1. They had intact dentitions in both arches from first permanent molar to first permanent molar. The teeth designated by the Plaque and Gingival Indices, were all uncrowned. 2. The mean Plaque Index of each subject was equal to or less than 1.0 at the commencement of the study. 3. The individual mean Gingival Index of each sub ject was equal to or less than 0.5 at the commencement of the study. 4. They had no systemic disease. 5. All the tobacco smokers had smoked ten or more filter-tipped cigarettes per day for at least the last year. The mean number of cigarettes smoked was 16; the range was from 10 to 20 cigarettes. Plaque deposits and the gingival status were recorded using the Plaque and Gingival Indices respectively. The measurements were applied to the teeth specified in the description of the indices. The subjects were assessed at the commencement of the study, prior to having any treatment. All the teeth were then scaled and polished following which the sub jects were instructed to refrain from all oral hygiene measures for 10 days. Further assessments were then performed at day 3, day 7 and day 10. On the 3rd, 7th and 10th days a sample of plaque was removed from a specified tooth surface. The surfaces were the distal of the lower left first premolar on day 3, the mesial of the lower left second premolar on day 7 and the distal of the lower left second premolar on day 10. The plaque samples were removed from the tooth surfaces by two vertical strokes with a periodontal hoe. The strokes were commenced as close to the dentogingival junction as possible without inflicting trauma to the soft tissues, and were terminated approximately 3 mm from the occlusal surface of the crown. The sample was placed in a drop of sterile phosphate buffered saline (pH = 7.3) on a glass slide. Dispersion of the plaque sample was achieved with the aid of a 1-mm wide metal spatula moved in a circular motion through the plaque sample for 30 seconds. The smears were gram stained and examined at 1000X magnification under a light microscope. A cross grid, microscope lens graticule:): was employed to assist in the counting of plaque bacteria. On each slide five selected unrelated areas were examined and the number of Gram-negative and Gram-positive microorganisms were counted. The selection of any one area was dependent upon the individual bacteria in that area being clearly discernible and not clumped together or overlapping one another. Areas of clumping or sparse bacterial distribution were not selected for counting. The organisms were assessed according to their tinctorial appearance and not their morphology.
Effects of Tobacco Smoking on Plaque Development and Gingivitis
12,13
by R . J . BASTIAAN*
I. M. WAITE† THE INFLUENCE of tobacco smoking on the etiology of periodontal disease is controversial. A number of work ers have found that smoking does not appear to increase the amount of dental plaque, and this finding has been supported by similar results with regard to gingival inflammation. When smokers and nonsmokers were matched with regard to age and oral hygiene levels, no difference was found between the two groups in the degree of periodon tal disease. In contrast, other studies using matched groups have found more severe gingivitis and periodon titis in smokers than nonsmokers. The lack of agree ment between the findings of these studies indicates a need for further research. The influence of tobacco smoking on the oral micro flora also has been investigated. Examination of plaque from the mouths of smokers and nonsmokers has dem onstrated that the ratios are similar for aerobic and anaerobic microorganisms in the two groups there are also similar proportions of bacteria as assessed after culturing. The aim of the present study was to compare the rate of plaque formation in a group of smokers with the rate of formation in a group of nonsmokers. All the subjects had minimal clinical signs of gingival inflammation ini tially. Over the period of the study the changes in gingival condition were also to be assessed. Samples of plaque were to be stained and examined during the course of the study to determine the ratio between grampositive and gram-negative bacteria. 1
12,13
2,3
4,5,6
3,7
8,9
10
11
MATERIALS AND METHODS
The participants in this study were 10 subjects who did not smoke tobacco (four males, six females) and 10 subjects who smoked 10 cigarettes or more daily (six males, four females). The mean age of the nonsmokers was 24 years (range 17 to 29 years) and that of the smokers was 22 years (range 18 to 28 years). The following criteria were used for the selection of subjects:
Repeatability * Royal Dental Hospital, Leicester Square, London W.C. 2, Eng land. †University College Hospital, Dental School, Mortimer Market, London W.C. 1, England. Formerly Royal Dental Hospital, London.
All measurements in this study were made by one X Graticule ElOa, Graticules Ltd., Sovereign Way, Tonbridge, Kent.
