Bacterial densities were determined according to the method of Yang et al.56 . Natural biofilms from each treatment (n = 24) were separately scraped off using sterile glass slides, suspended in AFSW, and fixed in formalin (final concentration = 5%). Suspensions containing bacteria and diatoms were vortexed for 60 s to ensure the homogeneous distribution of microorganisms before counting cell densities. Bacteria were stained with 0.1% acridine orange (Sigma Chemical Co., St Louis, Mo) solution. The suspended bacteria were filtered and collected on polycarbonate Nucleopore filters (pore size: 0.2 μm, Whatman 4.9 cm2, Whatman International Ltd, Maidstone, England). The bacterial densities of each stained sample (n = 3) were immediately determined with an Olympus BX51 epifluorescence microscope (Olympus, Tokyo, Japan) at 1000× magnification. The cell densities of suspended diatoms (n = 3) were evaluated using a hemocytometer under an Olympus BX51 epifluorescence microscope (Olympus, Tokyo, Japan) with 200× magnification. Ten random fields of view for each biofilm sample were counted, and the mean densities of bacteria and diatoms were calculated.
Bx51 epifluorescence microscope
Manufactured by Olympus
Sourced in Japan, Germany, United States
The BX51 is an epifluorescence microscope designed for fluorescence imaging applications. It features a high-intensity mercury vapor lamp as the illumination source and a specialized optical system to enable fluorescence observation and photomicrography.
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Lab products found in correlation
Pds 1000 he system, by Bio-Rad (5 mentions)
Dp71 digital camera, by Olympus (5 mentions)
Cellsens software, by Olympus (4 mentions)
Vectashield antifade solution, by Vector Laboratories (3 mentions)
Fitc pna, by Merck Group (3 mentions)
Bx51 microscope, by Olympus (2 mentions)
Dp72 camera, by Olympus (2 mentions)
Dp72 digital camera, by Olympus (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Calcofluor white m2r cw, by Thermo Fisher Scientific (2 mentions)
Dp 7 camera, by Olympus (2 mentions)
Slowfade gold antifade mountant with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (2 mentions)
Alkaline comet assays, by Cell Biolabs (2 mentions)
Celltiter 96 aqueous non radioactive cell proliferation assay kit, by Promega (2 mentions)
Methanol, by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Pectinase, by Merck Group (2 mentions)
Retiga exi fast 1394 ccd camera, by Olympus (2 mentions)
Anti digoxygenin rhodamine, by Roche (2 mentions)
118 protocols using bx51 epifluorescence microscope
1
Quantifying Bacterial and Diatom Densities
2
Fluorescent Nuclei Staining Protocol
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Cells were fixed for 15 min at room temperature with 4% formaldehyde (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in PBS buffer (137 mM NaCl, 8.10 mM NaHPO4, 2.68 mM KCl, 1.47 mM KH2PO4; pH 7.4). After three rinses with TBS buffer (20 mM Tris, 150 mM NaCl; pH 7.4) and centrifugation (3 min at 5000g), each sample was stained with DAPI (Sigma-Aldrich, St. Louis, MO, USA, 1 μg·mL–1 in TBS) for 30 min at room temperature. After three rinses with TBS and centrifugation (3 min at 5000g), the sample was mounted in 50% glycerol—TBS containing 0.1% p-phenylendiamine and observed under an Olympus BX51 epifluorescence microscope (Olympus Optical Co., Tokyo, Japan). At least 100 cells were observed in each of three independent experiments.
3
CARD-FISH Analysis of Amoebophrya Infection
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CARD-FISH samples were fixed with paraformaldehyde (1% final concentration) for 1 h (4 °C in the dark) before filtration on a 0.8 µm, polycarbonate filter with a vacuum pump (< 200 mm Hg). Filters were then dehydrated using successive 50%, 80%, and 100% ethanol solutions, dried and stored in the dark at −20 °C. FISH staining was then performed according to [8 (link)]. The prevalence was estimated from microscope observations with an Olympus BX-51 epifluorescence microscope (Olympus Optical) equipped with a mercury light source, a Wide Blue filter set (Chroma Technology, VT, USA) and fluorescence filter sets for PI (excitation: 536 nm; emission: 617 nm) and FITC (excitation: 495 nm; emission: 520 nm).
