Thorsten Mathias Auschill

Spatial distribution of vital and dead microorganisms in dental biofilms.

Abstract To examine the spatial structure of dental biofilms a vital fluorescence technique was combined with optical analysis of sections in a confocal laser scanning microscope (CLSM). Enamel slaps were worn in intraoral splints by three volunteers for five days to accumulate smooth-surface plaque. After vital staining with fluorescein diacetate and ethidium bromide the specimens were processed for CLSM examination. Optical sections 1 μm apart were analysed in the z-axis of these dental biofilms. One of the films was 15 μm high, sparse and showed low vitality, i.e.

Individual Vitality Pattern of in situ Dental Biofilms at Different Locations in the Oral Cavity

The aim of the study was to examine the three-dimensional vitality structure of dental biofilms grown simultaneously at different locations in the oral cavity over a 48-hour period. Eight healthy volunteers wore special acrylic appliances. On each buccal side of the upper and the lower jaw three glass slabs were inserted, allowing for growth of a biofilm mimicking approximal plaque. After 48 h, the specimens were removed and biofilms were stained using two fluorescent dyes which selectively stain vital bacteria green and dead bacteria red. Under the confocal laser scanning microscope optical sections of 1 µm throughout the biofilm were made. To assess the vitality values (proportion of vital bacteria) of the whole biofilm as well as the vitality distribution in the different plaque sections an image analysis program was used. Plaque from the different locations revealed mean vitality values between 64.4 and 75.7% in the upper jaw and between 64.3 and 76.8% in the lower jaw, which were not statistically different. However, a great variation of the vitality values for the different layers and among the 8 subjects was found. Nevertheless, the analysis of the data of each single volunteer revealed a very similar vitality pattern in all twelve locations.

Confusion over live/dead stainings for the detection of vital microorganisms in oral biofilms - which stain is suitable?

BackgroundThere is confusion over the definition of the term “viability state(s)” of microorganisms. “Viability staining” or “vital staining techniques” are used to distinguish live from dead bacteria. These stainings, first established on planctonic bacteria, may have serious shortcomings when applied to multispecies biofilms. Results of staining techniques should be compared with appropriate microbiological data.DiscussionMany terms describe “vitality states” of microorganisms, however, several of them are misleading. Authors define “viable” as “capable to grow”. Accordingly, staining methods are substitutes, since no staining can prove viability.The reliability of a commercial “viability” staining assay (Molecular Probes) is discussed based on the corresponding product information sheet: (I) Staining principle; (II) Concentrations of bacteria; (III) Calculation of live/dead proportions in vitro. Results of the “viability” kit are dependent on the stains’ concentration and on their relation to the number of bacteria in the test. Generally this staining system is not suitable for multispecies biofilms, thus incorrect statements have been published by users of this technique.To compare the results of the staining with bacterial parameters appropriate techniques should be selected. The assessment of Colony Forming Units is insufficient, rather the calculation of Plating Efficiency is necessary. Vital fluorescence staining with Fluorescein Diacetate and Ethidium Bromide seems to be the best proven and suitable method in biofilm research.Regarding the mutagenicity of staining components users should be aware that not only Ethidium Bromide might be harmful, but also a variety of other substances of which the toxicity and mutagenicity is not reported.Summary– The nomenclature regarding “viability” and “vitality” should be used carefully.– The manual of the commercial “viability” kit itself points out that the kit is not suitable for natural multispecies biofilm research, as supported by an array of literature.– Results obtained with various stains are influenced by the relationship between bacterial counts and the amount of stain used in the test. Corresponding vitality data are prone to artificial shifting.– As microbiological parameter the Plating Efficiency should be used for comparison.– Ethidium Bromide is mutagenic. Researchers should be aware that alternative staining compounds may also be or even are mutagenic.

The effect of different bleaching agents on the surface texture of restorative materials.

This blind in vitro study evaluated the effect of a home and an in-office bleaching agent on the surface texture of different tooth-colored restorative materials. Four composites (a hybrid, a flowable, a microhybrid and a nano-hybrid), an ormocer and a ceramic were used, and 2 bleaching agents were tested: 38% hydrogen peroxide and 15% carbamide peroxide. For 38% hydrogen peroxide, the surface morphology of the restorative materials was evaluated after the following time periods: before bleaching, after 15, 30 and 45 minutes of bleaching, 24 hours and 1 month after bleaching. For 15% carbamide peroxide, the time periods were: before bleaching, after 8 and 56 hours of bleaching and 24 hours and 1 month after bleaching. For the 4 composite materials and the ormocer, 2 samples groups were prepared; in 1 group, the specimens were polished and in the other, they stayed unpolished. For the ceramic group, polished samples were prepared. For every material, 3 samples per category and time period were prepared, respectively. Subsequently, the appropriate bleaching procedure was performed on samples of every group. Scanning electron micrographs were produced at 60x, 200x and 2000x magnifications of respective areas of the samples. The results showed that the effect of bleaching on the surface texture was material- and time-dependent. Within the limitations of this study, it was concluded that bleaching with 38% hydrogen peroxide and 15% carbamide peroxide did not cause major surface texture changes on the polished surfaces of the restorative materials.