480
Volume 49 Number 9
Influence of Smoking 481
examiner. A n initial training period was used to famil iarize the examiner with the assessment criteria. The accuracy of applying the assessment criteria was evalu ated by repeating the measurements on a number of patients with 1 hour between assessments. A repeatability of just over 86% for both the Plaque Index and Gingival Index was achieved prior to the commencement of the clinical study. The repeatability of the microbiological counts was evaluated by assessing the proportion of Gram-positive and Gram-negative bacteria at randomly selected areas on the same slide with 4 weeks between counts. For the pooled data the variation was 0.3% between counts. Although the ratio of microorganisms was similar at the two counts, there was a wide variation in absolute num bers of microorganisms counted in the different fields due to variation in dispersion. Statistical Analysis
rABLE 2. Mean Gingival Index per Tooth Surface for the Nonsmokers
and Smokers over the 10-Day Experimental Period Value Nonsmokers mean ± SE
0 (Prior to scaling)
0.15 ± 0 . 0 3
44.0
P > 0.05
0.19 ± 0 . 0 5
3
0.18 ± 0 . 0 4
50.0
P > 0.05
0.31 ± 0 . 0 5
7
0.41 ± 0.05
32.0
P > 0.05
0.54 ± 0.06
10
0.51 ± 0 . 0 7
46.5
P > 0.05
TABLE 3. Percentage of Gram-Positive and Gram-Negative Microorga nisms*
The mean Plaque Index scores for the nonsmokers and smokers at different time intervals during the exper iment are shown in Table 1. Plaque deposits accumulated rapidly on the teeth of all the subjects over the 10-day period. There was a trend for more plaque accumulation on the teeth of smokers than on nonsmokers, and this difference between the two groups increased with time. At none of the time intervals, however, was the difference between the smokers and nonsmokers significant. The mean Gingival Index scores are shown in Table 2. Slight variations between the two groups were evident at the different time intervals but these differences showed no consistent trend and were not statistically significant (P > 0.05). Increases in the Gingival Index
Value Smokers mean ± SE
3
Chi square test
Statistical significance
P< 0.001
Gram positive Gram negative
55.8 44.2
63.6 36.4
35.74
7
Gram positive Gram negative
57.3 42.7
55.9 44.1
1.03
P > 0.05
10
Gram positive Gram negative
59.3 40.7
58.9 41.1
0.02
P > 0.05
were not as rapid as the increases in the Plaque Index and by the end of the experimental period there were only early signs of gingival inflammation. Bacteriological Observations The proportional counts of the Gram stained smears of dental plaque during the experimental period are shown in Table 3. On the third day there was a statisti cally significant increase in the percentage of Grampositive to Gram-negative bacteria in the smokers com pared with the nonsmokers (P < 0.001). This difference was not maintained on days 7 and 10; at these assess ments it was found that the percentages of Gram-positive to Gram-negative bacteria in both groups were similar.
(MannWhit ney)
DISCUSSION
It has been reported that gingival inflammation is preventable if dental plaque is removed every 48 hours. The rate at which plaque develops varies between indi viduals, and it may be deduced that the rate of devel opment of gingival inflammation also will show varia tion. The reasons why such differences occur has not yet been fully explained, but several factors may act indi rectly and it is possible that one of these might be tobacco smoking. The effects of smoking on the rate of formation of dental plaque have not been reported previously. The results of the present study showed that there was a trend for plaque to form more rapidly in smokers than nonsmokers. These differences were not statistically sig15
of "U" Significance
16
days
1.04 ± 0 . 1 0
Smok ers
3
TABLE 1. Mean Plaque Index per Tooth Surface for the Nonsmokers and Smokers over the 10-Day Experimental Period
0 (Prior to scaling)
Nonsmok ers
* Results of counting 10 fields for each of 20 subjects.
Clinical Assessments
0.28 ± 0.06
Significance
days
RESULTS
Assessment at
of "U" (MannWhit ney)
0.16 ± 0 . 0 4
Day
The Gingival Index and Plaque Index measurements were ordinal, nonparametric measurements. Compari sons between the two groups of subjects at the various time intervals were made using the Mann-Whitney U Test. The bacteriological data were analyzed using the chi-square test.
