A portion of the prepared soil samples (about 5 g) placed in porcelain crucibles with lids were thermally treated at different temperatures (100, 200, 300, 400, and 500°C) for 30 min using a muffe furnace (KDF007Ex, Denken, Tokyo, Japan). The heating rate was 3°C min -1 . After cooling, the thermally treated samples were dried under reduced pressure. Select soil samples were designated as controls and were thus not subjected to thermal treatment. WSOM was extracted by shaking a solution of 1:10 sample/distilled water (w/w) at 190 rpm and 25°C for 24 h in darkness. The solution was then filtered through a membrane with a 0.45-μm pore diameter (mixed cellulose ester, ADVANTEC, Tokyo, Japan). 20
Mixed cellulose ester
Manufactured by Advantec
Sourced in Japan
Mixed cellulose ester is a type of laboratory filtration membrane. It is designed for the separation and isolation of various substances during analytical processes. The membrane is composed of a combination of cellulose esters, providing a versatile filtration medium for a range of applications.
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5 protocols using mixed cellulose ester
1
Thermal Soil Treatment Effects on WSOM
2
Quantifying E. coli and Enterococci in Water Samples
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Each water sample (upstream water, 100–2000 mL; downstream water, 10–100 mL) was filtered through a sterile 0.45 μm-pore membrane filter (47 mm diameter, mixed cellulose ester; Advantec, Tokyo, Japan) to capture microbial cells. Membranes were placed on CHROMagar ECC agar plates (CHROMagar, France) and incubated at 37 °C for 24 h. Blue colonies were considered to be E. coli. Enterococci were enumerated using the membrane filter method with membrane–Enterococcus indoxyl-β-D-glucoside agar (mEI) plates [21 ]. The samples passed through a membrane filter incubated on mEI agar plates at 41 °C for 24 h. After incubation, colonies on the filter that had blue halos were regarded as enterococci. Concentrations of E. coli and enterococci in each sample were expressed as mean CFUs per 100 mL of three replicates. The detection limit of the method of this analysis was 0.3 CFU/100 mL.
3
Bacterial Interactions and Fouling Potential
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To investigate the microbial interaction between fouling-causing bacteria (FCB) and fouling-enhancing bacteria (FEB), S26 was cultured with the supernatant of either S22 or S31, and its fouling potential was measured. For the cultivation of S26, M9 medium was made with the supernatant of bacterial culture (OD600 = 0.3) sterilized with syringe filter (0.2 μm, Mixed Cellulose Ester; Advantec Toyo; Tokyo, Japan) to check the effect of microbial interaction on fouling potential via bacterial secretion. S22 and S31 were also cultured with M9 medium made with the supernatant of S26 to confirm the effect of the supernatant of FCB.
In order to evaluate the effect of AHL on enhancement of fouling potential, S22 and S26 was cultivated with M9 medium containing 4.4 μM of N-octanoyl-L-homoserine lactone (C8-HSL). The bacterial cultivation and the fouling potential measurement were carried out as mentioned above.
In order to evaluate the effect of AHL on enhancement of fouling potential, S22 and S26 was cultivated with M9 medium containing 4.4 μM of N-octanoyl-L-homoserine lactone (C8-HSL). The bacterial cultivation and the fouling potential measurement were carried out as mentioned above.
4
Protein Purification by Ion Exchange Chromatography
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Biologic duoflow chromatography system (Bio-Rad, USA) was utilized as the protein purification instrument especially in wash, elution, and clean step as described by Gu et al. (2016) with slight modification (Gu et al., 2016 ). A peristaltic pump was also utilized to apply the sample to the column. A ResourceQ column containing quaternary ammonium cation resin was chosen as the anion exchanger. Tris-HCl buffer (buffer A) was used as the equilibration buffer solution and Tris-HCl with 0.5 M NaCl (buffer B) was used as the elution buffer in ion exchange chromatography. Before applying the sample to the column, the sample was filtered by using mixed cellulose ester having pore and diameter of 0,45 μm and 47 mm respectively (ADVANTEC, Japan) to prevent any contaminant from entering the column. The sample was applied to the column using peristaltic pump (EYELA MP-2000, Japan). After connecting the column to the purification instrument, sample was washed by isocratic flow mode of 100% buffer A. The purification process continued by setting the elution process to gradient linear mode of 100%–0% buffer A and 0%–100% buffer B. The system was kept at – 4 °C with a flow rate of 3.0 mL/min. Most peaks were detected from 12 min to 26 min of the process for all samples. Tens of fractions were collected and ready to be grouped by SDS PAGE.
5
Monitoring Heavy Metal Concentrations in Water
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After the initiation of the column test on August 23rd, 2019, solution samples were routinely taken twice a week, from each sampling port, for a period of 85 days to monitor pH and DO levels using MM-43X (TOA DKK) and HQ30d (HACH), respectively. These samples were filtered through a 0.45 μm membrane filter (mixed cellulose ester; Advantec) and then used to determine the concentrations of Cu, Zn, and Cd by ICP-OES (Agilent 5110 ICP-OES, Agilent Technologies Inc., Santa Clara, CA, USA). Concentrations of sulfate (SO42−) and acetate in the filtrate were quantified by ion chromatography (Dionex ICS-6000, Thermo Fisher Scientific Inc., Waltham, MA, USA). The concentration of sulfide (HS−) was measured by the methylene blue method [29 (link)]. The concentration of ethanol was determined by UPLC equipped with an ion exclusion column (IC-Pak Ion Exclusion Column 7 µm, 7.8 mm × 300 mm; Waters Corp., Milford, MA, USA). Total inorganic carbon (TIC) was quantified by a TOC analyzer (TOC-L, Shimadzu Corp., Kyoto, Japan). The detection limits for each chemical specie were as follows: Cu 0.18 (µg/L); Zn 0.08 (µg/L); Cd 0.06 (µg/L).
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