Green Fluorescent Nucleic Acid Research Articles

Comparison of techniques for counting prokaryotes in marine planktonic and biofilm samples

Though a large number of techniques are available for the study of aquatic bacteria, the aim of this study was to establish a technique for analysing free-living and biofilm prokaryotic cells through laboratory assays. In particular, we wished to analyse the efficiency of ultrasound to detach and disrupt biofilm, to obtain an efficient stain treatment for quantifying free-living and biofilm prokaryotes in flow cytometry (FC), and to compare epifluorescence microscopy (EFM), scanning electron microscopy (SEM) and FC for quantifying free-living and biofilm prokaryotes#. Marine-grade plywood substrates were immersed in natural marine water that was conditioned for 12 days. At 6 and 12 days, water aliquots and substrates were removed to estimate free-living and biofilm prokaryote density. Ultrasound efficiently removed marine biofilm from substrates (up to 94%) without cell damage. FC analysis (unstained) reliably quantified marine plankton and young or mature biofilm prokaryotes compared with other staining (acridine orange, 4′,6-diamidino-2-phenylindole, propidium iodide and green fluorescent nucleic acid), EFM or SEM techniques. FC and SEM achieved similar results, while a high variability was observed in the EFM technique. FC was faster and more precise than SEM because the count is not dependent on the observer.

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages.

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Rapid quantification of live/dead lactic acid bacteria in probiotic products using high-sensitivity flow cytometry

A laboratory-built high-sensitivity flow cytometer (HSFCM) was employed for the rapid and accurate detection of lactic acid bacteria (LAB) and their viability in probiotic products. LAB were stained with both the cell membrane-permeable SYTO 9 green-fluorescent nucleic acid stain and the red-fluorescent nucleic acid stain, propidium iodide, which penetrates only bacteria with compromised membranes. The side scatter and dual-color fluorescence signals of single bacteria were detected simultaneously by the HSFCM. Ultra-high temperature processing milk and skim milk spiked with Lactobacillus casei were used as the model systems for the optimization of sample pretreatment and staining. The viable LAB counts measured by the HSFCM were in good agreement with those of the plate count method, and the measured ratios between the live and dead LAB matched well with the theoretical ratios. The established method was successfully applied to the rapid quantification of live/dead LAB in yogurts and fermented milk beverages of different brands. Moreover, the concentration and viability status of LAB in ambient yogurt, a relatively new yet popular milk product in China, are also reported.

Macrophage phenotype modulation to control synovial inflammation

Macrophage phenotype modulation to control synovial inflammation

In Situ Chlorhexidine Substantivity on Saliva and Plaque‐Like Biofilm: Influence of Circadian Rhythm

The aim of the present study is to assess in situ substantivity of a single mouthrinse with 0.2% chlorhexidine (CHX) on saliva and on undisturbed de novo plaque-like biofilm (PL-biofilm), differentiating between two times of application: 1) CHX mouthrinse in the morning; and 2) CHX mouthrinse at night. The study participants were 10 healthy volunteers who wore an individualized splint with glass disks for 48 hours to boost the growth of PL-biofilm. Saliva samples were collected, and two disks were removed from each volunteer's splint at 8, 10, and 12 hours after performing a mouthrinse with 0.2% CHX at 7:00 am (M-0.2% CHX-diurnal) and 1:00 am (M-0.2% CHX-nocturnal). The saliva and plaque samples were analyzed by epifluorescence and confocal laser scanning microscopy, respectively, using a green fluorescent nucleic acid stain/propidium iodide staining. With M-0.2% CHX-diurnal, the frequency of vital bacteria in saliva was significantly higher than in the PL-biofilm at 8, 10, and 12 hours after mouthrinse. After M-0.2% CHX-nocturnal, the frequency of vital bacteria in saliva was significantly lower than in the PL-biofilm at 8 hours and higher than in the PL-biofilm at 12 hours after mouthrinse. These results support the more active physiologic dynamics of the salivary flora and the possible reservoir function associated with the structure of undisturbed de novo PL-biofilm.

Flexor Tendon Tissue Engineering: Acellularization of Human Flexor Tendons with Preservation of Biomechanical Properties and Biocompatibility

