L. Netuschil

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.

A PILOT STUDY OF CONFOCAL LASER SCANNING MICROSCOPY FOR THE ASSESSMENT OF UNDISTURBED DENTAL PLAQUE VITALITY AND TOPOGRAPHY

Confocal microscopy and vital fluorescence techniques were combined for the first time to investigate ex vivo human dental plaque. The vital fluorescence technique used discriminates vital from dead cells, while confocal laser scanning microscopy allows the optical sectioning of undisturbed biofilms leaving the samples intact during analysis. The concomitant use of both methods made an examination of the three-dimensional architecture of dental plaque possible. The topography of plaque biofilms that were allowed to accumulate in situ on glass and enamel was recorded. The distribution of plaque microflora vitality as well as its accumulation varied according to plaque age. A plaque thickness of up to 8, 35 and 45 microm was estimated ex vivo on enamel after 1, 2 and 3 days, respectively. Young and sparse plaque biofilms consisted mainly of dead material. Vital bacteria were observed on top of this dead layers.

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.

Clinical and antibacterial effect of tea tree oil--a pilot study.

Abstract The aim of this clinical pilot study was to compare the effect of tea tree oil with the effect of water and chlorhexidine on supragingival plaque formation and vitality. Eight subjects were asked to refrain from any kind of mechanical oral hygiene for 4 days after professional tooth cleaning (day 0), and to rinse with water instead for 1 week, with chlorhexidine in a second and tea tree oil in a third test week. The plaque index (PI), which was evaluated daily (days 1–4), served as a clinical control parameter. On the last day of the study (day 4), the plaque covering the front teeth was stained, photographed, and therefrom the plaque area (PA; %) was estimated using a digital measuring system. Each day of the study (days 1–4), the sampled plaque was examined using a vital fluorescence technique. Tea tree oil reduced neither the clinical parameters (PI and PA) nor the vitality of the plaque flora significantly. Within the limitations of the study design, it was determined that a solution with tea tree oil – utilized as ordinary mouthwash – has no positive effect on the quantity or quality of supragingival plaque.

An approach to differentiate between antibacterial and antiadhesive effects of mouthrinses in vivo

An experimental set-up allowing differentiation in vivo between antibacterial and antiadhesive properties of mouthrinses is described. The percentage of vital bacteria (= microbial vitality) and the bacterial counts were microscopically evaluated in saliva and in supragingival dental plaque both collected simultaneously at various times during de novo plaque formation. In a cross-over design, 12 healthy participants refrained from all oral hygiene for four separate periods of 2 x 4 h and 2 x 72 h after having rinsed with either an amine fluoride/stannous fluoride solution (Meridol) or 0.9% NaCl (placebo). Stimulated whole saliva was collected before and after the rinse. Together with whole-saliva samples, representative 4, 24 and 72-h-old plaque samples were separately taken from defined vestibular tooth surfaces that had been either exposed to the mouthrinse (unprotected sites) or temporarily covered with inert plastic films (protected sites) during rinsing. The pooled plaque and saliva were stained with fluorescent dyes to differentiate vital from dead micro-organisms which permitted the estimation of the percentages of vital bacteria. The total bacterial counts were quantified under the darkfield microscope. The Wilcoxon test was used for selected pairwise comparisons (alpha = 0.05). The percentage of vital bacteria in saliva fell significantly from 80-95% to about 50-60% as a result of the antibacterial activity of the test solution. These baseline values and those found in the presence of 4 and 24-h-old plaque were frequently lower than those recorded after the placebo rinse. In comparison to the placebo, microbial vitality was significantly reduced in early supragingival plaque formed on unprotected sites after applying the test solution. The similar total bacterial counts in 4-h-old plaque recorded after the use of the test solution on the unprotected and the protected areas did not point to an antiadhesive effect of the agent. It is concluded that this new experimental set-up allows decoding of the mode of action of a mouthrinse.

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.