Bacteria were observed by phase-contrast or epifluorescence using a microscope with a HBO 100 mercury lamp and the A filter (excitation filter BP 340–380, dichromatic mirror 400, and suppression filter LP 425) from Leica. The images were captured with a Leica DC 300F camera (Leica Microsystems, Heerbrugg, Switzerland) using a 100 × oil immersion N plan objective and processed using Leica IM 50-Image manager software.
Hbo 100 mercury lamp
Manufactured by Leica
Sourced in Germany
The HBO 100 mercury lamp is a high-intensity light source used in various microscopy and imaging applications. It provides a broad spectrum of illumination, with the majority of the light being emitted in the ultraviolet and visible wavelength ranges. The lamp operates using a mercury vapor discharge, allowing it to generate a high-intensity output suitable for various laboratory and research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Im50 image manager software, by Leica (2 mentions)
Dc300f, by Leica (1 mentions)
Gfp filter, by Leica (1 mentions)
Axio imager, by Zeiss (1 mentions)
Axiocam 503 mono microscope camera, by Zeiss (1 mentions)
Wga fitc, by Merck Group (1 mentions)
Sp8 lsm confocal microscope, by Leica (1 mentions)
Calcofluor white cfw, by Merck Group (1 mentions)
I3 filter set, by Leica (1 mentions)
Dc300f camera, by Leica (1 mentions)
Dlmb epifluorescence microscope, by Leica (1 mentions)
Gfp filter set, by Leica (1 mentions)
Victor3, by PerkinElmer (1 mentions)
H2dcfda, by Merck Group (1 mentions)
6 protocols using hbo 100 mercury lamp
1
Microscopic Observation of Bacteria
2
Plasma Membrane Permeability Assay
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Cells with a plasma-membrane permeabilized were discriminated using the membrane-impermeant SYTOX Green (SG) probe, as previously described (Machado and Soares, 2012a) . SG enters only in cells with compromised membrane and binds to DNA, exhibiting a green fluorescence (Haugland, 2005) .
Cells (1 £ 10 6 mL -1 ) were incubated with 0.5 μmol L -1 SG (Molecular Probes, Invitrogen), in the dark, at room temperature, for 20 min. Positive (cells with permeabilized membrane by heat treatment, at 65 • C, for 1 h) and negative control (cells not exposed to TCS) were used. Cells were observed with an epifluorescence microscope, equipped with an HBO-100 mercury lamp and a GFP filter from Leica. The experiment was repeated, independently, three times. In each experiment and for each TCS concentration, at least 400 cells were examined; therefore, for each TCS concentration, a total of ≥ 1200 cells were analyzed in randomly selected microscope fields.
Cells (1 £ 10 6 mL -1 ) were incubated with 0.5 μmol L -1 SG (Molecular Probes, Invitrogen), in the dark, at room temperature, for 20 min. Positive (cells with permeabilized membrane by heat treatment, at 65 • C, for 1 h) and negative control (cells not exposed to TCS) were used. Cells were observed with an epifluorescence microscope, equipped with an HBO-100 mercury lamp and a GFP filter from Leica. The experiment was repeated, independently, three times. In each experiment and for each TCS concentration, at least 400 cells were examined; therefore, for each TCS concentration, a total of ≥ 1200 cells were analyzed in randomly selected microscope fields.
3
Visualization of Candida albicans Cell Wall Components
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Total chitin visualization was performed by staining C. albicans cells with Calcofluor white (CFW) (Sigma-Aldrich), as described before [30 (link)], with modifications. Visualization of unmasked chitin was performed by staining C. albicans cells with wheat germ agglutinin conjugated with FITC (WGA-FITC) (Sigma-Aldrich), by modifying the protocol of Malavia et al. [34 (link)]. Unmasked β-glucans were stained according to the protocol of Wagener et al. [35 (link)]. In all cases, C. albicans cells were pelleted, washed twice with 0.9% saline (4 k × g, 5 min) and resuspended (to A600 = 1) in 0.9% saline with either 12.5 µM CFW, 50 µg/mL WGA-FITC or 5 µg/mL Fc–hDectin-1 (Invivogen). After 1h incubation cells were pelleted, washed twice with 0.9% saline (4 k × g, 5 min) and, in case of CFW and WGA-FITC staining, concentrated. Fc-hDectin-1 treated cells were incubated with 1:250 Alexa fluor 448-conjugated anti-human IgG Fc antibodies (Thermo fisher, Waltham, USA) for 1 h on ice. Afterwards, cells were pelleted, washed twice with 0.9% saline (4 k × g, 5 min) and concentrated. Preparations were observed under Zeiss Axio Imager (Oberkochen, Germany) A2 microscope equipped with a Zeiss Axiocam 503 mono microscope camera and a Zeiss HBO100 mercury lamp or in case of CFW-stained cells also under a Leica SP8 LSM confocal microscope.
4
Monitoring Algal Cell Cycle Distribution
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The distribution of algal cells among the reproductive cycle was monitored as previously described by Machado and Soares (2014) (link). Cells suspended in OECD medium at 3 × 10 6 cells mL -1 were treated with 1pentanol (70 %, v/v) for 1 h, to permeabilize cell membrane. Then, cells were incubated with 0.5 μmol L -1 SYTOX Green for 40 min, in the dark, at 25 °C and observed using an epifluorescence microscope, equipped with an HBO-100 mercury lamp and filter set GFP from Leica. Images were acquired and processed as described above. For each concentration, at least 100 cells were analysed in randomly selected microscope fields. This experiment was independently repeated four times; a total of ≥400 cells were analysed in control or each MET concentration, at each time point.
5
Epifluorescence Microscopy Protocol
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Samples were observed under an epifluorescence microscope equipped with a HBO 100 mercury lamp and the I3 filter set from Leica. Images were captured by Leica DC 300F camera (Leica Microsystems, Heerbrugg, Switzerland) and processed using Leica IM 50-image software.
6
ROS Detection in Yeast Cells
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ROS production was monitored with 2 0 ,7 0 -dichlorodihydrofluorescein diacetate (H 2 DCFDA; Sigma). Before metal treatment, cells were suspended at 1 9 10 7 cells/ml in MES buffer (10 mmol/l or 100 mmol/l in the case of the assay with AA, pH 6.8) and incubated at 25 °C for 20 min with 20 lmol/l H 2 DCFDA (final concentration). Subsequently, yeast cells were exposed to metal mixture (in the absence or presence of 10 mmol/l AA) or individual metals and placed in quadruplicate in a 96-well flat microplate, 200 ll per well. The fluorescence intensity was measured (relative fluorescence units, RFU) in a PerkinElmer (Vic-tor3) microplate reader at a fluorescence excitation of 485/14 nm and an emission of 535/25 nm. Fluorescence was corrected (subtracting cell, buffer and dye autofluorescence) and normalized considering the cell concentration. Cells were also observed using a Leica DLMB epifluorescence microscope equipped with an HBO 100 mercury lamp and a GFP filter set from Leica. The images were acquired with a Leica DC 300 F camera using a 1009 oil immersion N plan objective and processed using Leica IM 50-Image manager software.
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