Prevalence was determined by averaging infection counts on a minimum of 80 cells per replicate. Prevalence was characterized in: non-infected host cells, early stage (one or more dinospores of Amoebophrya sp. in the cytoplasm), and advanced stages (intermediate and beehive stages) as described in [42 (link)]. The progeny count (i.e., the number of dinospores per infected host) was estimated by dividing the maximal concentration of dinospores by the concentration of infected hosts in advanced stages.
Prevalence was determined by averaging infection counts on a minimum of 80 cells per replicate. Prevalence was characterized in: non-infected host cells, early stage (one or more dinospores of Amoebophrya sp. in the cytoplasm), and advanced stages (intermediate and beehive stages) as described in [42 (link)]. The progeny count (i.e., the number of dinospores per infected host) was estimated by dividing the maximal concentration of dinospores by the concentration of infected hosts in advanced stages.
4
Quantitative Immunofluorescence Analysis of Complement Proteins
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Immunofluorescence was performed on OCT-embedded frozen sections after fixing with ice-cold Methanol for 10 minutes at -20°C. The sections then were incubated with 3% Goat serum in PBS-Tween (0.01%) for 1 hour at room temperature in a humidified chamber to block nonspecific antibody binding. For co-staining, the primary antibodies, mouse anti-Crry antibody (Hycult Biotech, Wayne, PA, USA) and rabbit polyclonal anti-C3 (Abcam, Cambridge, MA, USA) were applied at 1:200 dilution overnight, followed by goat anti-mouse TX-red and anti-rabbit IgG Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA) for 1 hour at room temperature in a humidified chamber. Mouse and rabbit IgG (ready to use) (Dako, Glostrup, Denmark) was used as a negative control. Slides were then counterstained with Hoechst 33342 (Molecular Probes). Anti-fade mounting medium (Dako) was applied and slides were viewed and captured with a constant exposure time and aperture using a single threshold value under Olympus BX51 epifluorescence microscope, and images were recorded by a DP70 Digital Camera (Olympus Optical Co. Ltd., Tokyo, Japan). Subsequently, images were analyzed using ImageJ software and the numerical output of average intensity per nuclei was calculated.
5
Apoptotic Cell Quantification using Fluorescence
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Apoptotic cells qualification were carried out using Apoptosis and Necrotic Qualification Kit (Biotium Inc, Hayward, USA). Both treated and untreated cells were washed with PBS. Then, binding buffer (10x), Ethidium homodimer III, Annexin V-FITC (250 μL in TE buffer) and Hoechst 33342 (500 μg/ml in PBS) were added to the cells [27 (link)]. Olympus BX 51epifluorescence microscope, (Olympus, Wetzlar, Germany) were used to observe the samples using image analyser software [27 (link)]. Bright field images of tryphan blue stained cells were captured using Olympus BX 51microscope and the same parasites stained with FITC were viewed when the filter was changed (BP 450–480). Apoptotic cells were calculated with regard to the percentages of cells which are apoptotic in 100 cells [27 (link)].
6
Micronucleus and Nuclear Bud Assay
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All slides were coded before microscopic analysis. Samples were evaluated by the same researcher, and they were scored manually using an OLYMPUS BX51 epifluorescence microscope (Olympus, Tokyo, Japan) and an oil-immersion objective (100×). The number of spontaneous micronucleated erythrocytes (MNEs) and the nuclear bud formation in erythrocytes (NBEs) were determined in a total of 10,000 erythrocytes (TEs). The frequencies of micronucleated young erythrocytes (MNYEs) and NB young erythrocytes (NBYEs) were also determined in 1000 young erythrocytes (YEs); the proportion of YEs in 1000 TEs was counted to determine a decrease in the cellular division as a cytotoxic effect. Acridine orange stained the YEs red or orange, while normochromatic (mature) erythrocytes were dark green.
MNEs were counted only when the micronucleus was clearly separated from the main nucleus and had a round shape (Figure 1 a). NBEs were counted when an elongation originated from the nucleus, partially overlapping the nucleus, or it was in the form of a drop with an obvious (or presumed) strand connecting it to the nucleus (Figure 1 b).