Acellular human tendons are a candidate scaffold for tissue engineering flexor tendons of the hand. This study compared acellularization methods and their compatibility with allogeneic human cells. Human flexor tendons were pretreated with 0.1% ethylenediaminetetracetic acid (EDTA) for 4 h followed by 24 h treatments of 1% Triton X-100, 1% tri(n-butyl)phosphate, or 0.1% or 1% sodium dodecyl sulfate (SDS) in 0.1% EDTA. Outcomes were assessed histologically by hematoxylin and eosin and SYTO green fluorescent nucleic acid stains and biochemically by a QIAGEN DNeasy kit, Sircol collagen assay, and 1,9 dimethylmethylene blue glycosaminoglycan assay. Mechanical data were collected using a Materials Testing System to pull to failure tendons acellularized with 0.1% SDS. Acellularized tendons were re-seeded in a suspension of human dermal fibroblasts. Attachment of viable cells to acellularized tendon was assessed biochemically by a cell viability assay and histologically by a live/dead stain. Data are reported as mean±standard deviation. Compared with the DNA content of fresh tendons (551±212 ng DNA/mg tendon), only SDS treatments significantly decreased DNA content (1% SDS [202.8±37.4 ng DNA/mg dry weight tendon]; 0.1% SDS [189±104 ng DNA/mg tendon]). These findings were confirmed by histology. There was no decrease in glycosaminoglycans or collagen following acellularization with SDS. There was no difference in the ultimate tensile stress (55.3±19.2 [fresh] vs. 51.5±6.9 [0.1% SDS] MPa). Re-seeded tendons demonstrated attachment of viable cells to the tendon surface using a viability assay and histology. Human flexor tendons were acellularized with 0.1% SDS in 0.1% EDTA for 24 h with preservation of mechanical properties. Preservation of collagen and glycoaminoglycans and re-seeding with human cells suggest that this scaffold is biocompatible. This will provide a promising scaffold for future human flexor tendon tissue engineering studies to further assess biocompatibility through cell proliferation and in vivo studies.

Membrane damage of bacteria by silanols treatment

Antimicrobial action of silanols, a new class of antimicrobials, was investigated by transmission electron microscopy and fluorescent dye studies. Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa and Gram-positive bacteria, Staphylococcus aureus and Enterococcus faecalis were treated by silanols at concentration of less than 0.2 wt% for one hour. Membrane damage of the bacteria by the silanol treatment was clearly observed by transmission electron microscopy. Separation of the cytoplasmic membrane from the outer membrane for E. coli and disorganized cytoplasmic membrane of the Gram-positive bacteria were observed when compared to the control. Fluorescent dyes, green-fluorescent nucleic acid stain (Syto 9) and the red-fluorescent nucleic acid stain (Propidium iodide), were used to monitor membrane damage of the bacteria by Confocal microscopy and Spectrophotometer. A reduction of the green fluorescent emission was detected for silanol treated bacteria indicating membrane damage of the bacteria and supporting the hypothesis that their viability loss may be due to their membrane damage analogus to alcohols.

Enumeration of soil bacteria with the green fluorescent nucleic acid dye Sytox green in the presence of soil particles

Total counts in soils are usually determined using fluorescent dyes, such as DAPI or Sybr green, due to fluorescence enhancement if they are bound to nucleic acids. Unfortunately, these commonly used dyes stain soil particles as well. Therefore, besides fluorescence enhancement, sufficient spectral differentiation is also required. We present a new procedure that overcomes the problems of visualising bacteria on surfaces in soil and avoids the separation of soil particles to a large extent. Spectral differentiation between bacteria and soil matrix is achieved by using Sytox green and a suboptimal excitation wavelength. Bacteria exhibit a bright green fluorescence, while soil particles fluoresce blue or red. Slight homogenisation and sedimentation of the sand and coarse silt that were too big for microscopic investigations were the only separation steps required. We compared the proposed Sytox green staining with Sybr green staining. The recovery of Sybr green-stained cells amounted to 38%, whereas in samples stained by Sytox green 81% of the spiked cells were counted. Sytox green can also be combined with fluorescence in situ hybridisation (FISH) using deep red dyes such as Cy5.

Enhanced detection of live bacteria using a dendrimer thin film in an optical biosensor.

Here we describe the detection of live Pseudomonas aeruginosa using a sensing film containing a fourth-generation hydroxy-terminated polyamidoamine (PAMAM) dendrimer (i.e., G4-OH) and SYTOX Green fluorescent nucleic acid stain. The films are configured on simple, disposable plastic coupons or optical fibers and are interrogated using a miniature fiber-optic spectrometer. SYTOX Green is generally considered a dead cell stain because it is not able to cross the membranes of live cells. In the presence of PAMAM-OH (G4-OH) in water, the bacterial cell becomes permeable to the SYTOX dye and the fluorescence is significantly enhanced. The fluorescence increases with the bacteria concentration, and the intensity at 5.4 x 10(7) cells mL(-1) is 350% higher than the liquid controls without PAMAM-OH. We also demonstrate that dendrimers stabilize the sensing film. After drying and desiccation, the SYTOX Green/PAMAM-OH films are still able to quantitatively detect P. aeruginosa in water. Incorporation of glucose into the SYTOX Green/ PAMAM-OH film may improve the homogeneity of the film and enhances the fluorescence signal an additional 11-25%.

Utility of green fluorescent nucleic acid dyes and aluminum oxide membrane filters for rapid epifluorescence enumeration of soil and sediment bacteria.

High background fluorescence and unspecific staining hampered the epifluorescence enumeration of bacteria in 45% of the tested soil and sediment samples with 4',6-diamidino-2-phenylindole (DAPI) and polycarbonate membrane filters. These problems of the determination of total cell counts can be circumvented by using green fluorescent high-affinity nucleic acid dyes and aluminum oxide membrane filters. Due to the bright staining of cells, we recommend SYBR Green II as dye.