Oral biofilms: emerging concepts in microbial ecology.

Oral biofilms develop under a range of different conditions and different environments. This review will discuss emerging concepts in microbial ecology and how they relate to oral biofilm development and the treatment of oral diseases. Clues to how oral biofilms develop may lie in other complex systems, such as interactions between host and gut microbiota, and even in factors that affect biofilm development on leaf surfaces. Most of the conditions under which oral biofilms develop are tightly linked to the overall health and biology of the host. Advances in molecular techniques have led to a greater appreciation of the diversity of human microbiota, the extent of interactions with the human host, and how that relates to inter-individual variation. As a consequence, plaque development may no longer be thought of as a generic process, but rather as a highly individualized process, which has ramifications for the treatment of the diseases it causes.

Effect of various rinsing protocols after use of amine fluoride/stannous fluoride toothpaste on the bacterial composition of dental plaque.

This clinical study evaluated the effect of different oral hygiene protocols on the bacterial composition of dental plaque. After a 2-week period of using fluoride-free toothpaste, 30 participants followed three 1-week experimental protocols, each followed by 2-week fluoride-free washout periods in a randomized crossover examiner-blind controlled trial. The 1-week experimental protocols comprised the use of AmF/SnF(2) toothpaste twice daily, after which participants either (1) rinsed with tap water, (2) did not rinse but only spat out the toothpaste, or (3) rinsed with an AmF/SnF(2) mouthwash. In the fluoride-free washout periods, the participants brushed their teeth with fluoride-free toothpaste without further instructions. Six hours after the last brushing (+/- rinsing) of each period, buccal plaque samples in the upper molar region were taken. The microbiota composition of the plaque samples was analyzed by checkerboard DNA:DNA hybridization. A statistically significant reduction was found in the total amount of DNA of the 39 major plaque species measured, and in the proportions of some acid-producing bacterial strains after the period having used the AmF/SnF(2) toothpaste + AmF/SnF(2) mouthrinsing. The results indicate that using the AmF/SnF(2) toothpaste and rinse combination could result in plaque of lower cariogenicity.

Treatment of Streptococcus mutans biofilms with a nonthermal atmospheric plasma.

AIMS: A nonthermal atmospheric plasma, designed for biomedical applications, was tested for its antimicrobial activity against biofilm cultures of a key cariogenic bacterium Streptococcus mutans. METHODS AND RESULTS: The Strep. mutans biofilms were grown with and without 0.15% sucrose. A chlorhexidine digluconate rinse (0.2%) was used as a positive antimicrobial reference. The presence of sucrose and the frequency of plasma application during growth were shown to have a significant effect on the response to treatment and antibacterial activity. CONCLUSIONS: A single plasma treatment for 1 min on biofilms cultured without sucrose caused no re-growth within the observation period. However, with either single or repeated plasma treatments of 1 min, on biofilms cultured with 0.15% sucrose, growth was only reduced. SIGNIFICANCE AND IMPACT OF THE STUDY: In summary, there may be a role for nonthermal plasma therapies in dental procedures. Sucrose and associated growth conditions may be a factor in the survival of oral biofilms after treatment.

Microbiota of plaque microcosm biofilms: effect of three times daily sucrose pulses in different simulated oral environments.