Nonsmokers mean ± SE
Assessment at
Smokers mean ± SE
0.39 ± 0.06
33.5
P > 0.05
1.14 ± 0 . 1 1
47.5
P > 0.05
1.58 ± 0.12
7
1.75 ± 0 . 1 2
37.0
P > 0.05
1.79 ± 0 . 1 0
10
2.01 ± 0.09
31.5
P > 0.05
482
J. Periodontol. September, 1978
Bastiaan, Waite
nificant. It is possible that increasing the study period might have resulted in significant differences emerging. There was no significant difference in the Gingival Indices between the two groups at any of the time intervals. Unlike the results for the Plaque Index there was no trend for one group to have higher values than the other. This investigation once again has confirmed that the withdrawal of oral hygiene procedures results in rapid plaque accumulation followed by early signs of gingivitis in all subjects. Reintroduction of effective oral hy giene measures resulted in a rapid resolution of the induced experimental gingivitis. Although previous studies have been based on a dif ferent experimental design, their findings support those of the present study. Alexander reported a similar mean plaque score for smokers and nonsmokers and Sheiham could find no significant difference in the oral debris scores between the two groups in the patients he examined. With regard to the bacteriological study, the 3-day-old plaque samples taken from the two groups exhibited a significant difference in the relative numbers of Gram staining organisms, the smokers having a higher per centage of Gram-positive than Gram-negative bacteria. There were, however, no significant differences at the other sampling times. This difference in the staining characteristics between the plaque of smokers and nonsmokers which were seen only in the early stages of development, may have been due to an alteration in the oxidation-reduction potential. This finding remains to be confirmed and amplified by more sophisticated mi crobiological techniques before definitive conclusions can be made. 17,18
2,3
CONCLUSIONS
On the basis of the present study it seems justified to conclude that tobacco smoking has only a minor effect on the rate at which plaque forms. There appeared to be no effect on the development of gingivitis over a 10-day period of withdrawal of oral hygiene measures. The composition of the microflora of early plaque appeared to differ in the two groups, the smokers having a higher percentage of Gram-positive to Gram-negative bacteria. Further work to confirm the latter finding is indicated. SUMMARY
A study was undertaken to test the hypothesis that patients who smoke tobacco have a more rapid rate of plaque deposition and development of gingivitis than those patients who do not smoke tobacco. Twenty sub jects participated in this study, their age range was 17 to 30 years. Ten were smokers and 10 were nonsmokers. After an initial thorough removal of all deposits, oral hygiene measures were stopped for 10 days. On days 3, 7 and 10, plaque levels were evaluated using the Plaque
Index and the gingival status was evaluated using the Gingival Index. Samples of plaque were stained by the Gram technique and examined microscopically. Plaque levels appeared to be higher in smokers than nonsmokers but the differences were not statistically significant. No consistent differences were evident in the gingival status of the two groups. Microbiological anal ysis showed a statistically significant increase in the percentage of Gram-positive bacteria to Gram-negative bacteria in the smokers as compared to the nonsmokers on day 3, however these differences were not maintained in the plaque samples taken after the 3rd day. REFERENCES
1. Bastiaan, R. J., and Reade, P. C : The effects of tobacco smoking on oral and dental tissues. Aust Dent J 21: 308, 1976. 2. Alexander, A. G.: The relationship between tobacco smoking calculus and plaque accumulation and gingivitis. Dent Health 9: 6, 1970. 3. Sheiham, A.: Periodontal disease and oral cleanliness in tobacco smokers. J Periodontol 42: 259, 1971. 4. Pindborg, J. J.: Tobacco and gingivitis. J Dent Res 26: 261, 1947. 5. Ludwick, W., and Massler, M.: Relation of dental caries experience and gingivitis to cigarette smoking in males 17 to 21 years old. J Dent Res 31: 319, 1952. 6. Brandtzaeg, P., and Jamison, H. C : A study of periodon tal health and oral hygiene in Norwegian army recruits. J Periodontol 35: 302, 1964. 7. Lilienthal, B., Amerena, V., and Gregory, G.: An epide miological study of chronic periodontal disease. Arch Oral Biol 10: 553, 1965. 8. Arao, A., Schei, O., Lovdal, A., and Waerhaug, J.: Al veolar bone loss as a function of tobacco consumption. Acta Odontol Scand 17: 3, 1959. 9. Loftus, E. R., Kapur, K. K., Glass, R. L., and Alman, J. E.: Tobacco smoking and oral health. J Dent Res (IADR abstr.) 53: 92, 1974. 10. Kenney, E. B., Saxe, S. R., and Bowles, R. D.: The effect of cigarette smoking on anaerobis in the oral cavity. J Periodontol 46: 82, 1975. 11. Colman, G., Beighton, D., Chalk, A. J., and Wake, S.: Cigarette smoking and the microbial flora of the mouth. Aust Dent J 21: 111, 1976. 12. Silness, P., and Löe, H.: Periodontal disease in preg nancy; II. Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 22: 121, 1964. 13. Löe, H., and Silness, J.: Periodontal disease in preg nancy: I. Prevalence and severity. Acta Odontol Scand 21: 532, 1963. 14. Siegel, S.: Nonparametric Statistics for Behavioral Sci ences, pp. 116-120. New York, McGraw-Hill Book Company 1956. 15. Lang, N. P., Cumming, B. R., and Löe, H.: Toothbrushing frequency as it relates to plaque development and gingival health. J Periodontol 44: 396, 1973. 16. Genco, R. J., Evans, R. T., and Ellison, S. A.: Dental research in microbiology with emphasis on periodontal disease. J Am Dent Assoc 78: 1016, 1969. 17. Loe, H., Theilade, E., and Jensen, S. B.: Experimental gingivitis in man. J Periodontol 36: 177, 1965. 18. Theilade, E., Wright, W. H., Jensen, S. B., and Löe, H.: Experimental gingivitis in man. II. A longitudinal clinical and bacteriological investigation. J Periodont Res 1: 1, 1966.
