Biofilm inhibition capacity of phytate compounds was assessed semi-quantitatively by crystal violet (CV, Sigma Aldrich) staining and through the count of viable bacteria (colony forming unit, CFU) found in both the planktonic solution and bacterial biofilm. S. mutans CECT 479 was grown in brain heart infusion (BHI, NutriSelect® Plus, Sigma Aldrich) broth medium. Experiments were performed in triplicate with five different inoculums coming from a bacterial solution with an optical density of 0.1 registered at 600 nm (OD600 0.1), recorded in a spectrophotometer model Ultrospec 10 cell density meter (Amersham Biosciences). Bacteria stock inoculum was storage at − 80 °C in 15% (v:v) glycerol solution. PA, ZnPhy and SrPhy were solved at 100 µg/mL in Tris–HCl buffer 50 mM, pH ≈ 7. To do this, the phytate complexes were solved at 2 mg/mL in Tris–HCl buffer 1 M overnight. Subsequently, 5 mL of this solution were diluted in 100 mL of deionised water. The resulting solutions were adjusted to a final concentration of metal complexes of 100 µg/mL, 50 mM of Tris–HCl, and pH close to neutral. Finally, samples were sterilised by filtration with a 0.22 nylon filters. The control samples of these experiments was a S. mutans culture in BHI:Tris–HCl buffer 50 mM (1:1) without phytate compounds treatment.
Ultrospec 10 cell density meter
Manufactured by Cytiva
Sourced in United Kingdom
The Ultrospec 10 cell density meter is a compact and easy-to-use instrument designed for measuring the optical density of cell cultures. It provides a straightforward and reliable way to monitor cell growth and determine cell density in a variety of applications.
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Lab products found in correlation
Cellulose acetate filter, by Avantor (2 mentions)
Dionex ultimate 3000 hplc, by Thermo Fisher Scientific (2 mentions)
Variable wavelength uv detector, by Thermo Fisher Scientific (2 mentions)
Ri 101 detector, by Thermo Fisher Scientific (2 mentions)
Crystal violet, by Merck Group (1 mentions)
Nutriselect plus, by Merck Group (1 mentions)
Sheep blood, by BD (1 mentions)
Epoch 2, by Agilent Technologies (1 mentions)
Sampleprep, by SPEX (1 mentions)
Seveneasytm, by Mettler Toledo (1 mentions)
Inlab semi micro, by Mettler Toledo (1 mentions)
β estradiol, by Merck Group (1 mentions)
Hplc system, by Beckman Coulter (1 mentions)
15 protocols using ultrospec 10 cell density meter
1
Phytate Compounds Inhibit S. mutans Biofilm
2
Cultivating Human Pathogenic E. coli
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Human pathogenic E. coli (O6:K2:H1 CFT073) was cultivated in 35 mL
of NB medium suspension in an Erlenmeyer flask with shaking at 37
°C overnight. On the next day, 5 mL of cell suspension was centrifuged
(5000g, 5 min, 20 °C), and the optical density
OD600 was brought to 1.0 by diluting cells in PBS (pH 7.4) supplemented
with 1 mM CaCl2. OD600 was determined using
an Ultrospec10-Cell density meter (Amersham Biosciences).
of NB medium suspension in an Erlenmeyer flask with shaking at 37
°C overnight. On the next day, 5 mL of cell suspension was centrifuged
(5000g, 5 min, 20 °C), and the optical density
OD600 was brought to 1.0 by diluting cells in PBS (pH 7.4) supplemented
with 1 mM CaCl2. OD600 was determined using
an Ultrospec10-Cell density meter (Amersham Biosciences).
3
Automated Bacterial Growth Monitoring
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The optical density was measured using an Ultrospec 10-cell density meter (Amersham Biosciences, UK) at 600 nm. An OD600 of 1.0 corresponded with a cell dry weight of 366 mg L−1.
Furthermore, to enable real-time and automated monitoring of bacterial growth and growth rate [85 (link)], the Growth Profiler 960 (System Duetz, EnzyScreen BV, Heemstede, The Netherlands) was employed. Throughout the cultivations in the Growth Profiler, a constant temperature of 30°C and a shaking speed set to 250 rpm (revolutions per minute) were maintained. The cultivation was carried out in 96-well MTP plates (CR1496dg: polystyrene white square 96-half-deep well microtiter plates), which were covered with sandwich covers (CR1396: universal sandwich cover for 96-well MTPs). The entire run spanned 48 h.
The Growth Profiler recorded online green values, which were subsequently converted to OD600 values, depicting the growth of the bacteria [86 (link)]. To calculate growth rates, a MATLAB-based script was utilized in the analysis of the recorded data, which portrays exponential growth [87 (link)].