AIM: To explore the Ecological Plaque Hypothesis for dental caries. To test modification of the microbiota of dental plaque microcosm biofilms by sucrose pulsing during growth in two different simulated oral fluids, and with a urea-induced plaque pH elevation. METHODS: Plaque microcosm biofilms were cultured in an 'artificial mouth' with and without 6-min 5% w/v sucrose pulses every 8 h in an environment of continuously supplied saliva-like defined medium with mucin (DMM), or basal medium mucin (BMM, a high-peptone-yeast extract oral fluid analogue), and also in DMM + 10 mmol/l urea, with sucrose pulsing. Forty plaque species were quantified by checkerboard DNA:DNA hybridization analysis. RESULTS: Sucrose pulsing extended rapid plaque growth in DMM and BMM, inducing major microbiota changes in DMM but not in BMM. In DMM, some streptococci and lactobacilli were unaffected while others implicated in caries, together with Candida albicans and Capnocytophaga gingivalis, increased. Aerobic, microaerophilic and major anaerobic species decreased. Elevation of the pH(max) from 6.4 to 7.0 had almost no effect on the microbiota. BMM plaques were distinct from DMM plaques with particularly low levels of Candida albicans and Actinomyces. CONCLUSIONS: Modest sucrose exposure in a saliva-like environment causes profound changes in the developmental self-organization of plaque microcosms, supporting the Ecological Plaque Hypothesis. Nevertheless, there is significant stability in microbial composition with varying pH near neutrality. Increases in levels of specific bacteria in response to sucrose could be characteristic of organisms particularly important in caries.

Caries-related plaque microcosm biofilms developed in microplates.

In vivo dental plaque biofilms consist of complex communities of oral bacteria that are a challenge to replicate in vitro. The aim of this investigation was to establish human dental plaque microcosms in microplates to reflect conditions that are relevant to dental caries. Microcosm plaque biofilms were initiated from the saliva of two different donors, grown for up to 10 days in 24-welled microplates on Thermanox coverslips in various types of artificial saliva with and without sucrose, which were replaced daily. Microbiota composition of 40 species associated with oral health and dental caries was monitored in the plaques using Checkerboard DNA-DNA hybridization analysis. pH was measured as an indicator of cariogenic potential. The composition of the saliva inocula was different, and yielded plaque microcosms with different composition and growth responses to sucrose. Artificial saliva type and presence of sucrose, and the resulting growth and pH conditions, modified the growth of individual species and hence the ecological profile of the microplate plaques during development. Complex population shifts were observed during development, and older plaques comprised predominantly facultative anaerobic species. Sucrose supplementation limited the decline of Streptococci over time but did not increase the abundance of mutans Streptococci. Sucrose at 0.15% increased levels of caries-associated species including Lactobacillus fermentum, Lactobacillus acidophilus and Actinomyces gerensceriae; these were further increased with sucrose at 0.5%, in addition to Actinomyces israelii, Rothia dentocariosa and Capnocytophaga gingivalis. The microplate plaques demonstrated complex community dynamics that appeared to reflect the maturation of natural plaques, and sucrose induced a cariogenic plaque composition and pH.

Antimicrobial effects of essential oils in combination with chlorhexidine digluconate.

The aim of the present study was to compare antimicrobial effects of essential oils alone and in combination with chlorhexidine digluconate against planktonic and biofilm cultures of Streptococcus mutans and Lactobacillus plantarum. The essential oils included cinnamon, tea-tree (Melaleuca alternifola), manuka (Leptospermum scoparium), Leptospermum morrisonii, arnica, eucalyptus, grapefruit, the essential oil mouthrinse Cool Mint Listerine and two of its components, menthol and thymol. Cinnamon exhibited the greatest antimicrobial potency (1.25-2.5 mg/ml). Manuka, L. morrisonii, tea-tree oils, and thymol also showed antimicrobial potency but to a lesser extent. The combination effect of the essential oil-chlorhexidine was greater against biofilm cultures of both S. mutans and L. plantarum than against planktonic cultures. The amount of chlorhexidine required to achieve an equivalent growth inhibition against the biofilm cultures was reduced 4-10-fold in combination with cinnamon, manuka, L. morrisonii, thymol, and Listerine. We conclude that there may be a role for essential oils in the development of novel anticaries treatments.

Biofilm growth of Lactobacillus species is promoted by Actinomyces species and Streptococcus mutans.

