Oral microbiome research from a public health perspective and implications for oral health.

Advancing oral microbiome research has revealed the association between oral microbiome composition and oral disease. However, much of the research has predominantly focused on comparing health and disease conditions, overlooking the potential dental public health implications. This article examines the evolution of oral microbial research from inception, advancement, and current knowledge of health-associated microbiota. Specifically, we focus on two key aspects: the impact of lifestyle and environmental factors on the oral microbiome and using the oral microbes as a therapeutic modality. The complex interaction of host intrinsic, environmental, and lifestyle factors affects the occurrence and development of the oral microbiota. The article highlights the need for ongoing research that embraces population diversity to promote health equity in oral health research and integrate public health practices into microbiome-based research. The implication of population-level interventions and targeted approaches harnessing the oral microbiome as an intervention, such as oral microbiome transplantation, should be further explored.

Efficacy of low concentrations of sodium hypochlorite and low-powered Er,Cr:YSGG laser activated irrigation against an Enterococcus faecalis biofilm.

AIM: To establish the antibacterial efficacy of low concentrations of sodium hypochlorite with and without Er,Cr:YSGG laser activation on Enterococcus faecalis biofilms in extracted teeth. METHODOLOGY: The root canals of 96 decoronated single-rooted extracted human teeth were prepared to a size 40, 0.06 taper 1 mm beyond the apex. They were mounted within a flow cell, which was sterilized before pumping a nutrient media through the root canals. The flow cell was inoculated with E. faecalis (ATCC 700802) and cultivated for 4 weeks. The root-ends were sealed, and the roots were then subjected to one of six treatment groups: group 1: syringe irrigation (SI) with saline (control) using a 27 -gauge Monoject needle 1 mm from the apex for 2 min; group 2: as for group 1 but with 1% NaOCl; group 3: as for group 1 but with 4% NaOCl; group 4: 0.5% NaOCl irrigation for 15 s followed by laser-activated irrigation (LAI) with four 15-s cycles replenishing the irrigant between cycles; group 5: as for group 4 but with 1% NaOCl as the irrigant; group 6: as for group 4 but with 4% NaOCl as the irrigant. Following treatment, teeth were crushed and viable bacteria were quantitated by serial dilution and plating. The colony-forming unit values were compared between groups using one-way anova and Tukey-adjusted post hoc tests. A two-tailed P value of <0.05 was considered statistically significant. RESULTS: The mean number of cells recovered from the 1% NaOCl SI group was significantly higher than that from the 4% NaOCl LAI group (P = 0.02). CONCLUSION: Within the limitations of this laboratory study, low-powered (0.5 W) Er,Cr:YSGG laser activation did not improve the antibacterial effect of low concentrations of sodium hypochlorite.

Clonal diversity in biofilm formation by Enterococcus faecalis in response to environmental stress associated with endodontic irrigants and medicaments.

AIM: To determine whether clonal diversity within E. faecalis affects biofilm formation when exposed to antimicrobial compounds found in endodontic medicaments and irrigants. METHODOLOGY: Five human isolates of E. faecalis were compared; biofilms were grown in microtitre trays in the presence of sodium hypochlorite, calcium hydroxide, chlorhexidine, tetracycline or clindamycin. Biofilms were quantified by staining with crystal violet and optical density determined with a microplate reader. Slime production (an amorphous extracellular matrix comprising polysaccharides, glycoproteins and glycolipids loosely attached to the cell surface) was determined qualitatively by growth on Congo red agar plates. Linear mixed models were used to examine whether medicaments affected biofilm growth of the isolates in the presence of the medicaments or irrigants. RESULTS: Overall, different endodontic antimicrobials significantly altered biofilm growth in E. faecalis isolates. Two E. faecalis isolates significantly (P < 0.0001) increased biofilm formation in the presence of tetracycline and one in the presence of NaOCl (P = 0.018). Qualitatively, slime production also varied between isolates and correlated with biofilm production. CONCLUSIONS: When subjected to sub-minimum inhibitory concentration (MIC) levels of antimicrobial compounds found in endodontic medicaments, E. faecalis isolates demonstrated significant clonal variation in their capacity to form biofilms. Interestingly, there was a correlation between slime production and the ability of isolates to form a biofilm in the presence of antimicrobials. The results indicate that isolates of E. faecalis that form biofilms in response to endodontic medicaments may be more likely to survive endodontic treatment.