Effects of Tobacco Smoking on Plaque Development and Gingivitis
12,13
by R . J . BASTIAAN*
I. M. WAITE† THE INFLUENCE of tobacco smoking on the etiology of periodontal disease is controversial. A number of work ers have found that smoking does not appear to increase the amount of dental plaque, and this finding has been supported by similar results with regard to gingival inflammation. When smokers and nonsmokers were matched with regard to age and oral hygiene levels, no difference was found between the two groups in the degree of periodon tal disease. In contrast, other studies using matched groups have found more severe gingivitis and periodon titis in smokers than nonsmokers. The lack of agree ment between the findings of these studies indicates a need for further research. The influence of tobacco smoking on the oral micro flora also has been investigated. Examination of plaque from the mouths of smokers and nonsmokers has dem onstrated that the ratios are similar for aerobic and anaerobic microorganisms in the two groups there are also similar proportions of bacteria as assessed after culturing. The aim of the present study was to compare the rate of plaque formation in a group of smokers with the rate of formation in a group of nonsmokers. All the subjects had minimal clinical signs of gingival inflammation ini tially. Over the period of the study the changes in gingival condition were also to be assessed. Samples of plaque were to be stained and examined during the course of the study to determine the ratio between grampositive and gram-negative bacteria. 1
12,13
2,3
4,5,6
3,7
8,9
10
11
MATERIALS AND METHODS
The participants in this study were 10 subjects who did not smoke tobacco (four males, six females) and 10 subjects who smoked 10 cigarettes or more daily (six males, four females). The mean age of the nonsmokers was 24 years (range 17 to 29 years) and that of the smokers was 22 years (range 18 to 28 years). The following criteria were used for the selection of subjects:
Repeatability * Royal Dental Hospital, Leicester Square, London W.C. 2, Eng land. †University College Hospital, Dental School, Mortimer Market, London W.C. 1, England. Formerly Royal Dental Hospital, London.
All measurements in this study were made by one X Graticule ElOa, Graticules Ltd., Sovereign Way, Tonbridge, Kent.