The ability of oral bacteria to integrate within a biofilm is pivotal to their survival. A dependence on the amount of biofilm growth by noncoaggregating Lactobacillus rhamnosus and Lactobacillus plantarum on coculture with Actinomyces naeslundii, Actinomyces gerencseriae, Streptococcus mutans and Veillonella parvula was investigated using an artificial-mouth culture system. Biofilm formation by the lactobacilli in mono-culture was poor. In coculture with Actinomyces species the amount of L. rhamnosus increased 7-20 times and L. plantarum 4-7 times compared to its mono-culture biofilm. S. mutans also promoted substantial biofilm growth of lactobacilli but V. parvula had no effect. We conclude that these Actinomyces species promoted growth of key Lactobacillus species in a biofilm, as did S. mutans to a smaller extent, and that the ability of individual bacteria to form mono-culture biofilms is not necessarily an indicator of their survival and pathogenic potential in a complex multispecies biofilm community.

Minimizing prion risk without compromising the microbial composition of biofilms grown in vivo in a human plaque model.

AIMS: To determine whether the stringency of sterilization procedures for biological components of in vivo dental plaque-generating devices based on enamel can be increased to minimize prion risk without compromising natural biofilm composition. METHODS AND RESULTS: The composition of in vitro biofilms, grown on hypochlorite-treated and untreated autoclaved enamel surfaces, was determined using culture-based methods and checkerboard DNA: DNA hybridization analysis. No differences were found between biofilms recovered from either substrate. SIGNIFICANCE: Several in situ models allow generation of plaque in the oral cavity, followed by recovery of intact biofilms for experimentation. Approaches allowing plaque formation on natural tooth surfaces are most valuable, but present a possible infection risk to volunteers wearing plaque-collecting devices, particularly with respect to prions. Hypochlorite treatment of biological material, as an adjunct to autoclaving, reduces infection risk without compromising biofilm composition and should be adopted in all future studies using plaque-generating devices incorporating enamel, where there is a potential prion threat, and further investigated in other biological hard tissues.

Calcium phosphate deposition in human dental plaque microcosm biofilms induced by a ureolytic pH-rise procedure.

The objectives were to develop and characterize a procedure based on a ureolytic pH rise to deposit calcium phosphate into microcosm dental plaque biofilms and to test the importance of the plaque pH range. Plaque biofilms were cultured in a multiplaque culture system ('artificial mouth') with a continuous supply of a simulated oral fluid (basal medium mucin; BMM) with 146 mmol/l (5% w/v) sucrose periodically applied over 6 min every 8h. After initial plaque growth, the biofilms were periodically exposed for up to 16 days to 6-min applications of calcium phosphate monofluorophosphate urea (CPMU) solution containing 20 mmol/l CaCl(2), 12 mmol/l NaH(2)PO(4), 5 mmol/l monofluorophosphate and 500 mmol/l urea (pH 5.0). Three application regimes were examined, one included a sucrose-induced acidic pH fluctuation. Plaque hydrolysis of the urea in CPMU caused the pH to rise to between 8.2 and 8.8, depositing fluoridated and carbonated calcium phosphates, and possibly some calcium carbonate, into the plaque. Calcium, phosphate and fluoride deposition was rapid for about 4 days and then slowed. After 10 days' treatment under standard conditions (BMM containing 1 mmol/l urea and 1 mmol/l arginine), plaque calcium and phosphate concentrations had increased up to 50-fold and 10-fold to approximately 2-4 and 1-2 mmol/g plaque protein, respectively. The calcium, phosphate and fluoride content increased steadily. Calcium phosphate deposition was proportional to the plaque resting pH, increasing over four-fold when the BMM urea concentration was increased from 0 to 20 mmol/l, which raised the resting pH from 6.4 to 7.2 and yielded a mean plaque calcium concentration of 14.3 mmol/g protein, one subsample reaching 20.8 mmol/g protein. Supplementation of BMM with 20% human serum inhibited deposition. These results support the hypothesis that an alkaline pH in plaque is critical in promoting plaque mineralization and that mineral deposition is modulated by serum. These factors are likely to be important in regulating calculus formation.

Checkerboard DNA-DNA hybridisation technology focused on the analysis of Gram-positive cariogenic bacteria.