Furthermore, to enable real-time and automated monitoring of bacterial growth and growth rate [85 (link)], the Growth Profiler 960 (System Duetz, EnzyScreen BV, Heemstede, The Netherlands) was employed. Throughout the cultivations in the Growth Profiler, a constant temperature of 30°C and a shaking speed set to 250 rpm (revolutions per minute) were maintained. The cultivation was carried out in 96-well MTP plates (CR1496dg: polystyrene white square 96-half-deep well microtiter plates), which were covered with sandwich covers (CR1396: universal sandwich cover for 96-well MTPs). The entire run spanned 48 h.
The Growth Profiler recorded online green values, which were subsequently converted to OD600 values, depicting the growth of the bacteria [86 (link)]. To calculate growth rates, a MATLAB-based script was utilized in the analysis of the recorded data, which portrays exponential growth [87 (link)].
4
Analysis of Bioreactor Cultures
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All experiments were performed in duplicates. Shown is the arithmetic mean of the duplicates. Error bars and ±values indicate deviation from the mean.
From bioreactors, 5 mL of culture broth was taken for OD600 and HPLC analysis. When using CaCO3 as buffer, the CaCO3 in 1 mL culture broth was dissolved with HCl prior to further measurements. OD600 was determined in an Ultrospec 10 cell density meter (Amersham Biosciences, UK); samples were diluted to an OD600 between 0.1 and 0.8.
For HPLC analysis, centrifuged samples (13.000g, 5 min) were filtered through cellulose acetate filters (diameter 0.2 µm, VWR, Germany) prior to diluting 1:10 with distilled water. For analysis of glycerol and organic acids, a Dionex Ultimate 3000 HPLC (Dionex, USA) was used with an Organic Acid Resin column (CS-Chromatographie, Germany) at 75 °C, with a constant flow rate of 0.8 mL min−1 5 mM sulfuric acid as eluent. For detection, a Shodex RI 101 detector at 35 °C and a variable wavelength UV detector (Dionex, USA) at 210 nm were used.
Ammonium concentration was determined by a colorimetric assay according to Willis [55 (link)].
Calculation of the molar fraction of undissociated and dissociated species for malate was performed using CurTiPot [56 ].
From bioreactors, 5 mL of culture broth was taken for OD600 and HPLC analysis. When using CaCO3 as buffer, the CaCO3 in 1 mL culture broth was dissolved with HCl prior to further measurements. OD600 was determined in an Ultrospec 10 cell density meter (Amersham Biosciences, UK); samples were diluted to an OD600 between 0.1 and 0.8.
For HPLC analysis, centrifuged samples (13.000g, 5 min) were filtered through cellulose acetate filters (diameter 0.2 µm, VWR, Germany) prior to diluting 1:10 with distilled water. For analysis of glycerol and organic acids, a Dionex Ultimate 3000 HPLC (Dionex, USA) was used with an Organic Acid Resin column (CS-Chromatographie, Germany) at 75 °C, with a constant flow rate of 0.8 mL min−1 5 mM sulfuric acid as eluent. For detection, a Shodex RI 101 detector at 35 °C and a variable wavelength UV detector (Dionex, USA) at 210 nm were used.
Ammonium concentration was determined by a colorimetric assay according to Willis [55 (link)].
Calculation of the molar fraction of undissociated and dissociated species for malate was performed using CurTiPot [56 ].
5
Automated Bacterial Growth Curve Assay
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Strains were cultured from frozen stock onto tryptic soy agar (TSA) supplemented with 5% sheep blood (Becton, Dickinson, Vianen, the Netherlands) at 37°C overnight. Bacterial suspensions were prepared at an OD at 600 nm (OD600) of 1.00 ± 0.05 (Ultrospec 10 Cell Density Meter; Amersham Biosciences, United Kingdom) in trypticase soy broth (TSB) (Becton, Dickinson, Vianen, the Netherlands) representing approximately 109 CFU/mL. Subsequently, 10-fold serial dilutions were prepared in TSB in sterile U-bottom 96-well polystyrene (PS) microplates (Greiner Bio-One GmbH, Frickenhausen, Germany).
For classical growth curves, 10 μL of this logarithmic dilution series was added to a sterile U-bottom 96-well PS microplate filled with 190 μL TSB per well. Turbidity was measured every 10 min by OD600 in a microplate reader (Epoch 2, BioTek Instruments, VT) for at least 20 h. Before each measurement, the microplate was subjected to 1 min of double-orbital shaking at low speed.
For classical growth curves, 10 μL of this logarithmic dilution series was added to a sterile U-bottom 96-well PS microplate filled with 190 μL TSB per well. Turbidity was measured every 10 min by OD600 in a microplate reader (Epoch 2, BioTek Instruments, VT) for at least 20 h. Before each measurement, the microplate was subjected to 1 min of double-orbital shaking at low speed.