480
Volume 49 Number 9
Influence of Smoking 481
examiner. A n initial training period was used to famil iarize the examiner with the assessment criteria. The accuracy of applying the assessment criteria was evalu ated by repeating the measurements on a number of patients with 1 hour between assessments. A repeatability of just over 86% for both the Plaque Index and Gingival Index was achieved prior to the commencement of the clinical study. The repeatability of the microbiological counts was evaluated by assessing the proportion of Gram-positive and Gram-negative bacteria at randomly selected areas on the same slide with 4 weeks between counts. For the pooled data the variation was 0.3% between counts. Although the ratio of microorganisms was similar at the two counts, there was a wide variation in absolute num bers of microorganisms counted in the different fields due to variation in dispersion. Statistical Analysis
rABLE 2. Mean Gingival Index per Tooth Surface for the Nonsmokers
and Smokers over the 10-Day Experimental Period Value Nonsmokers mean ± SE
0 (Prior to scaling)
0.15 ± 0 . 0 3
44.0
P > 0.05
0.19 ± 0 . 0 5
3
0.18 ± 0 . 0 4
50.0
P > 0.05
0.31 ± 0 . 0 5
7
0.41 ± 0.05
32.0
P > 0.05
0.54 ± 0.06
10
0.51 ± 0 . 0 7
46.5
P > 0.05
TABLE 3. Percentage of Gram-Positive and Gram-Negative Microorga nisms*
The mean Plaque Index scores for the nonsmokers and smokers at different time intervals during the exper iment are shown in Table 1. Plaque deposits accumulated rapidly on the teeth of all the subjects over the 10-day period. There was a trend for more plaque accumulation on the teeth of smokers than on nonsmokers, and this difference between the two groups increased with time. At none of the time intervals, however, was the difference between the smokers and nonsmokers significant. The mean Gingival Index scores are shown in Table 2. Slight variations between the two groups were evident at the different time intervals but these differences showed no consistent trend and were not statistically significant (P > 0.05). Increases in the Gingival Index
Value Smokers mean ± SE
3
Chi square test
Statistical significance
P< 0.001
Gram positive Gram negative
55.8 44.2
63.6 36.4
35.74
7
Gram positive Gram negative
57.3 42.7
55.9 44.1
1.03
P > 0.05
10
Gram positive Gram negative
59.3 40.7
58.9 41.1
0.02
P > 0.05
were not as rapid as the increases in the Plaque Index and by the end of the experimental period there were only early signs of gingival inflammation. Bacteriological Observations The proportional counts of the Gram stained smears of dental plaque during the experimental period are shown in Table 3. On the third day there was a statisti cally significant increase in the percentage of Grampositive to Gram-negative bacteria in the smokers com pared with the nonsmokers (P < 0.001). This difference was not maintained on days 7 and 10; at these assess ments it was found that the percentages of Gram-positive to Gram-negative bacteria in both groups were similar.
(MannWhit ney)
DISCUSSION
It has been reported that gingival inflammation is preventable if dental plaque is removed every 48 hours. The rate at which plaque develops varies between indi viduals, and it may be deduced that the rate of devel opment of gingival inflammation also will show varia tion. The reasons why such differences occur has not yet been fully explained, but several factors may act indi rectly and it is possible that one of these might be tobacco smoking. The effects of smoking on the rate of formation of dental plaque have not been reported previously. The results of the present study showed that there was a trend for plaque to form more rapidly in smokers than nonsmokers. These differences were not statistically sig15
of "U" Significance
16
days
1.04 ± 0 . 1 0
Smok ers
3
TABLE 1. Mean Plaque Index per Tooth Surface for the Nonsmokers and Smokers over the 10-Day Experimental Period
0 (Prior to scaling)
Nonsmok ers
* Results of counting 10 fields for each of 20 subjects.
Clinical Assessments
0.28 ± 0.06
Significance
days
RESULTS
Assessment at
of "U" (MannWhit ney)
0.16 ± 0 . 0 4
Day
The Gingival Index and Plaque Index measurements were ordinal, nonparametric measurements. Compari sons between the two groups of subjects at the various time intervals were made using the Mann-Whitney U Test. The bacteriological data were analyzed using the chi-square test.
Nonsmokers mean ± SE
Assessment at
Smokers mean ± SE
0.39 ± 0.06
33.5
P > 0.05
1.14 ± 0 . 1 1
47.5
P > 0.05
1.58 ± 0.12
7
1.75 ± 0 . 1 2
37.0
P > 0.05
1.79 ± 0 . 1 0
10
2.01 ± 0.09
31.5
P > 0.05
482
J. Periodontol. September, 1978
Bastiaan, Waite
