Automated immunofluorescence for enumeration of selected taxa in supragingival dental plaque

The present study investigated a recently developed automated image analysis technique for its applicability to the enumeration of selected bacteria in supragingival dental plaque. Following initial calibration, the system is capable to count fluorescence-labeled target cells in up to 48 samples without user interference. Test samples contained a characteristic mixture of planktonic bacteria, small almost planar bacterial aggregates, and large, virtually indisruptable clumps with cells from multiple species. Due to their complex composition, these samples provided a challenging validation step for the image analysis system. Automated enumeration of target bacteria was compared with visual counting of the fluorescence-labeled bacteria. Results are shown for six taxa (Actinomyces naeslundii, Fusobacterium nucleatum, Prevotella intermedia/Prev. nigrescens, Streptococcus gordonii/Strep. oralis/Strep. sanguis, Strep. sobrinus, and Veillonella dispar/ V. parvula) with characteristic differences in abundance, cell morphology and aggregation behavior. Results revealed good correspondence between the two enumeration techniques (correlation coefficients ranging from 0.77 to 0.92) provided that the portion of target bacteria exceeded 0.05% of the total bacterial cell number. This work demonstrates the applicability and usefulness of fully automated immunofluorescence to analyze such complex ecosystems as supragingival dental plaque.     

The quantification of dental plaque using an image analysis system: reliability and validation

AIM: Established clinical indices used to measure dental plaque have limitations. The aim of this study was to develop and evaluate a new method of quantitative plaque measurement, with increased sensitivity and reproducibility, using a purpose designed image analysis system. METHOD: After plaque disclosure, the patient was positioned in a cephalometric head holder mounted in a custom-built frame with standard lighting and camera position. A 32-bit Digital Camera grabbed the image of the patient's teeth, which was analysed using Adobe Photoshop (V5.02, Adobe Systems Ltd, Europe) and Image Pro Plus software (V4.0, Media Cybernetics, USA). Measurements were made on 120 incisors in 15 patients and reproducibility was assessed using Fliess' Coefficient of Reliability. Sensitivity was assessed by comparison of results of treatment effects from a clinical trial involving 30 patients. Analysis of covariance was used to compare the image analysis system with the Turesky modification of the Quigley and Hein plaque index and the Addy plaque area index. RESULTS: The results were within the 'Excellent Reliability' category, for inter-operator reproducibility and intra-operator repeatability. CONCLUSIONS: This new system provided accurate quantitative measurements of dental plaque and labial tooth surfaces and was shown to have increased sensitivity when compared with the Turesky modification of the Quigley and Hein plaque index and the Addy plaque area index. The stored images can be re-measured for reliability and comparability between studies.     

Effects of bactericidal treatments on bacterial adherence and dental plaque formation

abstract — in vivo plaque formation was significantly reduced when tooth surfaces were subjected to topical applications of iodine (0.2% I₂ in 2.0% KI) twice daily for 3 d. Similarly, in vivo plaque formation was significantly reduced on enamel surfaces that were subjected to ultraviolet irradiation. Control experiments indicated that neither ultraviolet irradiation nor iodine treatment interfered with mechanisms for bacterial apposition to dental plaque. The results are interpreted to suggest that plaque grows in mass primarily by the division and multiplication in situ by plaque bacteria, not by a continued apposition of salivary microbes.     

Quantification of dental plaque on lingual tooth surfaces using image analysis: reliability and validation