6
Cryogenic Yeast Cell Lysis Protocol
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Cells were grown in YPD at 25°C to an OD600 of 0.6–0.8, as measured on an Ultrospec 10 Cell Density Meter (Amersham). Cells from 4 L of culture were harvested by centrifugation, washed in cold water, and centrifuged again. Standing moisture was removed from pellets and cells were flash frozen in liquid N2 and then stored at −80°C. Frozen cultures were lysed by mechanical shearing in a pre-chilled cryogenic grinder (SPEX SamplePrep) using a medium-sized SPEX vial that had been pre-chilled in liquid nitrogen. Milling consisted of a 5 min pre-chill followed by 6–10 cycles comprising 3 min of grinding and 1 min of rest. The sample vial remained submerged in liquid N2 throughout the milling process. Powdered lysate was collected in a 50 ml conical vial that had been pre-chilled in liquid N2. Lysate preparations stored at −80°C were stable for >6 months but were highly sensitive to temperature excursions.
7
Correlating Cell Density and Dry Weight
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The optical density at 600 nm (OD600) was measured using an Ultrospec 10 cell density meter (Amersham Biosciences, UK). A correlation between OD600 and cell dry weight (CDW) was established. An OD600 of 1.0 corresponds with a cell dry weight of 369 mg L−1.
8
Optical Density and pH Monitoring
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Biomass was monitored by optical density (λ = 600 nm) using a photometer (Amersham Bioscience, Ultrospec 10 cell density meter) by applying the biomass/optical density correlation from a previous study [62 (link)]. The pH was measured off-line with a pH meter (SevenEasyTM; Mettler Toledo, Columbus, OH, USA) connected to a pH electrode (InLab Semi-Micro; Mettler Toledo, Columbus, OH, USA).
9
Analytical Methods for Microbial Cultivation
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All experiments were performed in duplicates. Shown is the arithmetic mean of the duplicates. Error bars and ± values indicate deviation from the mean.
When using CaCO3 as buffer, 1 mL of culture broth was taken for OD600 determination and HPLC analysis. The CaCO3 was dissolved with HCl prior to further measurements. OD600 was determined in an Ultrospec 10 cell density meter (Amersham Biosciences, UK), samples were diluted to an OD600 between 0.1 and 0.8.
For HPLC analysis, centrifuged samples (13.000 g, 5 min) were filtered through cellulose acetate filters (diameter 0.2 µm, VWR, Germany) and subsequently diluted 1:10 with distilled water. Glycerol and organic acids were analyzed on a Dionex Ultimate 3000 HPLC (Dionex, USA) with an Organic Acid Resin column (CS–Chromatographie, Germany) kept at 75 °C, with a constant flow rate of 0.8 mL min−1 of 5 mM sulfuric acid as eluent. For detection, a Shodex RI 101 detector at 35 °C and a variable wavelength UV detector (Dionex, USA) at 210 nm were used.
Ammonium concentration was determined by a colorimetric assay according to Willis [38 (link)].
When using CaCO3 as buffer, 1 mL of culture broth was taken for OD600 determination and HPLC analysis. The CaCO3 was dissolved with HCl prior to further measurements. OD600 was determined in an Ultrospec 10 cell density meter (Amersham Biosciences, UK), samples were diluted to an OD600 between 0.1 and 0.8.
For HPLC analysis, centrifuged samples (13.000 g, 5 min) were filtered through cellulose acetate filters (diameter 0.2 µm, VWR, Germany) and subsequently diluted 1:10 with distilled water. Glycerol and organic acids were analyzed on a Dionex Ultimate 3000 HPLC (Dionex, USA) with an Organic Acid Resin column (CS–Chromatographie, Germany) kept at 75 °C, with a constant flow rate of 0.8 mL min−1 of 5 mM sulfuric acid as eluent. For detection, a Shodex RI 101 detector at 35 °C and a variable wavelength UV detector (Dionex, USA) at 210 nm were used.
Ammonium concentration was determined by a colorimetric assay according to Willis [38 (link)].
10
Fundamental Osmotic Stress Response in Yeast
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The diploid yeast strain Saccharomyces cerevisiae SKQ2n (ATCC 44827; a/alpha; ade1/+; ade2; +/his1) was used in all experiments. This strain has been previously used as a reference/model strain in fundamental studies of the osmotic stress response [51 (link),52 (link)]. In this study, yeast cells were stored at −80 °C in 20% glycerol until use. The cells were then cultivated in a medium (sterilized by filtration) containing 0.67% (wt/vol) yeast nitrogen base (YNB, Difco Laboratories, Detroit, MI, USA) with 5% ammonium sulfate, supplemented with 2% glucose, and grown at 30 °C in 10 mL tubes in a LabRollerTM Rotator at 30 rpm and maintained on plates with YPD agar (per L: 1−0 g of yeast extract, 20 g of peptone, 20 g of d -glucose and 20 g of agar). Optical densities were measured at 600 nm (OD600) using a ULTROSPEC 10 cell density meter (Amersham Biosciences, Little Chalfont, UK).
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