could be formulated into a simple taxonomic scheme that would permit the recognition of 29 species or groups of organisms in the plaque. This scheme, given in Table 2, was compiled from several references (2, 6,
TABLE 1. Composition of basal esculin nitrate broth Constituents"
Tryptone Yeast extract (Difco) Potassium nitrate Esculin (Difco) Glucose Sodium chloride Mineral salt solution no. 1' Mineral salt solution no 2' Distilled water
Amt 10 g 5g Ig 0.5 g 0.5 g 5g 75 ml 75 ml 800 ml
I ml Hemin (1-mg/ml stock solution)' 1 ml Menadione (0.5-mg/ml stock solution)Y 0.2 g Dithiothreitol (Sigma) 0.4 g Sodium carbonate (anhydrous) 50 ml Distilled water " The first nine constituents formed a medium which was autoclaved at 121'C for 15 min. A solution of the last five compounds was sterilized by means of a membrane filter (0.22-tLm pore size) and added to the sterile medium, cooled to 50'C. The solution should be freshly prepared each time the medium is made. bContained 1.2% NaCi, 1.2% (NH4)2SO4, 0.6% KH2PO4, and 0.25% MgSO4 in distilled water. Contained 0.6% K2HPO4 in distilled water. d Prepared by dissolving 0.2 g of hemin (Eastman Kodak Co., Rochester, N.Y.) in 200 ml of 0 iN KOH solution in 50% ethanol. The solution was stored in a screw-cap bottle in the refrigerator. e Prepared in 50% ethanol in distilled water, filter sterilized, and stored in the refrigerator. Commercial source of menadione: Nutritional Biochemical Corp., Cleveland, Ohio. 19) and, in the case of the actinomycetes, drew upon the numerical taxonomic study of Holmberg and Hallander (7) which separated Actinomyces viscosus and Actinomyces naeslundii by the single criterion of being catalase positive or negative. All other anaerobic gram-positive rods with branching Gram-stain morphology were categorized as Actinomyces israelii, with the exception of a group which was unable to hydrolyze esculin. These esculin-negative isolates are listed as unidentified actinomyces in the tables and would include those strains of Actinomyces odontolyticus that are esculin negative (32). Approximately 200 to 400 isolates were characterized each week, and during the entire study approximately 8,500 isolates were characterized. Plaque and gingivitis scores. The amount of plaque and gingivitis about each site was estimated by modifications of the plaque and gingivitis indexes (15). In the described plaque index, the amount of plaque is visually estimated on the tooth surface at the gingival margin and given a score. We used this score to assign an initial maximal value to the plaque. Because we physically removed all the plaque from the site, we were able to use the amount of plaque on the scaler to assign the final score, i.e., 0.5 or 1, 1.5 or 2, 2.5 or 3.0. Two examiners concurred on each score. The ability of this scoring system to reflect plaque wet weight had
VOL. 21, 1978
EFFECT OF TIME ON PLAQUE BACTERIA
823
TABLE 2. Identification to species ofgingivitis isolates Taxonomic criteria"
Group Gram stain
Gram-positive cocci Micrococci Streptococci S. mutans S. sanguis S. salivarius S. mitis (mitior) Enterococci
Colony type
Aero- Terminal bic pH in Catalase growth glucose
Nitrate Esculin reduc- hydroltion ysis
GLC acids"
Other'
+C + +
5.5 5.5
+
-
-
Heaped Adherent Mucoid
MOH+ MOH-
MOH+,
+
Gel V Anaerobic cocci Unidentified
Gram-negative cocci Neisseria Veillonella Gram-positive rods Actinomyces Viscous naeslundii israelii Unidentified Bacillus
Clostridium Corynebacterium Rothia
-C +
-
5.5 6.5
+ -,+
5-6 5-6 5-6 5-6 5-6 5-7
+
AP
+
+R
Branching
ALS + +
+
Spores Spores
_
+
Nocardia Bacterionema
Propionibacterium
-
+ +
+ +
-
V
+
+
+
-
V V
V V
+
+
_
+ + + +
+ + + +
+ + + +
GelGelGelGelGel+ Gel V Gel+ Gel+ GelGel-
Unidentified
Gram-negative rods
-R
Bacteroides Black
melaninogenicus Other F. nucleatum C. sputorum Unidentified
Speckled Motile
-
_ -
5-7 5.5 6-7 6.5
-
ABS
Indole V
ABL None
Indole V
+, Positive; -, negative; V, variable. "GLC, Gas-liquid chromatography. A, Acetic acid; P, propionic acid; L, lactic acid; S, succinic acid; B, butyric acid. ' MOH, Mannitol; gel, gelatin hydrolysis.