AIM: The aim of this study was to increase the versatility and further validate the method reported by Smith et al. (2001) by testing the reliability of plaque measurement against two well-known dental plaque quantification methodologies using image analysis in a clinical trial. METHOD: The teeth of 40 subjects were disclosed before digital images of the labial and lingual surfaces of their upper and lower incisors were acquired. The amount of plaque present was quantified using a modification of the method described by Smith et al. (2001). The method was modified for obtaining images of the lingual surfaces by incorporating the use of orthodontic occlusal mirrors and 5-mm pieces of moistened blue articulating paper used to enable calibration. Plaque measurements were made from 320 upper and lower anterior teeth from the 40 subjects by two operators. Fliess' coefficient of reliability was used to assess intra- and inter-operator reliability and the independent sample t test was used to assess statistical significance between test and control groups after checking the data for normality. For validation, measurements were recorded using the Turesky et al. (1970) (modification of the Quigley & Hein (1962) plaque index and the Addy et al. (1983) plaque area index. The results were compared with the image analysis method using Pearson's correlation coefficient. RESULTS: The results for reliability were within Fliess' range of "excellent" for both intra-operator repeatability and inter-operator reproducibility. Pearson's correlation coefficients showed highly significant values indicating the close similarity between all three methods. CONCLUSIONS: This method for the measurement of dental plaque on lingual surfaces of anterior teeth proved reliable. The combined results from the labial and lingual surfaces of anterior teeth using image analysis produced trial conclusions comparable with the alternate plaque quantification methods used, with less clinician time and further producing a permanent database of images for future use.     

不同性别刷牙效果的临床对比研究

目的 对不同性别人群刷牙效果进行对比研究。方法 实验对象按性别分组，均使用波浪型牙刷，刷牙前后各做一次菌斑检查，用O′Leaiy卡记录菌斑在牙面的分布。结果 女性比男性刷牙更彻底（P＜0．01），菌斑清除率相差10．42％。结论 性别对刷牙后菌斑清除效果有一定影响。     

Relative effects of sucrolytic enzymes in human dental plaque

abstract – The relative effects in human dental plaque material from the three main extracellular sucrolytic enzymes from bacterial origin, invertase, dextransucrase and levansucrase, have been investigated by means of quantitative determination of products with sucrose as the substrate. Twenty young men having carious lesions and harboring plaque material on the tooth surfaces, were selected. One gram (wet weight) of plaque material was obtained and divided in five samples, 0.2 g each, for different investigations and controls. Twice as much fructan as glucan was found in plaque. Invertase activity was found to dominate sucrolysis within plaque with 99.67 % of the total activity.     

In vivo effect of four antibacterial agents upon the acidogenicity of dental plaque

Abstract – The present study was carried out to compare the effect of chlorhexidine, iodine, alcohol and hydrogen peroxide on the acidogenicity of dental plaque in vivo . Plaque pH changes after application of sucrose were measured before and at various time intervals after the topical application of the agents. The MIC values of the different agents were assessed against plaque bacteria in vitro . The antimicrobial activity of chlorhexidine and iodine were similar whereas alcohol and hydrogen peroxide were less effective in the concentrations employed. The results showed that chlorhexidine 2%, iodine 2%, and alcohol 70% inhibited pH drops for a 24-h period after treatment whereas 3% hydrogen peroxide had no effect. Chlorhexidine 0.2% inhibited acid production to a greater extent than did alcohol 50% and iodine 0.2%. It is suggested that retention of chlorhexidine in plaque may explain the observed prolonged effect.     

Microbial vitality of supragingival dental plaque during initial stages of experimental gingivitis in humans

Although vital plaque micro-organisms are part of the natural ecosystem in the oral cavity they are also the key factor in the development of diseases induced by the human dental plaque. In a previous study (9) the portion of vital bacteria related to the total number of plaque micro-organisms (i.e. the microbial vitality) appeared low in small plaque samples. The objective of this investigation was to determine the exact relationship of microbial vitality and age of supragingival plaque during the early phases of human dental plaque formation. Between intervals of optimal oral hygiene, thirteen participants refrained from all oral hygiene measures for periods of 1, 2, 4, 8, 24 and 72 h. Plaque was completely sampled from a defined area situated on the vestibular surface of the teeth 13, 14, 15, 23, 24 and 25. The pooled plaque from these areas was immediately processed. Total bacterial counts (BC) as enumerated by darkfield microscopy, and colony-forming units (CFU) were recorded. The microbial vitality was calculated indirectly as plating efficiency (PE=CFU per BC) and directly assessed using a vital fluorescence (VF) technique. In the 1 h old plaque samples the median values of PE and VF were 29% and 18%, respectively. Thereafter, the microbial vitality increased significantly with plaque age. The 24 h old plaque samples yielded values of 77% (PE) and 62% (VF). It was concluded that the microbial vitality of the early dental plaque investigated was considerably lower compared to that of a more mature plaque.     

