Biofilm formation was assessed by CV assays in 96-well plates (untreated PS surfaces) (CytoOne; StarLab, Hamburg, Germany). ON cultures of strains were diluted 1:100 in fresh medium, and 150 μl was added per well (eight wells per strain and biological replicate). Plates were incubated at 12, 28, and 37°C for 48 h. After incubation, plates were washed three times with 200 μl dilution solution (8 g/liter NaCl, 1 g/liter peptone) per well and subsequently stained with 200 μl 0.1% CV solution (Sigma-Aldrich, Buchs, Switzerland) per well for 20 min. Staining was followed by three washes with double-distilled water (ddH2O), and biofilms were dissolved in 200 μl 96% ethanol (EtOH) per well. Biofilm formation was assessed by measurement of the optical density at 600 nm (OD600). The assay was performed in biological triplicate. Biofilm formation results are reported in Table 2 , using categories defined by OD/ODc ratios (79 (link)). Overall scores for media and strains are the sums of the category numbers in the columns and rows of the table, respectively. For comparison of wild-type K-12 MG1655 and its pFAM21845_1 and pFAM21845_2 transconjugants, triplicates of the values for ODstrain − ODnegative control were used (Table 4 ).
Cv solution
Manufactured by Merck Group
Sourced in United States, Germany
CV solution is a laboratory product manufactured by Merck Group. It is a standard solution used for calibrating and verifying the performance of conductivity meters and other conductivity measurement devices. The solution has a known conductivity value that allows for the accurate calibration and verification of the instrument's functionality.
Automatically generated - may contain errors
Lab products found in correlation
Methanol, by Merck Group (2 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Acetic acid, by Thermo Fisher Scientific (2 mentions)
Cytoone, by Starlab (1 mentions)
Evos floid imaging system, by Thermo Fisher Scientific (1 mentions)
Polarstar omega microplate reader, by BMG Labtech (1 mentions)
Hexane, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
Ethyl acetate, by Merck Group (1 mentions)
Acetone, by Merck Group (1 mentions)
Tryptone, by Avantor (1 mentions)
Sodium phosphate dibasic, by Merck Group (1 mentions)
Potassium phosphate monobasic, by Merck Group (1 mentions)
Tryptic soy broth tsb, by Neogen (1 mentions)
M9 minimal salts 5, by Merck Group (1 mentions)
Powerwave x52, by Agilent Technologies (1 mentions)
Nunclon delta surface, by Thermo Fisher Scientific (1 mentions)
12 protocols using cv solution
1
Biofilm Formation Quantification by CV Assay
2
Quantifying Biofilm Formation via Crystal Violet Assay
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The CV staining assay was performed according to the protocol used by Peeters et al. (2008 (link)), with minor modifications. After 24 h of biofilm development, 1 mL of 99% methanol (Merck Millipore, Billerica, MA) was added for 15 min for fixation of biofilms. After that, supernatants were removed and the plates were air-dried. Then, 2 mL of a 0.1% CV solution (Sigma-Aldrich, St. Louis, MO) was added to all wells. After 20 min, the excess CV was removed by washing the plates in sterile distilled water. Finally, bound CV was released by adding 2 mL of 95% ethanol (Synth, Diadema, São Paulo) (Li et al. 2003 ). The absorbance was measured at 570 nm and standardized in relation to the area of acrylic specimens (Abs/cm2). All steps were carried out at room temperature. Each group included two specimens, and the experiment was repeated six times.
3
Quantitative Biofilm Assessment Protocol
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To assess the extent of biofilm formation, the protocol described by Stepanovic and collaborators was used, with few modifications23 (link). Briefly, at the defined time points, the CFS-treated pathogen and untreated control samples were washed twice with saline solution and fixed with pure methanol for 15 min. Then, 150 μL/well of CV solution (Sigma-Aldrich) were added for 5 min to stain the dried biofilms. The excess amount of CV was removed by washing carefully with tap water. The images were acquired with an EVOS FLoid Imaging System (Thermo Fisher Scientific, Waltham, MA). 3D biofilm pictures were obtained using the ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/ , 1997–2018). Then, CV was dissolved with 33% acetic acid and quantified by measuring the absorbance at 570 nm using a Spark microplate reader. All experiments were replicated three times on separate days.
