Evaluation of fluorescent stains for visualizing extracellular DNA in biofilms

Okshevsky, Mira; Meyer, Rikke Louise

doi:10.1016/j.mimet.2014.07.010

Cited by 83 publications

(75 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…All bacterial treatments and controls were plated on 12-well plates (Thermo Scientific, Waltham, MA, USA) in triplicate; bacteria were also plated in duplicate on 35 mm glass bottom dishes (No. 1.5, uncoated; MatTek Corporation, Ashland, MA, USA) for extracellular DNA detection using the TOTO-1 Iodide 514/533 stain (Molecular Probes, Invitrogen) and counterstained for live cells using SYTO60 red (Molecular Probes, Invitrogen) [49]. Additionally, purified salmon sperm DNA (Invitrogen) was added to separate cultures at a final volume of 300 ng mL −1 .…”

Section: Methodsmentioning

confidence: 99%

Isolation and Characterization of a Shewanella Phage–Host System from the Gut of the Tunicate, Ciona intestinalis

Leigh

Karrer

Cannon

et al. 2017

Viruses

View full text Add to dashboard Cite

Outnumbering all other biological entities on earth, bacteriophages (phages) play critical roles in structuring microbial communities through bacterial infection and subsequent lysis, as well as through horizontal gene transfer. While numerous studies have examined the effects of phages on free-living bacterial cells, much less is known regarding the role of phage infection in host-associated biofilms, which help to stabilize adherent microbial communities. Here we report the cultivation and characterization of a novel strain of Shewanella fidelis from the gut of the marine tunicate Ciona intestinalis, inducible prophages from the S. fidelis genome, and a strain-specific lytic phage recovered from surrounding seawater. In vitro biofilm assays demonstrated that lytic phage infection affects biofilm formation in a process likely influenced by the accumulation and integration of the extracellular DNA released during cell lysis, similar to the mechanism that has been previously shown for prophage induction.

show abstract

Section: Methodsmentioning

confidence: 99%

Isolation and Characterization of a Shewanella Phage–Host System from the Gut of the Tunicate, Ciona intestinalis

Leigh

Karrer

Cannon

et al. 2017

Viruses

View full text Add to dashboard Cite

show abstract

“…FLBA and fluorescencelabeled probe analyses have to be coupled with CLSM analyses and specific cell fluorescent probes to provide 3-D images of the distribution of the EPS according to cell localization. Other matrix components can also be visualized by CLSM using specific stains, such as fluorescein isothiocyanate (FITC) or SYPRO Ruby staining for proteins (Daniels et al, 2013;Hochbaum et al, 2011), Thioflavin T or antibody-labeling of amyloids (Larsen et al, 2008) or cell-impermeant nucleic acid stains for eDNA (Okshevsky & Meyer, 2014;Wang et al, 2015;Webb et al, 2003). The compound 7-Hydroxy-9H-(1,3-Dichloro-9,9-Dimethylacridin-2-one (DDAO) has been the compound of choice for eDNA staining in many publications, but a recent report systematically compared different eDNA staining techniques and concluded that the dye TOTO-1 provides the most reproducible and sensitive detection of eDNA (Okshevsky & Meyer, 2014).…”

Section: Measurement Of Eps Componentsmentioning

confidence: 99%

“…Other matrix components can also be visualized by CLSM using specific stains, such as fluorescein isothiocyanate (FITC) or SYPRO Ruby staining for proteins (Daniels et al, 2013;Hochbaum et al, 2011), Thioflavin T or antibody-labeling of amyloids (Larsen et al, 2008) or cell-impermeant nucleic acid stains for eDNA (Okshevsky & Meyer, 2014;Wang et al, 2015;Webb et al, 2003). The compound 7-Hydroxy-9H-(1,3-Dichloro-9,9-Dimethylacridin-2-one (DDAO) has been the compound of choice for eDNA staining in many publications, but a recent report systematically compared different eDNA staining techniques and concluded that the dye TOTO-1 provides the most reproducible and sensitive detection of eDNA (Okshevsky & Meyer, 2014). Alternatively, some specific fibrous strands of exopolysaccharides in biofilms could be detected using specific antibodies (Choi et al, 2009;Cramton et al, 1999;Darby et al, 2002;Gerke et al, 1998;Jarrett et al, 2004).…”

Section: Measurement Of Eps Componentsmentioning

confidence: 99%

Critical review on biofilm methods

Azeredo

Azevedo

Briandet

et al. 2016

Critical Reviews in Microbiology

Self Cite

784

679

View full text Add to dashboard Cite

Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms. ARTICLE HISTORY

show abstract

“…TOTO ® -1 is a dimeric derivative of the cyanine dye thiazole orange that fluoresces upon intercalation with DNA [Rye et al, 1993]. Due to its size and charge, TOTO ® -1 is membrane impermeant, and it was evaluated to be an optimal stain for eDNA visualization by Okshevsky and Meyer [2014]. Bacterial cells were counterstained with SYTO ® 60 (20 μL, 10 μ M , 15-min incubation time; Thermo Fisher Scientific).…”

Section: Visualization Of Ednamentioning

confidence: 99%

“…Images were 1,024 × 1,024 pixels in size (101.52 μm 2 ) and acquired with a pixel dwell time of 1.58 μs, line average 2, zoom 1, with the pinhole set to 1 Airy unit. For a detailed description of the method employed for eDNA staining, see Okshevsky and Meyer [2014]. 438 Statistical Analysis A sample size of 8 subjects was calculated to detect differences of 25% in biovolume upon treatment, with a standard deviation of 25%, 80% power, and a type I error rate of 5%.…”

Section: Visualization Of Ednamentioning

confidence: 99%

Extracellular DNA Contributes to Dental Biofilm Stability

Schlafer¹,

Dige²,

Regina

2017

Caries Res

View full text Add to dashboard Cite

Extracellular DNA (eDNA) is a major matrix component of many bacterial biofilms. While the presence of eDNA and its role in biofilm stability have been demonstrated for several laboratory biofilms of oral bacteria, there is no data available on the presence and function of eDNA in in vivo grown dental biofilms. This study aimed to determine whether eDNA was part of the matrix in biofilms grown in situ in the absence of sucrose and whether treatment with DNase dispersed biofilms grown for 2.5, 5, 7.5, 16.5, or 24 h. Three hundred biofilms from 10 study participants were collected and treated with either DNase or heat-inactivated DNase for 1 h. The bacterial biovolume was determined with digital image analysis. Staining with TOTO®-1 allowed visualization of eDNA both on bacterial cell surfaces and, with a cloud-like appearance, in the intercellular space. DNase treatment strongly reduced the amount of biofilm in very early stages of growth (up to 7.5 h), but the treatment effect decreased with increasing biofilm age. This study proves the involvement of eDNA in dental biofilm formation and its importance for biofilm stability in the earliest stages. Further research is required to uncover the interplay of eDNA and other matrix components and to explore the therapeutic potential of DNase treatment for biofilm control.

show abstract

Evaluation of fluorescent stains for visualizing extracellular DNA in biofilms

Cited by 83 publications

References 12 publications

Isolation and Characterization of a Shewanella Phage–Host System from the Gut of the Tunicate, Ciona intestinalis

Isolation and Characterization of a Shewanella Phage–Host System from the Gut of the Tunicate, Ciona intestinalis

Critical review on biofilm methods

Extracellular DNA Contributes to Dental Biofilm Stability

Contact Info

Product

Resources

About