Checkerboard DNA-DNA hybridisation enabled the quantitative analysis of plaque samples against 40 microbial species simultaneously, using digoxygenin-labelled, whole-genome DNA probes. This technique was initially developed to study the predominantly Gram-negative sub-gingival plaque microbiota. The aim of this study was to apply it to a suite of predominantly Gram-positive microorganisms, such as those implicated in cariogenesis. To specifically target Gram-positive species (and Candida albicans) required optimisation and modification of DNA extraction, prehybridisation, hybridisation, and antibody detection conditions. The suitability of the revised technique for clinical and epidemiological studies was confirmed using interproximal plaque from small groups of 5- to 6-year-old children of high (decayed, missing, or filled teeth (dmft)> or =5, n=8) and zero (n=5) caries rates.

The effect of sucrose application frequency and basal nutrient conditions on the calcium and phosphate content of experimental dental plaque.

A reduced pool of calcium in dental plaque would be expected to increase the ability of plaque fluid to dissolve the underlying enamel when the pH falls during sugar exposure. We have examined the relationship between frequency of sugar application and Ca and P(i) concentrations in artificial mouth plaque microcosm biofilms. Ten plaques were grown simultaneously from a human saliva inoculum using a continuous flow of simulated saliva, DMM, supplemented with either urea or glucose to modulate the resting pH. In addition the plaques received sucrose applications of varying frequency: 12-, 8-, 6-, or 4-hourly, or not at all. After 15 days the plaques were sampled by taking 4 full-thickness specimens of each, and acid-extractable Ca and P(i), and alkali-soluble protein and carbohydrate were determined. Ca and P(i) concentrations were in a range comparable with those in human plaque, except in the DMM + urea plaque receiving no sucrose, when concentrations were higher. Plaque Ca concentration decreased significantly as sucrose application frequency increased. Increasing sucrose application frequency also reduced the protein, i.e. the cell biomass, content of the plaques and, in the case of DMM + urea plaques, increased the water-insoluble hexose content, presumably extracellular polysaccharide. Reduced biomass was partly due to the bulking of plaque with extracellular polysaccharide, but the marked effect of urea on polysaccharide formation is not understood. This study shows that increasing frequency of sugar application alters dental plaque by reducing its mineral protection capacity.

Inhibitory effect of ZnCl(2) on glycolysis in human oral microbes.

Although the inhibition of bacterial glycolysis by zinc ions might be expected to moderate dental caries, there has not been a comparison of the effect of Zn on different organisms under both fixed pH and free-fall conditions. Here, the effect of ZnCl(2) on Streptococcus salivarius, Strep. mutans, Strep. sobrinus, Actinomyces naeslundii and Lactobacillus casei, as well as on mixtures of oral organisms outgrown from human dental plaque and saliva, was surveyed. pH-stat experiments were performed at pH 7, 6 or 5 in a solution containing 5% glucose and a suspension of the test organism; pH-fall experiments started at pH 7. In both cases, acid production was monitored for 60 min, when samples were taken for Zn and lactate determinations. Under pH-stat conditions, acid production was inhibited by Zn most strongly in Strep. sobrinus and Strep. salivarius. In terms of total acid production averted, however, the effect of Zn under both pH-stat and pH-fall conditions was clearly greatest with Strep. salivarius. A. naeslundii was inhibited the least strongly under pH-stat conditions. Cultured oral organism mixtures were more sensitive to moderate concentrations of zinc (0.2-0.3mM initial concentration) than were the single species to higher concentrations (1mM). Packed cell layers responded to Zn quite differently from suspensions, the pH often falling in the presence of 1mM Zn at a rate similar to the no Zn control. As streptococci had the highest acidogenesis rates in both pH-stat and pH-fall experiments, it seems likely that inhibition of acid production with these organisms would be of more value in moderating caries than the inhibition of less acidogenic organisms such as A. naeslundii.

Plaque mineral induction and inhibition properties in the formation of supragingival calculus.