nificant. It is possible that increasing the study period might have resulted in significant differences emerging. There was no significant difference in the Gingival Indices between the two groups at any of the time intervals. Unlike the results for the Plaque Index there was no trend for one group to have higher values than the other. This investigation once again has confirmed that the withdrawal of oral hygiene procedures results in rapid plaque accumulation followed by early signs of gingivitis in all subjects. Reintroduction of effective oral hy giene measures resulted in a rapid resolution of the induced experimental gingivitis. Although previous studies have been based on a dif ferent experimental design, their findings support those of the present study. Alexander reported a similar mean plaque score for smokers and nonsmokers and Sheiham could find no significant difference in the oral debris scores between the two groups in the patients he examined. With regard to the bacteriological study, the 3-day-old plaque samples taken from the two groups exhibited a significant difference in the relative numbers of Gram staining organisms, the smokers having a higher per centage of Gram-positive than Gram-negative bacteria. There were, however, no significant differences at the other sampling times. This difference in the staining characteristics between the plaque of smokers and nonsmokers which were seen only in the early stages of development, may have been due to an alteration in the oxidation-reduction potential. This finding remains to be confirmed and amplified by more sophisticated mi crobiological techniques before definitive conclusions can be made. 17,18
2,3
CONCLUSIONS
On the basis of the present study it seems justified to conclude that tobacco smoking has only a minor effect on the rate at which plaque forms. There appeared to be no effect on the development of gingivitis over a 10-day period of withdrawal of oral hygiene measures. The composition of the microflora of early plaque appeared to differ in the two groups, the smokers having a higher percentage of Gram-positive to Gram-negative bacteria. Further work to confirm the latter finding is indicated. SUMMARY
A study was undertaken to test the hypothesis that patients who smoke tobacco have a more rapid rate of plaque deposition and development of gingivitis than those patients who do not smoke tobacco. Twenty sub jects participated in this study, their age range was 17 to 30 years. Ten were smokers and 10 were nonsmokers. After an initial thorough removal of all deposits, oral hygiene measures were stopped for 10 days. On days 3, 7 and 10, plaque levels were evaluated using the Plaque
Index and the gingival status was evaluated using the Gingival Index. Samples of plaque were stained by the Gram technique and examined microscopically. Plaque levels appeared to be higher in smokers than nonsmokers but the differences were not statistically significant. No consistent differences were evident in the gingival status of the two groups. Microbiological anal ysis showed a statistically significant increase in the percentage of Gram-positive bacteria to Gram-negative bacteria in the smokers as compared to the nonsmokers on day 3, however these differences were not maintained in the plaque samples taken after the 3rd day. REFERENCES
1. Bastiaan, R. J., and Reade, P. C : The effects of tobacco smoking on oral and dental tissues. Aust Dent J 21: 308, 1976. 2. Alexander, A. G.: The relationship between tobacco smoking calculus and plaque accumulation and gingivitis. Dent Health 9: 6, 1970. 3. Sheiham, A.: Periodontal disease and oral cleanliness in tobacco smokers. J Periodontol 42: 259, 1971. 4. Pindborg, J. J.: Tobacco and gingivitis. J Dent Res 26: 261, 1947. 5. Ludwick, W., and Massler, M.: Relation of dental caries experience and gingivitis to cigarette smoking in males 17 to 21 years old. J Dent Res 31: 319, 1952. 6. Brandtzaeg, P., and Jamison, H. C : A study of periodon tal health and oral hygiene in Norwegian army recruits. J Periodontol 35: 302, 1964. 7. Lilienthal, B., Amerena, V., and Gregory, G.: An epide miological study of chronic periodontal disease. Arch Oral Biol 10: 553, 1965. 8. Arao, A., Schei, O., Lovdal, A., and Waerhaug, J.: Al veolar bone loss as a function of tobacco consumption. Acta Odontol Scand 17: 3, 1959. 9. Loftus, E. R., Kapur, K. K., Glass, R. L., and Alman, J. E.: Tobacco smoking and oral health. J Dent Res (IADR abstr.) 53: 92, 1974. 10. Kenney, E. B., Saxe, S. R., and Bowles, R. D.: The effect of cigarette smoking on anaerobis in the oral cavity. J Periodontol 46: 82, 1975. 11. Colman, G., Beighton, D., Chalk, A. J., and Wake, S.: Cigarette smoking and the microbial flora of the mouth. Aust Dent J 21: 111, 1976. 12. Silness, P., and Löe, H.: Periodontal disease in preg nancy; II. Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 22: 121, 1964. 13. Löe, H., and Silness, J.: Periodontal disease in preg nancy: I. Prevalence and severity. Acta Odontol Scand 21: 532, 1963. 14. Siegel, S.: Nonparametric Statistics for Behavioral Sci ences, pp. 116-120. New York, McGraw-Hill Book Company 1956. 15. Lang, N. P., Cumming, B. R., and Löe, H.: Toothbrushing frequency as it relates to plaque development and gingival health. J Periodontol 44: 396, 1973. 16. Genco, R. J., Evans, R. T., and Ellison, S. A.: Dental research in microbiology with emphasis on periodontal disease. J Am Dent Assoc 78: 1016, 1969. 17. Loe, H., Theilade, E., and Jensen, S. B.: Experimental gingivitis in man. J Periodontol 36: 177, 1965. 18. Theilade, E., Wright, W. H., Jensen, S. B., and Löe, H.: Experimental gingivitis in man. II. A longitudinal clinical and bacteriological investigation. J Periodont Res 1: 1, 1966.
