A combined immunofluorescence and fluorescent in situ hybridization assay for single cell analyses of dental plaque microorganisms

Gmür, Rudolf; Lüthi-Schaller, Helga

doi:10.1016/j.mimet.2006.12.012

Cited by 27 publications

(16 citation statements)

References 17 publications

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“…The use of PCR‐based assays for the detection of specific periodontopathic bacteria is becoming state of the art but might lead to an overestimation of the actual bacterial load. Previously proposed solutions using ethidium monoazide or PMA intercalation, combined immunofluorescence and fluorescence in situ hybridization or RNA‐oligonucleotide quantification have proven their validity. Yet, most of those methods require samples to be immediately processed, special expertise and equipment.…”

Section: Resultsmentioning

confidence: 99%

“…Moreover, as we are dealing with a pool of many different bacterial species, a single preservation method might not be suitable and/or could be selective for certain species. Other possible solutions such as combined immunofluorescence and fluorescence in situ hybridization or RNA‐oligonucleotide quantification appear valuable, but technically demanding and therefore more suitable for research purposes than clinical application. Here we propose a simplified RNA method to provide basic insight into the viable microbial load within a patient's plaque sample.…”

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confidence: 99%

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Assessment of viable periodontal pathogens by reverse transcription quantitative polymerase chain reaction

Polonyi¹,

Prenninger²,

Arweiler

et al. 2013

J of Periodontal Research

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Assessment of viable periodontal pathogens by reverse transcription quantitative polymerase chain reaction

Polonyi¹,

Prenninger²,

Arweiler

et al. 2013

J of Periodontal Research

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“…The probe was used at a final concentration of 5 ng ؒ ml -1 in the presence of 30% formamide in the hybridization buffer. FISH was performed in 50-ml plastic centrifuge tubes at 46 ° C as described [Thurnheer et al, 2001] except for the following modifications [Gmur and Luthi-Schaller, 2007]: (1) the slides were not dehydrated in ethanol prior to hybridization; (2) sections were covered for 60 min at 37 ° C with protectRNA RNase inhibitor (Sigma-Aldrich, St. Louis, Mo., USA, diluted 1: 500 in 0.9% NaCl) prior to hybridization.…”

Section: Fluorescent In Situ Hybridizationmentioning

confidence: 99%

Dental Caries in Rats Associated with <i>Candida albicans</i>

et al. 2011

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In addition to occasional opportunistic colonization of the oral mucosa, Candida albicans is frequently found in carious dentin. The yeast’s potential to induce dental caries as a consequence of its pronounced ability to produce and tolerate acids was investigated. Eighty caries-active Osborne-Mendel rats were raised on an ampicillin-supplemented diet and exposed to C. albicans and/or Streptococcus mutans, except for controls. Throughout the 28-day test period, the animals were offered the modified cariogenic diet 2000a, containing 40% various sugars. Subsequently, maxillary molars were scored for plaque extent. After dissection, the mandibular molars were evaluated for smooth surface and fissure caries. Test animals exposed to C. albicans displayed considerably more advanced fissure lesions (p < 0.001) than non-exposed controls. While S. mutans yielded similar results, a combined association of C. albicans and S. mutans had no effect on occlusal caries incidence. Substituting dietary sucrose by glucose did not modify caries induction by C. albicans. However, animals fed a diet containing 20% of both sugars showed no differences to non-infected controls. Smooth surface caries was not generated by the yeast. This study provides experimental evidence that C. albicans is capable of causing occlusal caries in rats at a high rate.

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“…Fluorescence intensity depends on cellular ribosome content, in situ probe accessibility to the probe target region, and rRNA stability [25]. Several procedures to maximize the performance of FISH probes have been described [15,16,26,27]. They alter the 3-dimensional structure of the target region by using helper probes, optimize probe length and hybridization conditions, improve binding affinity by modifying the probes' backbone with LNA substitutions, or inhibit enzymatic rRNA degradation.…”

Section: Resultsmentioning

confidence: 99%

“…To improve cell wall permeability each well selected for FISH of lactobacilli was treated individually at room temperature first for 5 min with 9 μl of lysozyme (1 mg ml -1 ; Sigma-Aldrich L-7651) and achromopeptidase (1 mg ml -1 ; Sigma-Aldrich A-7550) in Tris-HCl (pH 7.5) with 5 mM EDTA, and then for 30 min with 9 μ l of lipase (Sigma-Aldrich L-1754; at 25 mg ml -1 in water the lipase suspension was centrifuged for 5 min at 16'000 × g after which the supernatant was used). Thereafter, to limit unspecific FISH probe binding all wells were covered for 30 min at 37 °C with 9 μ l of PBS containing Denhardt's solution (Fluka 30915; diluted 1:50) in the presence of protectRNA RNase inhibitor (Sigma-Aldrich R-7397; diluted 1:500) [15,16,26,27]. At the end of the respective incubation periods the solutions were carefully aspirated and the slides briefly washed in wash-buffer (0.9% NaCl, 0.05% Tween 20, 0.01% NaN 3 ), dipped in water, and air-dried.…”

Section: Methodsmentioning

confidence: 99%

Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms

et al. 2011

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BackgroundThe purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella.ResultsAs lactobacilli are known for notorious resistance to probe penetration, probe-specific assay protocols were experimentally developed to provide maximum cell wall permeability, probe accessibility, hybridization stringency, and fluorescence intensity. The new assays were then applied in a pilot study to three biofilm samples harvested from variably demineralized bovine enamel discs that had been carried in situ for 10 days by different volunteers. Best probe penetration and fluorescent labeling of reference strains were obtained after combined lysozyme and achromopeptidase treatment followed by exposure to lipase. Hybridization stringency had to be established strictly for each probe. Thereafter all probes showed the expected specificity with reference strains and labeled the anticipated morphotypes in dental plaques. Applied to in situ grown biofilms the set of probes detected only Lactobacillus fermentum and bacteria of the Lactobacillus casei group. The most cariogenic biofilm contained two orders of magnitude higher L. fermentum cell numbers than the other biofilms. Abiotrophia/Granulicatella and streptococci from the mitis group were found in all samples at high levels, whereas Streptococcus mutans was detected in only one sample in very low numbers.ConclusionsApplication of these new group- and species-specific FISH probes to oral biofilm-forming lactic acid bacteria will allow a clearer understanding of the supragingival biome, its spatial architecture and of structure-function relationships implicated during plaque homeostasis and caries development. The probes should prove of value far beyond the field of oral microbiology, as many of them detect non-oral species and phylogenetic groups of importance in a variety of medical conditions and the food industry.

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A combined immunofluorescence and fluorescent in situ hybridization assay for single cell analyses of dental plaque microorganisms

Cited by 27 publications

References 17 publications

Assessment of viable periodontal pathogens by reverse transcription quantitative polymerase chain reaction

Assessment of viable periodontal pathogens by reverse transcription quantitative polymerase chain reaction

Dental Caries in Rats Associated with <i>Candida albicans</i>

Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms

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