Several individual species of dental plaque bacteria have the ability to initiate the precipitation of calcium phosphate minerals in vitro; other plaque components have been shown to inhibit mineralisation. We have examined subjects' overall plaque mineralisation promoter and inhibitor properties, and have attempted to correlate them with supragingival calculus development over 6 months. Three-day-old plaque was collected from 22 adult subjects at the start and end of the study. To detect promoter activity, the plaque was placed in a suspension of brushite, the liquid phase of which was supersaturated with respect to hydroxyapatite. The extent of mineralisation was determined by the rise in phosphate concentration over 4 days. To detect inhibitor activity, plaque was placed in a similar suspension, which also contained hydroxyapatite. Promoter activity was compared with that hydroxyapatite, and inhibitor activity was compared with polyaspartate. The subjects' teeth were scaled at the start of the study, and calculus deposition was measured at the end using the Volpe Manhold method. Most plaque samples showed some promoter or inhibitor activity, or both, but no significant correlation existed between these activities and a subject's development of calculus. A significant inverse correlation existed between plaque mineralisation promoter activity and its inhibitor activity at the start of the study. Our results suggest that the nucleating and mineralisation inhibitory properties of young plaque will probably not be a useful target for a practical preventive methodology for supragingival calculus.

Development of multi-species consortia biofilms of oral bacteria as an enamel and root caries model system.

The aim was to establish defined-species consortium plaque biofilms to investigate enamel and root caries in an artificial mouth. Strains of the putative enamel and root caries pathogens, Streptococcus mutans, Strep. sobrinus, Actinomyces naeslundii and Lactobacillus rhamnosus, were screened in batch culture for potential cariogenic properties: a low terminal pH, ability to aggregate, and catabolic diversity. The strains selected were grown as monoculture biofilms and as consortium plaque biofilms in a multiplaque artificial mouth. The biofilms were supplied with a constant flow of a simulated oral fluid and were given periodic sucrose (and in some instances glucose) to simulate meals. All the bacteria except L. rhamnosus formed large, monospecies biofilms with resting pH in the range 5.3-5.8. The consortia biofilms were larger and had a resting pH of 4.9-5.3. The consortia biofilms supplied with 8-hourly carbohydrate comprised mainly 'mutans' streptococci (58, SD 5.5%) and L. rhamnosus (42, SD 5.7%). A. naeslundii characteristically was absent or present in a low percentage (up to 4% colony-forming units). All biofilms demineralized polished bovine enamel and dentine blocks, as assessed by microradiography and enamel-surface microhardness measurement. The consortia also demineralized intact enamel and tooth roots; they were more cariogenic to enamel than any of the monoculture biofilms, as measured by enamel-surface softening, but variation in lesion depth was proportional to biofilm wet weight irrespective of acidogen composition (r = 0.93, p < 0.05). Enamel lesions had a well-mineralized intact surface and a zone of subsurface demineralization, typical of early natural lesions. Dentine and root lesions showed extensive demineralization but lacked a pronounced surface mineralized zone. Substitution of glucose for sucrose had no effect on the cariogenicity of the consortium to bovine enamel or human roots and had no major effect on the plaque composition. Continuously supplied fluoride (19 parts/10(6)) resulted in a substantially reduced enamel surface softening and subsurface demineralization of intact roots. It was concluded that consortia biofilms of selected caries pathogens generate realistic caries lesions in all tooth hard tissues under controlled growth conditions in the artificial mouth. This in vitro caries experimental model may prove useful for the study of interrelations between the plaque biofilm, tooth tissues and the oral environment, and for the development of procedures to modify the course of caries development.

Suppression of urease levels in Streptococcus salivarius by cysteine, related compounds and by sulfide.

