HCT 116 and HT-29 cells were seeded in 6-well plates at a density of 2.5 × 105 cells per well and grown overnight. After treatment with 10, 20, or 40 µg/mL CRCE or DMSO as a control for 24 h, cells were trypsinized, counted, and 1000 cells per well were re-seeded in 6-well plates. Cells were further cultivated for 7 days with the medium refreshed every other day to allow colonies to grow. Obtained colonies were washed with PBS, fixed with methanol for 10 min at room temperature (RT, 20 to 25 °C), washed with PBS, and stained with a 0.5% crystal violet solution (Junsei Chemicals, Tokyo, Japan). After washing in water and adequate drying, colonies on the six-well plates were counted, and the seeding efficiency (SE) and surviving fraction (SF) were evaluated with the previously described method [54 (link)].
Crystal violet solution
Manufactured by Junsei
Sourced in Japan
Crystal violet solution is a laboratory reagent used for various applications in the field of biology and microscopy. It is a deep purple dye that can be used for staining cells and tissues. The solution is typically prepared in a concentration suitable for the specific application.
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Lab products found in correlation
3 protocols using crystal violet solution
1
Colony Formation Assay for HCT 116 and HT-29 Cells
2
Biofilm Removal on Dental Resins
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The 60 resin specimens prepared with the same fabrication technique were randomly assigned to six groups (n = 10). After biofilm formation, the resin specimens were gently washed with PBS and treated according to each group's corresponding protocol. The concentration and application time of each group's agents were determined based on previous studies or the manufacturer's instructions: PBS group, PBS for 10 min; Polident group, Polident 5-Minute (GlaxoSmithKline) for 5 min (manufacturer's instructions), CHX group, 0.12% (w/v) CHX for 10 min; H2O2 group, 3% (v/v) H2O2 for 10 min; DM group, co-treatment of 3 mg/mL of DM and 3% H2O2 for 5 min; and negative control (no contamination to verify asepsis of the experiment), PBS for 10 min [16 (link), 23 (link)].
After each treatment, the remaining biofilms were quantified by crystal violet assay [26 (link)]. The resin specimens were washed with PBS and incubated with 1 mL of 1% (w/v) crystal violet solution (Junsei Chemical) for 10 min to stain the remaining biofilm on the resin specimens. After that, the specimens were rinsed three times with PBS to remove the residual dye. The remaining crystal violet dye in the biofilms was extracted by using 95% ethanol. The dissolved crystal violet dye's optical density was quantified using a microplate reader (Epoch 2; Bio-Tek Instruments) at 570 nm.
After each treatment, the remaining biofilms were quantified by crystal violet assay [26 (link)]. The resin specimens were washed with PBS and incubated with 1 mL of 1% (w/v) crystal violet solution (Junsei Chemical) for 10 min to stain the remaining biofilm on the resin specimens. After that, the specimens were rinsed three times with PBS to remove the residual dye. The remaining crystal violet dye in the biofilms was extracted by using 95% ethanol. The dissolved crystal violet dye's optical density was quantified using a microplate reader (Epoch 2; Bio-Tek Instruments) at 570 nm.
3
Quantifying Anti-Biofilm Efficacy of Treatments
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A total of 50 sterile brackets were prepared and randomly assigned to 5 groups for crystal violet assay (n = 10). Of these, 10 specimens were placed in 200 mL of sterile broth as a negative control group, and the specimens of the other 4 groups were incubated with S. mutans suspension as previously described to form biofilms. After biofilm formation, the orthodontic brackets were gently washed with phosphate-buffered saline (PBS, pH = 7.4) and transferred to a new 96-well tissue culture plate. Next, orthodontic brackets were treated either with PBS (PBS group), 0.12% (w/v) CHX (Bukwang Pharmaceutical Co., Ltd, Seoul, Korea) (CHX group), 3% (v/v) H2O2 (H2O2; Sigma-Aldrich, St. Louis, MO) (H2O2 group), or co-treatment with 3 mg/mL of DM and 3% H2O2 (DM group) for 2 min.
After removing non-adherent cells by washing with PBS, the remaining biofilms on the brackets were quantified using a crystal violet assay [19 (link)]. The brackets were stained with 1 mL of 1% (w/v) crystal violet solution (Junsei Chemical, Tokyo, Japan) for 10 min and washed with PBS thrice. The crystal violet dye in the remaining biofilms was released using 95% ethanol. Subsequently, 200 μL of the solution was transferred to a microtiter plate, and the optical density (OD) of the dissolved crystal violet dye was measured using a microplate reader (Epoch 2; Bio-Tek Instruments, Winooski, VT, USA) at 570 nm.
After removing non-adherent cells by washing with PBS, the remaining biofilms on the brackets were quantified using a crystal violet assay [19 (link)]. The brackets were stained with 1 mL of 1% (w/v) crystal violet solution (Junsei Chemical, Tokyo, Japan) for 10 min and washed with PBS thrice. The crystal violet dye in the remaining biofilms was released using 95% ethanol. Subsequently, 200 μL of the solution was transferred to a microtiter plate, and the optical density (OD) of the dissolved crystal violet dye was measured using a microplate reader (Epoch 2; Bio-Tek Instruments, Winooski, VT, USA) at 570 nm.
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