Proteomic identification of proteinase inhibitors in the porcine enamel matrix derivative, EMD(®).

BACKGROUND AND OBJECTIVE: The porcine enamel matrix derivative, EMD(®), which is the active component of Emdogain(®), is used widely in periodontics because of its ability to promote the regeneration of soft and hard tissues and to reduce inflammation. Previous studies have used indirect methods to explain its angiogenic and proliferative effects on cells associated with wound healing. In this study we used proteomic techniques to identify proteins in EMD other than amelogenins. MATERIAL AND METHODS: Proteins in EMD were separated by two-dimensional gel electrophoresis and were identified using mass spectrometry. Proteomic results were validated by western blot analysis of Emdogain. RESULTS: Fourteen proteins of porcine origin were identified and included the serine and cysteine proteinase inhibitors alpha1-antichymotrypsin and fetuin A, respectively. Alpha1-antichymotrypsin is an acute-phase factor that has been reported to indirectly down-regulate the expression of the gelatinase MMP-9. Fetuin A, a major glycoprotein component of bone and teeth, is a potent inhibitor of ectopic calcification of vascular and soft tissues and has been implicated in both osteogenesis and bone resorption. It also facilitates plasma membrane repair in damaged fibroblasts. CONCLUSION: EMD contains a number of high-molecular-weight compounds which include the proteinase inhibitors, fetuin A and alpha1-antichymotrypsin.

Dengue virus infection induces upregulation of GRP78, which acts to chaperone viral antigen production.

Dengue virus (DENV) pathogenesis is related to the host responses to viral infection within target cells, and therefore, this study assessed intracellular changes in host proteins following DENV infection. Two-dimensional gel electrophoresis and mass spectrometry identified upregulation of the host endoplasmic reticulum (ER) chaperone GRP78 in K562 cells following DENV infection, in the absence of virus-induced cell death. Upregulation of GRP78 in DENV-infected cells was confirmed by immunostaining and confocal microscopy and by Western blot analysis and was also observed in DENV-infected primary monocyte-derived macrophages, a natural target cell type for DENV infection. GRP78 was upregulated in both DENV antigen-positive and -negative cells in the DENV-infected culture, suggesting a bystander effect, with the highest GRP78 levels coincident with high-level DENV antigen production and infectious-virus release. Transfection of target cells to express GRP78 prior to DENV challenge did not affect subsequent DENV infection, but cleavage of GRP78 with the SubAB toxin, during an established DENV infection, yielded a 10- to 100-fold decrease in infectious-virus release, loss of intracellular DENV particles, and a dramatic decrease in intracellular DENV antigen. However, DENV RNA levels were unchanged, indicating normal DENV RNA replication but altered DENV antigen levels in the absence of GRP78. Thus, GRP78 is upregulated by DENV infection and is necessary for DENV antigen production and/or accumulation. This may be a common requirement for viruses such as flaviviruses that depend heavily on the ER for coordinated protein production and processing.

Effect of dietary omega-3 polyunsaturated fatty acids on experimental periodontitis in the mouse.

