To detect the clonogenicity of BM-MSCs, a colony-forming unit–fibroblastic (CFU-F) test was applied. BM-MSCs were plated in a 6-well plate (triplicate) at a seeding density of 250 cells per well. Following the 14 days of culturing in GM at standard conditions, cells were washed two times with PBS and fixed using ice-cold methanol for 5 min at room temperature. After fixation, cells were stained with 0.3% crystal violet (Carlo Erba reagents S.A.S., Emmendingen, Germany) for 15 min, when the cells were washed using distilled water. The number of colonies was determined by a light microscope (Olympus, Tokyo, Japan). Only colonies that were larger than 2 mm in diameter and consisted of more than 50 cells were counted. The ratio of the number of colonies to the number of cells plated was denoted as colony- forming efficiency.
Crystal violet
Manufactured by Carlo Erba
Sourced in Italy, France
Crystal violet is a synthetic dye commonly used in various laboratory applications. It is a dark purple, crystalline powder that is soluble in water and alcohol. Crystal violet is a staining agent used in microscopy, histology, and microbiology to visualize and identify cellular structures and microorganisms.
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Lab products found in correlation
Crystal violet, by Merck Group (2 mentions)
Multiskan fc, by Thermo Fisher Scientific (2 mentions)
Light microscopy, by Olympus (1 mentions)
Formaldehyde, by Carlo Erba (1 mentions)
Glacial acetic acid, by Carlo Erba (1 mentions)
Paraformaldehyde (pfa), by Carlo Erba (1 mentions)
96 well plate, by Corning (1 mentions)
Model 550, by Bio-Rad (1 mentions)
9 protocols using crystal violet
1
Clonogenicity Evaluation of BM-MSCs
2
Clonogenic Potential of BM-MSCs
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To confirm the clonogenic potential of isolated BM-MSCs, their ability to form CFU–F colonies was estimated as described in a previous report [26 (link)]. Briefly, cells were plated in triplicate in 24-well plates at a seeding density of 250 cells/well. After 14 days of standard cultivation, cells were washed twice with PBS and fixed using ice-cold methanol. After fixation, cells were stained with 0.3% crystal violet (Carlo Erba reagents S.A.S., Val de Reuil, France) for 15 min, and then washed using distilled water. CFU–Fs that consisted of more than 50 cells and had a diameter lager than 2 mm were observed using light microscopy (Olympus, Tokyo, Japan).
3
Quantifying Microbial Adhesion on Electrospun Structures
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The evaluation of microbial adhesion to the electrospun structures with and without mucins was assessed using a crystal violet assay at different time points (24 h, 48 h, 72 h, and 7 days) [30 (link)]. The culture medium was first removed, and then non-adherent planktonic microorganisms were eliminated by washing the wells three times with 1 mL of PBS. Microbial biofilms on the electrospun structures were stained using 2 mL of 0.1% w/v crystal violet (Carlo Erba, Milan, Italy) for 30 min at RT [30 (link)]. Wells were washed three times with 1 mL of deionized water and covered with 2 mL of absolute ethanol for 15 min at RT to solubilize the crystal violet. Aliquots of ethanol–crystal violet solution (200 μL) were transferred to a 96-well plate and the optical density at 570 nm (OD570) was measured using a microplate reader (Multiskan FC, Thermo Fisher Scientific, Waltham, MA, USA) [31 (link),32 (link)]. The absorbance was adjusted by subtracting the mean OD570 of the sterile controls to the OD570 from each sample.
4
Quantifying Bacterial Biofilm Biomass with Crystal Violet
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crystal violet is a basic dye that binds to negatively charged molecules, such as those in biofilm EPS matrix and on bacterial membrane surface,43 (link) thus staining the biofilm biomass. S. aureus suspensions at 1 × 106 cfu/mL were incubated in a 96-well microtitre clear flat-bottomed polystyrene plate (Corning) for 24 h at 37°C. After a washing step with MHB to remove non-adherent bacteria, preformed biofilms were incubated in the absence or presence of 2-fold serial dilutions of [G1K,K8R]cGm, ranging from 0.78 to 50 μM, for 4 h at 37°C. Untreated biofilms were used as a control. Biofilms were washed twice with MHB and incubated with 0.25% (v/v) crystal violet (Sigma–Aldrich) in sterile Milli-Q water, for 30 min at room temperature. After washing each sample three times with MHB, crystal violet was solubilized with 95% (v/v) ethanol (Carlo Erba Reagents S.A.S., France) by repeated pipetting. Biofilm biomass was quantified by measuring the absorbance at 590 nm of each sample in an Infinite® M200 microplate reader (Tecan). The percentage of crystal violet staining was determined relatively to the control, after blank [0.25% (v/v) crystal violet in sterile Milli-Q water] correction.