Relationship between bacterial counts, microbial vitality and the accumulation of supragingival dental plaque in humans

Comparisons between plaque index (PlI) and bacterial counts have been made already; however, these did not take into account the area used for plaque sampling. The objective of the present study was to determine the correlation between the PlI score, the number of bacteria and the bacterial plaque vitality when dental plaque was repeatedly sampled from the same area in each subject during early plaque accumulation. Between intervals of optimal oral hygiene, 10 participants refrained from all oral hygiene measures for periods of 1, 2, 3, 4 and 5 days. The PlI was recorded on the vestibular surface of all first premolars as local PlI:LS. For statistical reasons, the scores of the independent variable LS were added for each subject giving LS* values ranging from 0 to 8. The plaque sampled from this specific surface was circumscribed by the marginal gingiva and an acrylic splint, giving reproducible areas for plaque collection. Total microscopic bacterial counts (BC), colony forming units of anaerobes (CFUan) and aerobes (CFUae), and proportions of vital bacteria (VF) were compared with LS* values. BC and LS* values were strongly correlated. CFUan and CFUae increased significantly with LS*, but this increase was higher for LS* 0 to 4 than for LS* 4 to 8. The ratio between vital and dead microorganisms, assessed by two different methods, was low when an LS* of 0 was recorded, with higher ratios registered for LS* values 4 and 8.     

Effect of chlorhexidine on acidogenicity of dental plaque in vivo

Abstract— In order to evaluate the effect of chlorhexidine on the acidogenicity of dental plaque, pH changes in plaque were measured in situ after sucrose applications. The results showed that a 0.2% chlorhexidine mouthrinse inhibited acid production for a period of 24 h, whereas a 0.05% chlorhexidine rinse showed an inhibitory effect for 4 h. It is suggested that the mechanism involved may be related to the retention of chlorhexidine in the mouth and in plaque providing a bacteriostatic milieu which may be of importance in the observed long-term effect.     

A digital SLR or intra-oral camera: preference for acquisition within an image analysis system for measurement of disclosed dental plaque area within clinical trials

Objectives:  The aim of this investigation was to compare the accuracy and reproducibility of images acquired from two types of camera (digital SLR camera, Kodak DCS410, KJP, UK and an intra-oral camera, Schick Technologies, UK) for application within an imaging system used for the quantification of disclosed dental plaque.
Methods:  Subjects refrained from brushing their teeth for 24 h. Their teeth were then disclosed with Erythrosin FDC Red 3 and duplicate sets of images were obtained by each examiner of the upper central and lateral incisors. Images were then saved and measured for dental plaque area. These data were used to calculate reliability.
Results:  No statistically significant bias in the measurements of plaque area was found. The reliability results showed the method was reliable. However, the image analysis system incorporating the 35 mm SRL camera was more reliable for both operators, as demonstrated by their intra-operator results. This was also collaborated by the inter-operator results.
Conclusion:  In this investigation the digital SLR camera combined with the image analysis system and frame permitted greater reliability of dental plaque surface area measurements than the digital intra-oral camera. This conclusion was derived both from the reliability data and from the perceived ease of use and flexibility of both camera types. When combined with the use of the custom-made frame, the images using the digital SLR camera were highly reproducible, confirming the systems application within clinical trials.     