BACKGROUND AND OBJECTIVE: Periodontitis is an infective disease caused predominantly by gram-negative anerobes. The host inflammatory response to these bacteria causes alveolar bone loss, which characterizes periodontitis. Omega-3 polyunsaturated fatty acids have recognized anti-inflammatory effects; their oxygenated derivatives are key mediators in reducing inflammation. In this study we tested the hypothesis that dietary supplementation with tuna fish oil rich in the n-3 polyunsaturated fatty acid, docosahexaenoic acid, would reduce alveolar bone loss in mice inoculated with periodontopathic bacteria. MATERIAL AND METHODS: Adult mice were fed experimental diets containing either 10% tuna oil or Sunola oil for 57 d. After 14 d, 35 mice on each diet were inoculated orally with Porphyromonas gingivalis, with a mixture of P. gingivalis and Fusobacterium nucleatum, with carboxymethylcellulose or remained untreated. The mice were killed, and soft tissue biopsies from the oral cavity of treated mice were used to determine the polyunsaturated fatty acid concentrations. The maxilla was removed, stained and digitally imaged to assess bone loss around the upper molars. RESULTS: n-3 polyunsaturated fatty acid levels were significantly higher in oral soft tissues of mice fed tuna oil compared with the control group. Mice fed tuna oil and inoculated with P. gingivalis or with the combination of F. nucleatum and P. gingivalis exhibited 72% and 54% less alveolar bone loss respectively, compared with the treatment control group. CONCLUSION: Alveolar bone loss was inversely related to n-3 polyunsaturated fatty acid tissue levels. In conclusion, fish oil dietary supplementation may have potential benefits as a host modulatory agent in the prevention and/or adjunctive management of periodontitis.

The use of live-animal micro-computed tomography to determine the effect of a novel phospholipase A2 inhibitor on alveolar bone loss in an in vivo mouse model of periodontitis.

BACKGROUND AND OBJECTIVE: Live-animal micro-computed tomography is a new and promising technique that can be used to quantify changes in bone volume for periodontal disease models. The major aim of this study was to develop the methodology of live-animal micro-computed tomography and to determine the effect of a novel secretory phospholipase A2 inhibitor on alveolar bone loss. MATERIAL AND METHODS: Periodontitis was induced in mice by oral infection with Porphyromonas gingivalis over a period of 13 wk, and live-animal micro-computed tomography scans were taken at different time-points to determine bone volume changes with disease progression. This enabled conclusions to be made as to when treatment was most likely to be effective. In addition, the model was used to investigate a novel drug, the secretory phospholipase A2 inhibitor, KHO64, and its potential ability to inhibit osteoclast bone resorption and treat periodontitis. RESULTS: The results from live-animal micro-computed tomography scans revealed greater, statistically significant, bone volume loss in diseased mice compared with normal mice (p < 0.05). This corresponded to a larger area from the cemento-enamel junction to the alveolar bone crest, as assessed by stereo imaging (p < 0.001). These techniques can therefore detect and quantify alveolar bone loss. Both methods revealed that KHO64 had no significant effect on the volume of bone resorption. CONCLUSION: Live-animal micro-computed tomography is a robust, reproducible technique that clearly demonstrates significant time-dependent changes in alveolar bone volume in a small-animal model of periodontitis.

The inability of Streptococcus mutans and Lactobacillus acidophilus to form a biofilm in vitro on dentine pretreated with ozone.

BACKGROUND: The use of ozone therapy in the treatment of dental caries is equivocal. The aim of this study was to use an in vitro model to determine the effects of prior ozone application to dentine on biofilm formation and to measure any associated reduction in bacteria viability. METHODS: Twenty dentine discs were bonded to the bases of 5 mL polycarbonate screw top vials. Ten dentine discs were infused with ozone for 40 seconds, 10 samples remained untreated as a control. The vials were filled with nutrient medium, sterilized and placed into the outflow from a continuous chemostat culture of Streptococcus mutans and Lactobacillus acidophilus for four weeks. At the conclusion of the experiment bacterial growth was monitored by taking optical density readings of the growth medium in each vial and the outer surface of the dentine specimens were examined by scanning electron microscopy as shown by SEM analysis. RESULTS: Ozone infusion prevented biofilm formation on all the treated samples while there was substantial biofilm present on the control specimens. While the average optical density of the control specimens was almost twice that of the ozone infused dentine (0.710 for the control with a SD of 0.288 and 0.446 for the ozonated samples with a SD of 0.371), the results were not significant (p > 0.05). CONCLUSIONS: This preliminary study has shown that the infusion of ozone into non-carious dentine prevented biofilm formation in vitro from S. mutans and L. acidophilus over a four-week period. The possibility exists that ozone treatment may alter the surface wettability of dentine through reaction with organic constituents.

Investigation of the effect of rapid and slow external pH increases on Enterococcus faecalis biofilm grown on dentine.