Effects of commonly used food preservatives on biofilm formation of Streptococcus mutans in vitro

OBJECTIVE Sodium benzoate (SB), potassium sorbate (PS) and sodium nitrite (SN) are commonly used food preservatives. In this in vitro study, the effects of these substances on biofilm formation of Streptococcus mutans were analysed. METHODS In addition to the microtiter plate test (MPT), a biofilm reactor containing bovine enamel slabs (BES) was used to study the influence of food preservatives on biofilm formation in 5 independent periods of 4 days each. These included one period with chlorhexidine digluconate (CHX) as a positive control as well as a period with growth medium alone as a negative control. The vitality of the biofilm on BES was detected using live/dead staining and confocal laser scanning microscopy. Additionally, the number of colony forming units (CFU) was determined. RESULTS In MPT 0.12% SN significantly reduced the biofilm formation. PS at a concentration of 0.4% tended to inhibit biofilm formation, whereas the inhibition for 0.8% PS was significant. Less inhibition was caused by 0.8% SB. In the biofilm reactor 0.06% of SN, 0.1% of SB and 0.1% PS significantly reduced the covering grade as well as the CFU of the biofilm. Biofilm vitality was reduced significantly by CHX to a level of 32.5% compared to the control. Only SB reduced the vitality to a level of 19.1%. SN and PS showed no influence on biofilm vitality. CONCLUSION This study indicates the potential of food preservatives as inhibitory agents in S. mutans biofilm formation, which should be kept in mind when studying the effects of conserved food on dental plaque biofilm in situ.

Occurrence and Causing Stimuli of Postoperative Sensitivity in Composite Restorations

Despite improvements in composite treatments over the past decade, postoperative sensitivity still remains a problem. Therefore, this clinical study evaluated the appearance of postoperative sensitivity after composite treatments and the stimuli that may have caused it. A total of 600 teeth in 231 patients was included in this study. All treatments were performed by dental students working under close supervision following standard procedures and using the bonding system Optibond FL and the nanofilled composite Ceram X. At baseline (visit 1), the restorations were grouped according to the following criteria: use of anesthesia, use of a rubber dam, indication for the restoration treatment, cavity class and clinical dimension of the cavity. After approximately two weeks (at visit 2), all the restorations were assessed and failure was defined if one of the following criteria occurred: a negative reaction to the vitality test, postoperative pain from masticatory forces or reported postoperative sensitivity by the patient. The reported postoperative sensitivity was specified with a visual analogue scale into hot/cold-sensitivity, sweet/soursensitivity, sharp/dull-sensitivity, spontaneous sensitivity and blistering/stinging-sensitivity. Failure was observed in 6% of the restorations. The statistical analysis showed that the clinical cavity depth turned out to be the only factor to have a significant influence on the appearance of postoperative sensitivity: caries profunda showed a four times higher risk of failure, while cavities with pulp exposure had a 14 times higher failure risk compared to restorations that were localized in the dentin. With regard to the type of sensitivity, no patients reported sensitivity to sweet/sour; most of them described their sensitivity as sharp/dull.

Erratum to “Spatial distribution of vital and dead microorganisms in dental biofilms”

To examine the spatial structure of dental biofilms a vital fluorescence technique was combined with optical analysis of sections in a confocal laser scanning microscope (CLSM). Enamel slaps were worn in intraoral splints by three volunteers for five days to accumulate smooth-surface plaque. After vital staining with fluorescein diacetate and ethidium bromide the specimens were processed for CLSM examination. Optical sections 1 microm apart were analysed in the z-axis of these dental biofilms. One of the films was 15 microm high, sparse and showed low vitality, i.e. <16%, while the others were taller (25 and 31 microm) and more vital, i.e. up to 30 and 69%, respectively. In all instances the bacterial vitality increased from the enamel surface to the central part of the plaque and decreased again in the outer parts of the biofilm. The spatial arrangement of the microorganisms in the biofilm showed voids outlined by layers of vital bacteria, which themselves were packed in layers of dead material.