Urease synthesis in Streptococcus salivarius is induced by an acid environment, carbohydrate and a high growth rate. We now report that both cysteine and sulfide above 1 mM strongly suppress S. salivarius urease levels. Close structural relatives of cysteine (cysteamine, ethanedithiol and penicillamine) at 5 mM buffered to pH 7.0 also caused urease suppression, but thiols in general (2-mercaptoethanol, dithiothreitol and glutathione) did not. In cultures buffered below pH 5.9, the cysteine-induced urease suppression was lifted substantially, but the sulfide suppression increased, suggesting involvement of different processes. Urease activity was inhibited 50% by 5 mM mercaptoethanol but unaffected by 5 mM cysteine or sulfide, hence modification of enzyme activity by thiols is not directly related to suppression of their levels in culture. Cysteine, arising primarily through protein hydrolysis which also raises the pH, could be a surrogate pH feedback signal for nearby alkaline conditions, and sulfide may reflect activity of periodontopathic plaques.

Fluoride distribution in sound and carious root tissues of human teeth.

Proton probe analysis has been used to provide for the first time quantitative F concentration data in carious root tissues from subjects consuming water containing 1 ppm F. In small lesions at the neck of the tooth with minimal tissue loss the F concentration was significantly higher at the outer lesion edge than at the outer edge of adjacent sound root tissue. In one sample with high F values the lesion edge had 19,000 ppm F and the adjacent sound root surface 5,400 ppm F microg F/g apatite). In large lesions with extensive cavitation F was again concentrated in the outer edge of the lesion and was significantly higher (1,800-4,100 ppm) than in adjacent sound inner dentine (190-290 ppm). Fluoride concentrations varied markedly along the outer edge of both normal and carious root tissues. Fluoride increase at the lesion edge is not an effect of tissue shrinkage but probably a result of remineralisation events during caries. This additional F may be expected to increase tissue resistance to further acid attacks.

Factors affecting the resting pH of in vitro human microcosm dental plaque and Streptococcus mutans biofilms.

The aim was to examine factors that potentially control the resting pH, defined as the pH unaffected by meals, of microcosm dental plaques and Streptococcus mutans biofilms under standard conditions, and to examine the effect of supplying urea at concentrations found intraorally. Microcosm plaques were cultured from plaque bacteria-enriched saliva in an 'artificial mouth' with a continuous supply of a medium including 0.25% mucin [Basal Medium Mucin, (BMM), 3.6 ml/hr per plaque] and a periodic supply of sucrose. The steady-state resting pH was 6.4 (range +/- 0.1) in BMM containing no urea and supplied at the standard flowrate. This is a robust property of the ecosystem. In one experiment with a replicated (n = 9) set of measurements, the resting pH was approx. pH 6.3, 6.4, 6.7 and 7.3 with 0, 1, 5 and 20 mmol/l urea in the BMM. The magnitude of sucrose- and urea-induced pH responses was unaffected by elevating the resting pH to produce parallel pH curves. The sucrose-induced pH curves were analogous to those classically reported by Stephan that showed an association between caries activity and increasingly acidic plaque pH responses to glucose. Stopping the BMM flow caused a pH rise, indicating continuing net alkali generation from BMM components in the absence of a fluid flow. Step. mutans monoculture biofilms had an acidic resting pH of 5.0 to 5.3, which increased to 6.8 following an adventitious superinfection by Bacillus cereus. It was concluded that the resting pH in plaque results from a delicate balance between alkali and acid generation, which is in turn dependent both on the bacterial composition of the plaque and on the supply of substrates and buffers from, and metabolite clearance into, flowing oral fluid. In vivo the resting pH will vary with site-specific changing saliva flows. Urea continuously supplied at concentrations normal for saliva and gingival crevicular fluid can raise the resting pH of microcosm plaque by an amount tat in vivo would probably be significant in reducing dental caries.

Inhibition of purified enolases from oral bacteria by fluoride.