BACKGROUND: Calcium hydroxide is a common endodontic medicament and has an antimicrobial effect by increasing the localized pH within the root canal. However, Enterococcus faecalis has shown some resistance to calcium hydroxide. METHODS: A flow cell apparatus was used to grow an E. faecalis biofilm on dentine discs. Following 4 weeks growth in Todd Hewitt Broth, flow cells were exposed to either a rapid or slow increase to pH 11.5 or 12.5. Cellular viability was determined using serial plating and the number of colony-forming units was normalized against the cellular protein content. Scanning electron microscopy was carried out to qualitatively observe the effects of the different rates of pH increase. RESULTS: A significant difference in viability between the pH rapid and slow groups was not shown in this study. Compared with pH 11.5 solutions, pH 12.5 solutions were more effective at killing bacteria although some E. faecalis still survived. CONCLUSIONS: Enterococcus faecalis did not adapt and develop a greater resistance to high pH following a slow rise in pH compared with a rapid rise in pH. As expected, pH 12.5 was more effective in reducing bacterial numbers compared with pH 11.5 although E. faecalis was not completely eliminated.

Comparative efficacy of endodontic medicaments and sodium hypochlorite against Enterococcus faecalis biofilms.

BACKGROUND: Many studies have investigated the effectiveness of root canal irrigants and medicaments against Enterococcus faecalis. The aim of this study was to compare the efficacy of commonly used medicaments against E. faecalis cultured as a biofilm on dentine substrate. METHODS: An E. faecalis biofilm was established on human dentine slices using a continuous flow cell. Each test medicament (Ledermix, Ca(OH)2 , Odontopaste, 0.2% chlorhexidine and 50:50 combinations of Ledermix/Ca(OH)2 and Odontopaste/Ca(OH)2 ) was introduced into the flow cell and biofilms were harvested and quantitated by determining cellular protein. Cellular viability was determined using serial plating and the number of colony-forming units was normalized against cellular protein to allow treatment protocols to be compared. Qualitative scanning electron microscopy analyses of the biofilm were performed after a 48-h exposure to each test agent. RESULTS: Sodium hypochlorite achieved total bacterial elimination. Ledermix and Odontopaste had no significant effect on the E. faecalis biofilm. Ca(OH)2 and 50:50 combinations of Ca(OH)2 /Ledermix or Ca(OH)2 /Odontopaste reduced the viability by more than 99% while 0.2% chlorhexidine reduced bacterial numbers by 97%. CONCLUSIONS: Sodium hypochlorite remains the gold standard for bacterial elimination in root canal therapy. However, Ca(OH)2 in isolation and combined with Ledermix, and Odontopaste was highly effective in reducing bacterial viability.

A colourimetric evaluation of the effect of bacterial contamination on teeth stained with blood in vitro: Evaluation of the efficacy of two different bleaching regimes.

BACKGROUND: Tooth discolouration could occur due to bacterial contamination in traumatized teeth. Hydrogen peroxide is the commonly used bleaching agent. However, due to concerns over safety, alternative bleaching regimes such as sodium perborate (S) and thiourea-hydrogen peroxide (T) have been investigated. METHODS: Apices resected and pulp extirpated 99 premolars were divided into two groups. Group 1 and 2 was injected with blood and blood/bacteria, stored anaerobically for 35 days. The two groups were treated by bleaching with water, S or T. Teeth were rebleached after 7 days. Colourimetric evaluation was assessed using digital imaging, CasMatch standardization and CIE L*a*b colour system preoperatively, 35 days of staining and 7 and 14 of bleaching. A linear mixed model with fixed effects of time, group and bleach was used to examine colour difference. RESULTS: Blood-stained teeth were significantly redder and darker on day 35 compared with blood/bacteria-stained teeth. After bleaching, blood-stained teeth retained significant redness compared with blood/bacteria-stained teeth using either S or T. T produced a significantly whiter shade in both the groups after 14 days. CONCLUSIONS: Blood-stained teeth were significantly darker and red compared with blood/bacteria-stained teeth. T bleaching regime was more effective than S.

An in vitro investigation of marginal dentine caries abutting composite resin and glass ionomer cement restorations.