4
Quantification of Biofilm Biomass
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After a further 24 h of incubation at 37 °C in the presence of substances or only BHI with 1% glucose (control), the microplates were turned upside down and tapped so that broth and most of the planktonic bacteria fell out onto the absorbent mat57 (link). The biofilms were then rinsed twice with 200 μL of sterile phosphate-buffered saline per well. Two hundred microlitres of CV solution (Sigma-Aldrich, 0.05% w/v) was added to each well and left for 3 min at 25 °C. The excess dye was then rinsed with 200 μL of sterile PBS, the wells were air-dried naturally, then 200 μL of 96% (w/v) ethanol was added to each well to resolubilise the residual dye. The biofilm’s biomass was measured using a microplate reader (CLARIOstar Plus) with the value of absorbance set at 595 nm (OD595).
5
Motility Assays for Bacterial Cultures
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For motility assays, bacterial cultures grown overnight were diluted to a density of 105 CFU/mL and transferred to Petri dishes containing Tryptic Soy Agar (TSA) medium, with different concentrations of agar (0.3%, 0.8% and 2% for swimming, swarming, and twitching motility, respectively). The bacterial suspension was transferred into the agar medium by puncture using a pipette tip (at 1/2 depth for swimming and swarming motility and at full depth for twitching motility) [35 ,36 ]. The plates were then incubated at 37 °C for 24 h (swimming and swarming motility) and 48 h (twitching motility). After the incubation period, the diameter of the growth zones (mm) were measured; in case of swimming and swarming motility, the measurements were made directly, while in case of twitching motility, the agar layer was removed and the bottom of the plates were stained directly with 0.01% CV solution (Sigma-Aldrich, St. Louis, MO, USA) [35 ,36 ]. All experiments were carried out in triplicate.
6
Quantifying Bacterial Biofilm Biomass
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The total biofilm biomass production of the strains was analyzed by the CV staining protocol [37 (link)]. Briefly, an initial 106 CFU/mL bacterial inoculum prepared in 3 mL of Mueller Hinton (MH) Broth (Pronadisa, Conda, Spain) was inoculated on the PLA Petri dishes, and subsequently incubated at 37 °C during 24 h to obtain a mature biofilm. The medium was removed at the end of the incubation period, and the biofilm was washed with phosphate buffer saline (PBS) and fixed with methanol during 15 min at room temperature. Plates were dried during 10 min at room temperature after removing the methanol. Then, 3 mL of CV solution was added (Sigma, final concentration 10% in PBS) and incubated at room temperature during 20 min. The CV excess was removed under running water and dried. Finally, the cell bound crystal violet was dissolved in 3 mL of acetic acid 66% v/v, incubated at room temperature for 1 h and quantified by absorbance at 590 nm. Measures were performed on a plate reader (POLAR star Omega microplate reader, BMG Labtech). Three PLA Petri dishes with no treatment were included as control in all assays. Four PLA Petri dishes were used per each treatment and microorganism. ANOVA test has been realized to determine if there are statistically significant differences on biofilm formation between coated and untreated PLA Petri dishes.
7
Synthesis and Characterization of Polymeric Hydrogels
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Gelatin powder (type A,
300 bloom), SPH, sodium hydroxide, hydrochloric acid, methanol, ethyl
acetate, hexane, acetone, sodium chloride, potassium chloride, sodium
phosphate dibasic, potassium phosphate monobasic, hydrochloric acid,
glucose, M9 minimal salts (5×), and CV solution (1%) were purchased
from Sigma-Aldrich (Milwaukee, WI). TA was purchased from Alfa Aesar
(Thermo Fisher, Belgium). Anhydrous sodium thiosulfate, 0.1 N iodine
standard solution, and 0.1 N sodium thiosulfate standard solution
were purchased from VWR Chemicals (Radnor, PA). Tryptic soy broth
(TSB) was purchased from Neogen (Lansing, MI), and tryptic soy agar
(TSA) was purchased from bioWorld (Dublin, OH). Tween 20 was purchased
from ChemImpex (Wood Dale, IL). Rifampicin was purchased from Thomas
Scientific LLC (NJ, USA). Tryptone was purchased from Amresco (Solon,
OH). Clorox bleach solution with a free chlorine content of 8.0% was
produced by Clorox Co., Ltd. (Oakland, CA, USA). LDPE sheets were
purchased from the Henta Corporation. DI water was used in the materials
fabrication and tests.