"
been shown in a previous study, where the correlation coefficient for plaque wet weight and plaque score was r = 0.84 (20). The majority of the plaque samples removed in the present study were too small to weigh reliably. However, some estimate of plaque mass was obtained by measuring the plaque DNA content with ethidium bromide using a fluorometric procedure (3). Sufficient numbers of sample sites had a degree of gingivitis that was intermediate to the described criteria. Accordingly, if a site was not perfectly healthy, i.e. gingivitis score = 0, but failed to show the edema and redness associated with a gingivitis score of 1.0, a value of 0.5 was assigned. If a site bled by spotting and did not flow along the gingival margin, a score of 1.5 was given. A score of 2.0 was given when the blood flowed along the gingival margin. These intermediate values increased the sensitivity of our clinical disease measurements and, since they represented the concurrence of two examiners, were thought to be more
accurate than scoring systems involving only one examiner. Statistical analysis. The various clinical and bacteriological data were obtained for each visit and recorded for statistical analysis using the MIDAS statistical package contained in the Michigan Terminal System. The compiling of the bacteriological data was independent of the clinical data. Analyses of variance were performed on the data, stratified according to age of plaque, plaque score, and gingivitis score. Whenever significance was found, comparisons between entries were performed using the Scheffe test. The Scheffe test is a method for testing the significance of one or more comparisons of mean values arising in an analysis of variance where the entire experimental error rate is taken into consideration (29). The same data set, again stratified according to age of plaque, plaque score, and gingivitis score, was analyzed using the nonparametric Kruskal-Walhis ranking test (31).
INFECT. IMMUN.
SYED AND LOESCHE
824
RESULTS No subject withdrew from the experiment, nor complained of any inconveniences caused by the period of no oral hygiene. No systemic antibiotics were taken by the subjects for medical reasons during the experimental period. There was an obvious clinical increase in plaque about all the teeth in the subjects' mouths, but the number of gingival sites at 3 weeks which exhibited bleeding were few. This was particularly so among the sample sites, where only 6 of 24 sites had a gingivitis that bled with a flow upon probing (Table 3). Additional gingival sites were sampled at 3 weeks from maxillary anterior teeth so as to increase the number of plaques associated with gingival bleeding (Table 3). The correlation between plaque and gingivitis scores with each other and with the total count, DNA content, and plaque age was determined in those plaques with a measurable amount of DNA (Table 4). The plaque score correlated significantly with plaque age, gingivitis score, total count, and DNA content. The gingivitis score correlated significantly with plaque age and with the plaque score, but showed no relationship with DNA content and total bacterial count (Table 4). At 0 time, the plaque score averaged 0.35 and the gingivitis score averaged 0.25 (Table 5). Approximately 3 x 106 organisms could be cultured from the tooth surface sites, even though half the sites had a plaque score of 0.0 (Table 3). After 1 week of no brushing, the plaque significantly increased to about 70 to 100% of its 3-week-old value as judged by the increase in plaque scores, DNA content in miTABLE 3. Frequency distribution of plaque and gingivitis scores as a function of time Time Score 0
Plaque 0.0
week
1
week 2 weeks
3 weeks"
score
0.5 1.0 1.5 2.0 2.5
13 9 3 0 0 0
1 0 8 7 8 0
0 0 2 9 13 0
17 4 4 0 0
2
1 2 14 6 1
0 0 0 9 11 4
(3) (2) (13) (6)
Gingivitis score
0.0 0.5 1.0 1.5 2.0
8 13 1 0
Figures in parentheses sites sampled at 3 weeks.
are
0 3 (3)
12 (5) 3 (4) 6 (12)
number of additional
TABLE 4. Correlation coefficient of plaque and gingivitis scores with various plaque parameters' Plaque score Parameter
Age of plaque Plaque score Gingivitis score DNA content (jig) Total bacterial
Gingivitis score
r coeffi- Signifi- r coeffi- Significient
cance
cient
cance
0.66