BACKGROUND: There are a number of studies citing the primary reason for replacing auto cure glass ionomer cements was due to recurrent caries. The purpose of this study was to use an in vitro model to measure caries at the dentine restoration interface of bonded composite resin and auto cure glass ionomer cement restorations and to measure the amount of surface degradation occurring in the restorative materials. METHODS: Specimens of auto cure glass ionomer cements (Riva Fast, Fuji IX Fast, Ketac Molar Quick and Fuji VII) and bonded composite resin restorations (Ice, SDI) were placed separately at the dentino-enamel junction of 10 recently extracted human third molar teeth, disinfected and placed into the overflow from a continuous culture of S. mutans for two weeks. Restorations were sectioned and prepared for scanning electron microscopy (SEM) and electron probe microanalysis (EPMA). Restoration tooth interfaces were photographed and the distance from the surface of the teeth to the surface of the restorations measured. EPMA of percentage weights of calcium, phosphorous and fluoride were made outwards from the restoration surface 130pm at a depth of 10 microm below the surface of the dentine. RESULTS: There were significant differences between the surface heights of composite resin, auto cure glass ionomer cements compared to teeth surfaces. Percentage weights of calcium and phosphorus levels were similar to non-demineralized dentine in the auto cure glass ionomer cement samples but there were significant reductions in mineral content of dentine adjacent to bonded composite resin restorations. Fluoride levels were mixed. CONCLUSIONS: This study shows that placing a bonded composite resin restoration into dentine affords little protection to the surrounding tooth from caries attack although insignificant degradation of the restorative surface occurs. Placing a glass ionomer cement restoration into dentine protects the surrounding tooth from caries but degradation of the restoration surface occurs.

Differences between normal and demineralized dentine pretreated with silver fluoride and potassium iodide after an in vitro challenge by Streptococcus mutans.

BACKGROUND: The application of diamine silver fluoride (Ag(NH3)2F) and potassium iodide (KI) to demineralized dentine has been shown to inhibit the growth of Streptococcus mutans. The purpose of this study was to observe the differences between demineralized and non-demineralized dentine treated with AgF/KI. METHODS: Thirty-five dentine discs were bonded to the bases of 5 mL polycarbonate screw top vials which were filled with nutrient medium, sterilized and placed into the overflow from a continuous culture of S. mutans. Samples were divided as follows: 10 samples of demineralized dentine; 10 samples of demineralized dentine treated with AgF/KI; 5 samples of non-demineralized dentine; and 10 samples of non-demineralized dentine treated with AgF/KI. Following two weeks connected to the Chemostat, an electron probe microanalysis (EPMA) of percentage weights and penetration depths of calcium, phosphorous silver and fluoride was conducted. Bacterial growth was monitored by taking optical density readings of the growth medium in each vial and outer surfaces of the specimens were examined by scanning electron microscopy (SEM). RESULTS: AgF/KI treatment of demineralized and non-demineralized dentine prevented biofilm formation and reduced further demineralization by S. mutans. AgF/KI treatment of demineralized dentine was more effective in reducing dentine breakdown and the growth of S. mutans. Significantly higher levels of silver and fluoride were deposited within demineralized dentine. CONCLUSIONS: A topical treatment with AgF/KI on dentine reduced in vitro caries development and inhibited surface biofilm formation. Reduction of in vitro caries development and viability of S. mutans was more pronounced on the dentine samples that had been demineralized prior to the application of

An in vitro model to measure the effect of a silver fluoride and potassium iodide treatment on the permeability of demineralized dentine to Streptococcus mutans.