MNEs were counted only when the micronucleus was clearly separated from the main nucleus and had a round shape (
7
Measuring Sperm Total Length in Diverse Species
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Samples (at least five individuals of each species) were anesthetized with alcohol at a concentration of 75%, and dissected with a fine scalpel blade under a binocular microscope (Olympus SZX16, Olympus Corporation, Tokyo, Japan) to obtain the seminal vesicles from which spermatozoa were recovered. For measuring the sperm total length, sperm samples were placed in 1% bisbenzimidazole Hoechst 33258, a cell-permeable adenine–cytosine binding epifluorescent dye used to stain DNA (Sakaluk and O’Day 1984 (link)), for 1 min, then rinsed in three changes of 0.1 M phosphate buffered saline (PBS , pH 7.2). Spermatozoa were evenly spread on a microscope slide and covered with a coverslip. Slides were examined with an Olympus BX-51 epifluorescence microscope (Olympus Corporation, Tokyo, Japan) at a wavelength of 343 nm. Digital images of 50–100 spermatozoa were randomly captured from each species under the same microscope with an Olympus DP72 camera (Olympus Corporation, Tokyo, Japan). Sperm lengths were measured using the Olympus DP2-BSW software version 2.1.
8
Microscopic Observation of Wheat Fungal Infection
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For penetration branches observation, glumes and lemmas were collected from inoculated wheat spikelets 24 h post-inoculation (hpi), and fixed with 4% glutaraldehyde in 0.1 M phosphate buffer (pH 6.8) overnight at 4°C [69 (link)]. After dehydration in a series of 30, 50, 70, 80, 90, and 100% acetone, the samples were coated with gold-palladium and examined with a JEOL 6360 scanning electron microscope (SEM) as described [69 (link)]. For assaying infectious hyphae, three-day-old seedlings of wheat cultivar Norm were inoculated as described [56 (link)]. In brief, the top 2 to 3 mm of the coleoptiles were removed with a razor blade and the wounded seedlings were wrapped in 4 mm X 1 cm cotton strips soaked with F. graminearum conidia (105 spores/ml). After inoculation, the seedlings were grown at 25°C in a growth chamber with 95% humidity and examined at 48 or 72 hpi with a BX51 Olympus epifluorescence microscope. The experiments were repeated at least 3 times. In each experiment, we examined at least ten samples for each strain.
9
Construction and Visualization of GFP Fusion Proteins
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To generate the GFP fusion constructs, genomic fragments containing the promoter and coding region of the target genes were amplified and cloned into pFL2 vector by the yeast gap repair approach [67 (link)]. The resulting in-frame GFP fusion constructs of CDC2A, CDC2B, and FgCAK1 were confirmed by DNA sequencing and transformed into protoplasts of the corresponding mutants. The resulting transformants harboring the fusion construct were identified by PCR and further confirmed by the presence of GFP signals. GFP signals in conidia, germ tubes, ascospores and infectious hyphae were observed with a BX51 Olympus epifluorescence microscope or an Olympus FV1000 confocal microscope. Nuclei were stained with Hoechst 33258 (Beyotime, China).
10
Propolis Effect on Candida Filamentation
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To evaluate the effect of propolis against Candida species filamentation four C. albicans, four C. parapsilosis and four C. tropicalis clinical isolates, and their respective references strains were tested. The clinical isolates were chosen randomly. Candida cells were grown overnight in YPD (1% yeast extract; 2% peptone; 2% dextrose) medium. And then, 1 × 10 6 yeasts/ml were incubated in RPMI 1640 medium with 10% fetal bovine serum (FBS), in the presence or absence of PE (450 μg/ml of TPC, selected in order to use a concordant concentration to all species in accordance with its MICs values), at 37°C for 4 h. Blastospore and filamentous forms were counted by observation under a phase contrast microscope, according to the criteria described by Toenjes et al. (2005) [39] . More than 100 cells were counted, in duplicate, for each strain. Additionally, images of cell morphologies were obtained, after staining the microorganisms with calcofluor white (Sigma-Aldrich, St Louis, Missouri, EUA). The cells were visualized with BX51 Olympus epifluorescence microscope coupled with a DP72 digital camera (Olympus Portugal SA, Porto, Portugal). All images were acquired using the Olympus Cell-B software.
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