Some aspects of the dynamics of Cu and Zn retained in plaque as related to their effect on plaque pH

Abstract – The effect of glucose on acid production in dental plaque was measured in vivo after oral rinses with CuSO₄ (1.0 mM) or Zn (Ac)₂ (20 mM) and compared with the retention of the metals. Plaque samples were analyzed for copper and zinc by neutron activation analysis. A rinse with the copper salt solution significantly depressed acid formation in plaque for a period of 6 h whereas a rinse with the zinc solution was effective for 2 h. Plaque concentrations of the metals increased from less than 75 p.p.m. to more than 1000 p.p.m. 2 h after a rinse with the metal salts. Twelve hours after a rinse the mean plaque concentrations were 190 p.p.m. Cu or 358 p.p.m. Zn respectively. Reducing the pH of the copper sulfate solution from 5.7 to 2.0 yielded lower Cu concentrations in plaque 2 h after a rinse, indicating lower initial retention. The results clearly demonstrate a high and long lasting retention of metal ions in dental plaque and their biological effect in this environment.     

Effect of Listerine® on dental plaque and gingivitis

abstract — The present study was performed in 10 adults in order to evaluate the effect of an antiseptic mouthrinse (Listerine®) on the rate of dental plaque formation and gingivitis development during a 2-week period when all efforts towards active mechanical oral hygiene were withdrawn. The study was performed as a crossover study and was carried out during four consecutive 2-week periods. During the first and third periods (preparatory periods) the participants were subjected to repeated professional tooth cleanings in order to establish plaque- and gingivitis-free dentitions. During the second and fourth periods (test and control periods) the participants were not allowed to brush their teeth but rinsed their mouths three times a day with Listerine or a placebo mouthwash. Plaque Index, Gingival Index, gingival fluid flow, and crevicular leukocytes were assessed on d 0, 2, 4, 7, and 14. On d 7 and 14, dental plaque was removed from the right and left jaws respectively and the wet weights determined. The chemotactic activity elaborated by the plaques was studied in Boyden chambers. During the Listerine test period, significantly lower Plaque and Gingival Index values were scored and lower amounts of plaque could be sampled in comparison to the control period.     

Percent of plaque on individual tooth surfaces and differences in plaque area between adjacent teeth in healthy adults

Abstract Objectives: To apply a planimetric method to determine the percentage of the tooth area covered by plaque on individual tooth surfaces and to compare the percentage of plaque between adjacent tooth surfaces. Methods: Forty‐three adults, mean age 43.0 years, with no signs of periodontal disease, participated. The subjects underwent comprehensive professional cleaning of all teeth. The subjects were then instructed to maintain their ordinary oral hygiene habits. Plaque was registered after 1 month, on each individual tooth surface by use of P%I. Results: The pattern of plaque accumulation was in large symmetrical, with differences in mean P%I between several adjacent tooth surfaces, e.g. for the maxillary buccal and lingual tooth surfaces between the first and second molars (P < 0.001) and for the lingual tooth surfaces between the canines and the first premolars (P < 0.01). Conclusion: In the present study, it was possible to identify and describe adjacent tooth surfaces with the most pronounced plaque accumulation, to make further improvements of oral hygiene possible.     

Acid anion profiles in dental plaque following consumption of cereal-based foods and fruits

The aim of this study was to investigate the acid anions produced in plaque after chewing various cereal-based foods and fruits for one minute. Test foods were oranges, apples, bananas, Cornflakes®. Branflakes®, Weetabix®. Alpert®. white bread, wholemeal bread, rice and spaghetti, plus positive and negative controls of 10% sucrose and 10% sorbitol. 4 males and 3 females, aged 22–37 years, participated in the study. 7 min from the start of chewing. 48-h plaque was collected from all accessible smooth surfaces, with no attempt to collect interproximal plaque, and centrifuged. Plaque fluid was withdrawn and analyzed by isotachophoresis for formate, succinate, lactate. acetate and propionate. At rest, acetate was the major anion present in plaque fluid, whereas following carbohydrate consumption, highest levels of lactate were detected followed by acetate. The amount of lactate only, detected in plaque fluid, was significantly correlated to the carbohydrate present in the food. It was concluded that important information regarding the acidogenicity of test foods is gained by studying the acid anion profile of plaque fluid.     