5
Clonogenic Assay of Trachinus vipera Venom
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To evaluate clonogenic survival, HCT116 cells were seeded at two different concentrations (100 and 200 cells/well) in 24-well plates. Twenty-four hours later, cells were treated with different concentrations of the Trachinus vipera purified venom (50, 100, 500 and 1000 μg/ml) for one week. Colonies were then fixed/stained with an aqueous solution containing 0.25% (w/v) crystal violet, 70% (v/v) methanol and 3% (v/v) formaldehyde (Carlo Erba Reagents) and counted. Only colonies made of >30 cells were included in the quantification. For each treatment, the survival fraction (SF) was estimated according to the formula: SF = number of colonies formed/number of cells seeded.
6
Quantifying Biofilm Formation by Crystal Violet
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The planktonic cells were removed from each well, and the plates were rinsed three times with deionised water. Excess moisture was removed by tapping the microplates on sterile napkins, and the plates were dried for 15 min. Then, biofilm formation was quantified by crystal violet assay, as reported by Sabaeifard et al. (2014 ), with some modifications. In detail, the wells were stained with 200 µL of 0.1% crystal violet (Sigma-Aldrich) solution at room temperature for 30 min, and then the microplates were washed with deionised water to remove the stain. After drying, crystal violet was solubilised in 250 µL of 30% acetic acid glacial (Carlo Erba Reagenti SpA, Milan, Italy).
Five absorbance values obtained for each strain were used to calculate means and standard deviations, by subtracting the value of control from each mean value.
The absorbance at 590 nm [optical density (OD)590] was measured, and the strains were grouped into: OD590<0.1, non-producers (NP); OD590=0.1-1.0, weak producers (WP); OD590=1.1-3.0, moderate producers (MP); and OD590>3.0, strong producers (SP).
Five absorbance values obtained for each strain were used to calculate means and standard deviations, by subtracting the value of control from each mean value.
The absorbance at 590 nm [optical density (OD)590] was measured, and the strains were grouped into: OD590<0.1, non-producers (NP); OD590=0.1-1.0, weak producers (WP); OD590=1.1-3.0, moderate producers (MP); and OD590>3.0, strong producers (SP).
7
Planktonic Cell Analysis Protocol
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To analyze planktonic cells, the strain was inoculated as described above and aliquots were withdrawn after 2, 4, 6, and 8 h using a sterile 10 µL loop and a drop was placed on a microscope glass slide (SuperFrost, Thermo Scientific, Rodano, Italy). The drop was then fixed by covering with paraformaldehyde 4% (Carlo Erba) and dried on a hot plate at 50 °C. Cells were then stained with crystal violet (Carlo Erba) and analyzed by an upright optical microscope (Olympus CX43) with a 40× objective. For each time point, three drops were withdrawn from each tube. The experiment was performed in triplicate for each strain. Five random images were acquired from each sample.
8
Quantification of Staphylococcal Biofilm Formation
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The identification of biofilm-forming strains was carried out by the microtiter plate (MtP) assay, as previously described [34 (link),35 (link)]. Briefly, after growing in BHI broth at 37 °C for 24 h, pure staphylococcal cultures were 1:100 diluted in fresh Trypticase broth (Oxoid, Italy) + 1% glucose (TSBg) and seeded in 96 well-plates (Corning, USA). After 24 h of incubation at 37 °C, planktonic bacteria were washed out and biofilm was stained with Crystal violet (Carlo Erba, Italy). Negative controls consist of TSBg only. Each strain was analyzed in triplicate on the same plate and three independent plates were used. The absorbance (570 nm) of negative controls was used to set the optical density cut-off (ODc) as three standard deviations above the mean OD of the negative control. Strains were classified as follows: Not adherent OD ≤ ODc; weakly adherent ODc < OD ≤ 2 × ODc; moderately adherent ODc < OD ≤ 4 × ODc; strongly adherent OD > 4 × ODc.
9
Crystal Violet Assay for Microbial Adhesion
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To evaluate the adhesion of microorganisms to the electrospun gelatin structures, the crystal violet assay was performed46 (link). The culture medium was removed and non-adherent planktonic microorganisms were eliminated by washing wells three times with 1 ml of PBS. Microbial biofilms were stained by using 2 ml of 0.1% w/v crystal violet (Carlo Erba, Italy) for 30 min at RT46 (link). The wells were washed three times with 1 ml of deionized water and covered with 2 ml of absolute ethanol for 15 min at RT to solubilize the crystal violet. Aliquots of ethanol-crystal violet solution (200 μl) were transferred to a 96-wells plate and the optical density at 570 nm (OD570) was measured by using a microplate reader (Biorad model 550, Biorad, USA)47 (link),48 (link). The absorbance was adjusted by subtracting the mean OD570 of the sterility controls to the obtained OD570 of each sample.
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