Enolase activity in strains of oral streptococci previously has been found to be inhibited by 50% (Ki) by fluoride concentrations ranging from 50 to 300 microM or more in the presence of 0.5 to 1.0 mM inorganic phosphate ions. In this study, enolase was extracted and partly purified by a two-step process from five oral bacterial species and the effect of fluoride on the kinetics of enolase examined. The molecular weight of the putative enolase proteins was 46-48 kDa. The Vmax values ranged from 20 to 323 IU/mg and K(m) for glycerate-2-phosphate from 0.22 to 0.74 mM. Enolase activity was inhibited competitively by fluoride, with Ki values ranging from 16 to 54 microM in the presence of 5 mM inorganic phosphate ions. Ki values for phosphate ranged from 2 to 8 mM. The enolase from Streptococcus sanguis ATCC 10556 was more sensitive to fluoride (Ki = 16 +/- 2) than was enolase from Streptococcus salivarius ATCC 10575 (Ki = 19 +/- 2) or Streptococcus mutans NCTC 10449 (Ki = 40 +/- 4) and all three streptococcal strains were more sensitive to fluoride than either Actinomyces naeslundii WVU 627 (Ki = 46 +/- 6) or Lactobacillus rhamnosus ATCC 7469 (Ki = 54 +/- 6) enolases. The levels of fluoride found to inhibit the streptococcal enolases in this study are much lower than previously reported and are likely to be present in plaque, especially during acidogenesis, and could exert an anti-glycolytic effect.

Artificial dental plaque biofilm model systems.

Difficulties with in vivo studies of natural plaque and its complex, heterogeneous structure have led to development of laboratory biofilm plaque model systems. Technologies for their culture are outlined, and the rationale, strengths, and relative uses of two complementary approaches to microbial models with a focus on plaque biodiversity are analyzed. Construction of synthetic consortia biofilms of major plaque species has established a variety of bacterial interactions important in plaque development. In particular, the 'Marsh' nine-species biofilm consortia systems are powerful quasi steady-state models which can be closely specified, modified, and analyzed. In the second approach, microcosm plaque biofilms are evolved in vitro from the natural oral microflora to the laboratory model most closely related to plaque in vivo. Functionally reproducible microcosm plaques are attainable with a biodiverse microbiota, heterogeneous structure, and pH behavior consistent with those of natural plaque. The resting pH can be controlled by urea supply. Their growth patterns, pH gradient formation, control of urease levels by environmental effectors, and plaque mineralization have been investigated. Microcosm biofilms may be the only useful in vitro systems where the identity of the microbes and processes involved is uncertain. Together, these two approaches begin to capture the complexity of plaque biofilm development, ecology, behavior, and pathology. They facilitate hypothesis testing across almost the whole range of plaque biology and the investigation of antiplaque procedures yielding accurate predictions of plaque behavior in vivo.

Inhibition by ethanol of the growth of biofilm and dispersed microcosm dental plaques.

Inhibition of microcosm plaque biofilm growth by periodic application of ethanol was compared with the minimum inhibitory concentration (MIC) and bactericidal effects of ethanol on liquid cultures of dispersed plaque bacteria. Microcosm plaques were cultured from saliva in a multiplaque 'artificial mouth' and their growth in wet weight measured daily. Nutrient conditions included: a continuous supply of a medium containing 0.25 percent mucin, and 8-hourly 5 percent (w/v) sucrose (1.5 ml over 6 min). Plaque biofilm growth was strongly inhibited by exposure to 40 percent (v/v) ethanol applied in volumes of 3.75 ml over 15 min, six times daily. Application of 1.5 ml over 6 min inhibited much less or not at all. Ethanol concentrations lower than 40 percent caused less inhibition, with 10 percent having almost no effect. The pH response to sucrose was unchanged by prior application of 40 percent ethanol for 30 min. Some evidence was obtained for either bacterial adaptation to ethanol or selection of ethanol-resistant bacteria. The MIC and bactericidal effects of ethanol were assessed by growth of dispersed plaque in liquid culture; the bactericidal effect was measured as the induced delay in growth. The aerobic and anaerobic MIC of ethanol for growth was 10 percent and 8 percent; 50 percent inhibition of growth rate occurred at 3.7 percent and 2.8 percent. Ethanol (40 percent) was bactericidal within 1-2 min, but 10 percent had almost no effect. It was concluded that, despite the well-known high ethanol sensitivity of dispersed plaque bacteria, prolonged application of ethanol concentrations in the order of 40 percent are necessary to inhibit growth of plaque biofilms.