BACKGROUND: Diamine silver fluoride (Ag(NH3)2F), referred to as AgF, has been used to reduce the incidence of caries in primary dentitions but has been limited by the associated staining of both teeth and restorative materials. The application of potassium iodide (KI), following AgF prevents staining but its effects on the ability of AgF to reduce caries are not known. The aim of this study was to develop an in vitro model that would provide an indication of the permeability of demineralized dentine to Streptococcus mutans after treatment of the dentine with AgF followed by KI. METHODS: Forty dentine discs were bonded to the base of forty 5mL polycarbonate screw top vials (that had had their bases removed), filled with nutrient medium, sterilized and placed into a continuous culture of S. mutans. Samples were divided into four groups as follows: 10 samples of demineralized dentine as a control, 10 samples of demineralized dentine treated with AgF/KI, 10 samples of demineralized dentine treated with KI and 10 samples of demineralized dentine treated with AgF. After two weeks the optical density of the growth medium chambers was measured to determine bacterial penetration and growth. Cultures were plated out to determine migration through the discs by S. mutans. RESULTS: S. mutans migrated through all dentine discs. However, the samples treated with AgF and AgF/KI had significantly lower optical densities than the corresponding controls. The range of optical densities was least amongst demineralized samples treated with AgF/KI. CONCLUSIONS: Under the conditions of this study, treatment of demineralized dentine discs with AgF followed by KI allowed the penetration of S. mutans. Based on optical density measurements, the treatment resulted in significantly fewer microorganisms being present subjacent to the discs treated with AgF and KI than the control discs at the end of the experimental period.

The response to oxidative stress of Fusobacterium nucleatum grown in continuous culture.

Fusobacterium nucleatum ATCC 10953 was grown in continuous culture and the atmosphere changed stepwise from nitrogen (anaerobiosis) to a mixture of air: oxygen (40:60). No significant differences in biomass were observed and the baseline low level of superoxide dismutase increased only slightly; catalase and peroxidase activities were never detected but the level of NADH oxidase increased more than three-fold when oxygen was introduced into the system. In relation to acidic end-products, the relative proportion of acetate increased while that of butyrate decreased. Due mainly, it would seem, to NADH oxidase activity, the culture maintained a low redox potential (E(h)=-274 mV) even under an atmosphere of 40% oxygen in air and dissolved oxygen was not detected. This may, in part, explain the protective role of F. nucleatum for anaerobes in both biofilm and planktonic phases of aerated, mixed cultures of oral bacteria.

The effect of growth rate on the adhesion of the oral bacteria Streptococcus mutans and Streptococcus milleri.

As a preliminary to measuring the hydrophobicity of continuous-culture cells, batch-grown cells of a number of Streptococcus mutans strains were tested for their ability to adhere to hexadecane. The hydrophobic properties of such cells were markedly affected by experimental variables such as the composition of the growth medium and the buffer in which the cells were subsequently suspended. For example, the replacement of glucose by fructose in a chemically-defined growth medium (CDM) increased cell hydrophobicity. Strep. mutans B13 and Streptococcus milleri B448 were separately grown glucose-limited in the CDM at various dilution rates from D = 0.04 h-1 to D = 0.7 h-1, corresponding to mean generation times of 17 and 1 h. Slow-growing cells of both strains were more hydrophobic than fast-growing cells, which, in conjunction with previous studies, supports the suggestion that hydrophobic bonding may play a role in bacterial adherence.

Microbiological evaluation of endodontic files after cleaning and steam sterilization procedures.

BACKGROUND: Infection control procedures are essential for modern dental practice and they are continually evolving to meet the dental profession's high standards. The present study evaluated the efficacy of two cleaning procedures to reduce bacterial numbers on endodontic files, and evaluated the effect of biological debris on the subsequent sterilization of files. METHODS: Stainless steel and nickel-titanium (NiTi) files were examined upon removal from the manufacturer's packaging, after instrumentation in root canals of human teeth inoculated with a broth containing two anaerobic species and one facultative anaerobic species of bacteria, and after instrumentation and cleaning with either an ultrasonic bath or a thermal disinfector. For each file, the bacterial numbers were quantified using routine microbiological techniques in an anaerobic chamber. RESULTS: No bacteria were detected from files direct from their packets. The size, taper and type of file did not affect the ability of either of the cleaning procedures to reduce bacterial numbers. However, an absence of bacteria was more likely when files were cleaned in the thermal disinfector. No bacteria were detected from files that were-subjected to steam sterilization irrespective of the type of prior cleaning procedure. CONCLUSIONS: Steam sterilization eliminated all bacteria from the endodontic files irrespective of the presence of biological debris. The majority of bacteria were eliminated from endodontic files after either ultrasonic cleaning or using a thermal disinfector.