微型pH电极直接测试人牙菌斑pH的应用研究

目的：进一步探讨微型ｐＨ电极在原位检测人牙菌斑ｐＨ的可行性，了解漱糖后对菌斑ｐＨ的影响。方法：采用微型蜂式ｐＨ指示电极直接测试人牙邻面菌斑ｐＨ变化。结果：微型ｐＨ电极与玻璃ｐＨ大电极所测结果基本一致；漱糖后菌斑ｐＨ呈下降趋势，２０ｍｉｎ时达到最低点；无龋组与龋敏感组之间的ｐＨ改变无显著性差异。结论：微型ｐＨ电极适用于在原位直接测试菌斑ｐＨ；漱糖后菌斑ｐＨ发生改变，但龋坏与否的个体之间未见明显不同     

Cariostat测试法中菌斑孵育时间选择的探讨

目的：了解机体龋病活跃性与其患龋状况的关系；分析Ｃａｒｉｏｓｔａｔ法２４ｈ、４８ｈ两种菌斑孵育时间所获的结果及其间的转换状态，确定合适的孵育时间。方法：对３９１名３岁～５岁幼儿进行龋病检查和Ｃａｒｉｏｓｔａｔ法龋病活跃性测试，观察菌斑孵育２４ｈ、４８ｈ的检测结果。结果：Ｃａｒｉｏｓｔａｔ法中菌斑孵育２４ｈ、４８ｈ的各度和患龋率、ｄｆｔ、ＣＳＩ均有高度相关性（Ｐ＜０．００１），４８ｈ的相关数值更大；孵育４８ｈ者各度患龋状况的差异分析亦比孵育２４ｈ者明显；孵育２４ｈ呈１度者有４６．５０％在４８ｈ时呈２度，４８ｈ呈２度者有６３．８５％在２４ｈ时呈０度或１度。结论：Ｃａｒｉｏｓｔａｔ能反映机体之患龋现状，其４８ｈ的菌斑孵育结果更能确切地体现机体的龋病活跃性     

Detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in dental plaque samples from children 2 to 12 years of age

The purpose of this study was to detect the presence of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in plaque samples from 104 children, collected from their toothbrushes using a polymerase chain reaction (PCR). The age range of all subjects was 2-12 years. 21, 73 and 10 children with healthy gingiva, gingivitis and periodontitis respectively were selected. Plaque samples were collected from all erupted teeth sites using a sterile toothbrush. The mean concentration of DNA recovered from brushing plaque samples was approximately 660 microg/ml, which was sufficient for performing a PCR-based survey. Both A. actinomycetemcomitans and P. gingivalis were detected in the primary and mixed dentition. The prevalence of A. actinomycetemcomitans in healthy subjects was 4.8%, and those with gingivitis and periodontitis was 6.8% and 20.0% respectively, while the prevalence of P. gingivalis was 4.8% in healthy subjects, and 9.6% and 20.0% in those with gingivitis and periodontitis, respectively. Our survey, using a toothbrush, indicated that A. actinomycetemcomitans and P. gingivalis are rarely present in oral cavities of healthy children.     

Formation and ecology of plaque at different locations in the mouth

Some recent findings concerning microbial colonization of smooth surfaces of teeth, gingival crevices, occlusal fissures and removable dentures are reviewed considering the many ecologic factors of importance for the oral microbial communities. The oral microbiota is extremely complex comprising at least 200 taxa. In spile of interindividual and site-to-site variations, each oral habitat has a characteristic microbiota ranging from Gram-positive, facultatively anaerobic cocci and rods in occlusal fissures to predominance of Gram-negative, strictly anaerobic rods and spirochetes in deep periodontal pockets.