300 bloom), SPH, sodium hydroxide, hydrochloric acid, methanol, ethyl
acetate, hexane, acetone, sodium chloride, potassium chloride, sodium
phosphate dibasic, potassium phosphate monobasic, hydrochloric acid,
glucose, M9 minimal salts (5×), and CV solution (1%) were purchased
from Sigma-Aldrich (Milwaukee, WI). TA was purchased from Alfa Aesar
(Thermo Fisher, Belgium). Anhydrous sodium thiosulfate, 0.1 N iodine
standard solution, and 0.1 N sodium thiosulfate standard solution
were purchased from VWR Chemicals (Radnor, PA). Tryptic soy broth
(TSB) was purchased from Neogen (Lansing, MI), and tryptic soy agar
(TSA) was purchased from bioWorld (Dublin, OH). Tween 20 was purchased
from ChemImpex (Wood Dale, IL). Rifampicin was purchased from Thomas
Scientific LLC (NJ, USA). Tryptone was purchased from Amresco (Solon,
OH). Clorox bleach solution with a free chlorine content of 8.0% was
produced by Clorox Co., Ltd. (Oakland, CA, USA). LDPE sheets were
purchased from the Henta Corporation. DI water was used in the materials
fabrication and tests.
8
Quantifying Biofilm Crystal Violet Staining
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The biofilms were dried for 30 min at 37°C and stained with 200 μL of CV solution (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at room temperature. Unbound stain was removed by rinsing with demineralized water, after which the plates were emptied and dried for 30 min at 37°C. Then 200 μL of acetic acid (33%) was added for 30 min at room temperature to extract the CV stain from the biofilms. 50 µL liquid from each well was transferred to and diluted in 150 μL acetic acid (33%). Absorbance was measured with a plate reader (BioTek PowerWave X52) in flat bottom 96-well plates (Nunclon Delta Surface, Thermo Fisher Scientific) at 590 nm. Experiments were performed in technical quadruplicates and at least in biological triplicates.
9
Biofilm Formation Assay with Vitamin D
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To assess the biofilm formation ability in the two different media, crystal violet (CV) staining of the biofilm produced by S. aureus and LJO02 cultivated in their conventional media and in DMEM 10% FBS with different concentrations of vitamin D was performed. S. aureus and LJO02 were respectively plated at an OD600 of 0.035 and 0.05 into 48-well plates and immediately treated with vitamin D, as described above. A plate for each bacterium and endpoint of 24, 48, and 72 h was used. At the defined endpoints, the supernatant was removed, and the biofilm was fixed with pure methanol (Sigma-Aldrich) for 15 min at RT. Then, the dried biofilm was stained with 1% CV solution (Sigma-Aldrich) for 5 min at RT. After removing the CV excess, the biofilm images were acquired with EVOS FLoidTM Cell Imaging Station (Thermo Fisher Scientific). To quantify the biofilm amount, a 33% acetic acid solution (Sigma-Aldrich) was used to dissolve the CV and the absorbance was read at 570 nm with a Spark microplate reader. Each experiment was conducted with four replicates per condition and repeated three times independently.
10
Quantifying Biofilm Biomass using Crystal Violet
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Biofilms biomass was quantifyed using the crystal violet (CV) method, as described before [42 (link)]. Briefly, biofilms were fixation with 100% (v/v) methanol (Thermo Fisher Scientific) for 20 min and then were stained with CV solution at 1% (v/v) (Merck, Darmstadt, Germany) for another 20 min. Biofilms were washed twice with phosphate-buffered saline (PBS). The CV stained was then recovered from the biofilm with the addition of 33% (v/v) acetic acid (Thermo Fisher Scientific), that was then transferred to a new plate before optical density (OD) at 595 nm was assessed. These assays were repeated three times on separate days, with four technical replicates assessed each time.
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