A SEM evaluation of debris removal from endodontic files after cleaning and steam sterilization procedures.

BACKGROUND: In recent times, it has been proposed to classify endodontic files as single-use items due to a perceived inability to adequately clean the instruments. The purpose of the present study was to quantify the surface debris on files removed from the manufacturer's packaging, and after cleaning using an ultrasonic bath or a thermal disinfector. METHODS: Stainless steel and rotary nickel-titanium files were examined after removal from the manufacturer's packaging, after instrumentation in broth-contaminated human teeth, and after various cleaning procedures. The cleaning procedures consisted of either a thermal disinfector cycle, ultrasonication with the files placed in a perforated container or ultrasonication with the files loosely placed in a beaker. The presence of manufacturing debris and biological debris was evaluated using scanning electron microscopy and quantified using image analysis software. RESULTS: The effectiveness of cleaning was not affected by variation in the size or taper of the files when an effective cleaning procedure was used. Cleaning the files in a thermal disinfector or by ultrasonication within a container did not consistently achieve complete removal of biological debris. Placing the files loosely in the ultrasonic bath achieved the most effective cleaning, an average of 98.33 per cent of the file surface area was freed of any biological debris. CONCLUSIONS: A conventional cleaning method is capable of effectively removing biological debris from endodontic files. The efficacy of ultrasonic cleaning was impaired when the files were placed within a perforated container.

Effects of pulsing with xylitol on mixed continuous cultures of oral streptococci.

Continuous culture is a means whereby organisms can be grown at rates approaching those occurring naturally. Moreover, the effect of adding transient excesses of various nutrients to the culture vessel ('pulsing') simulates the effect of dietary challenge on dental plaque organisms. Mixed cultures of Streptococcus mutans T8 and Streptococcus milleri B448 were grown glucose-limited in a chemically defined medium under an atmosphere of 5 per cent carbon dioxide in nitrogen, at a dilution rate of D = 0.1 h-1 and controlled pH of 7.0. The level of arginine in the medium reservoir was adjusted so that Strep. milleri predominated over Strep. mutans in a stable coexistence. After equilibration, the culture vessel was pulsed with various carbohydrates to a final concentration of 5 x 10(-2)mol/L. Samples were then taken at regular intervals and differential viable counts of Strep. mutans and Strep. milleri were done on mitissalivarius agar. Results demonstrated that pulsing with glucose, fructose, 'coupling sugar', lactose, xylose and sorbitol gave Strep. mutans a clear ecological advantage. In direct contrast, pulsing with xylitol resulted in a marked antimicrobial effect on Strep. mutans while Strep. milleri was essentially unaffected. This supports recent findings by other workers that uptake of this pentitol by Strep. mutans in batch culture sets up a 'futile cycle', leading to depressed growth or even cell death.

The influence of intracellular polyglucose and prior growth rate on the survival of Fusobacterium nucleatum under starvation conditions.

Grown in a chemically defined medium containing glucose at a dilution rate of D = 0.065 h-1, Fusobacterium nucleatum D212B-2 produced large amounts of intracellular polyglucose. Aliquots of this culture were starved by anaerobic incubation at 37 degrees C and at various times, assayed for intracellular polyglucose content and viability. This protocol was repeated using cells grown under the same conditions in a chemically defined medium, a medium lacking carbohydrate and in which the organism produced no intracellular polyglucose. Both cultures had 50% survival time values of about 1.5 h and were not eliminated even after 32 h of starvation. It was, therefore concluded that starvation-survival is not influenced by intracellular polyglucose. Starvation-survival was also determined for cells grown in a chemically defined medium at D = 0.048 h-1 and D = 0.12 h-1. The faster-grown cells had a 50% survival time of 3.8 h but were completely eliminated after 8-16 h of starvation. In contrast, slower-grown cells had a 50% survival time of 1.5 h but were not completely eliminated after 32 h of starvation. This illustrates the importance of cell history and technique standardization in comparing the starvation-survival of different organisms.