Bacteria were concentrated from water samples by membrane filtration through a 0.45 or 0.65 µm sterile filter membrane (Sartorius cellulose nitrate membrane filter, diameter 47 mm). Depending on the water turbidity, adapted volumes of 100–500 ml were filtered and the filter was placed retentate-up on mCCDA plates (Oxoid; PO5091A). The filter was removed from the agar surface after 18–20 h of incubation at 42 °C in sealed jars (Anaerojar 2.5; Oxoid, AG0025A) under microaerobic conditions (CampyGen gas-generating system; Oxoid, CN0025). After 24–72 h of supplementary incubation under microaerobic conditions, Campylobacter
Campylobacter
Cellulose nitrate membrane filter
Manufactured by Sartorius
Sourced in Germany
The cellulose nitrate membrane filter is a laboratory filtration device used for the separation and isolation of particles, microorganisms, and other suspended matter from liquid samples. It is a thin, porous membrane made of cellulose nitrate, a type of cellulose that has been chemically modified. The filter's pore size can be customized to meet specific filtration requirements.
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Lab products found in correlation
Gel extraction kit, by Qiagen (1 mentions)
Truseq dna pcr free sample preparation kit, by Illumina (1 mentions)
Hiseq2500 pe250 sequencing system, by Illumina (1 mentions)
E z n a water dna kit, by Omega Bio-Tek (1 mentions)
Phusion high fidelity pcr master mix, by New England Biolabs (1 mentions)
Cp 3800, by Agilent Technologies (1 mentions)
Dipalmitoylphosphatidylcholine dppc, by Lipoid (1 mentions)
Tryptic soy broth (tsb), by Merck Group (1 mentions)
Spd 10a vp uv vis detector, by Shimadzu (1 mentions)
Security guard c18 guard cartridge, by Phenomenex (1 mentions)
Lc 10atvp chromatographic pump, by Shimadzu (1 mentions)
Kinetex, by Phenomenex (1 mentions)
Filter holder manifold, by Merck Group (1 mentions)
Buffered peptone water (bpw), by Scharlab (1 mentions)
Cellulose acetate membrane filter, by Sartorius (1 mentions)
Polyethersulfone syringe filter, by Avantor (1 mentions)
Po5091a, by Thermo Fisher Scientific (1 mentions)
Ag0025a, by Thermo Fisher Scientific (1 mentions)
Po5090a, by Thermo Fisher Scientific (1 mentions)
Agar gsp, by Merck Group (1 mentions)
17 protocols using cellulose nitrate membrane filter
1
Isolation and Identification of Campylobacter
2
Seawater Microbiome Sequencing Protocol
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Seawater samples were prefiltered through sterile Whatman no. 2 filter papers to remove suspended particles and the filtrate was subsequently filtered through 0.2 μm sterile cellulose nitrate membrane filters (Sartorius, Stedim Biotech., Gottingen, Germany) [4 (link)]. DNA was extracted from seawater samples using E.Z.N.A®Water DNA kit (Omega Bio-tek, Inc., Norcross, GA, USA), according to the manufacturer’s instruction. The V4 variable region of the 16S rRNA gene was amplified by using the 515F and 806R specific primer set with the barcodes [34 (link), 35 (link)]. PCR reactions were carried out with Phusion® High-Fidelity PCR Master Mix (NEB, Ipswitch, MA, USA). The PCR products were purified using a Qiagen gel extraction kit (Qiagen, Inc., Valencia, CA, USA). The libraries were generated with TruSeq® DNA PCR-Free sample preparation kit (Illumina, Inc., San Diego, CA, USA), and analyzed by HiSeq2500 PE250 sequencing system (Illumina, Inc., San Diego, CA, USA), according to the manufacturer’s instructions. Negative controls (sterile water) were carried out through amplification and sequencing. Data was returned as fastq files and deposited in the Sequence Read Archive of the National Center for Biotechnology Information under BioProject accession number PRJNA530863 (SRA: SRP190963).
3
Protein Extraction from Microbial Cells
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Two mL of exponentially growing cells were harvested by vacuum filtration (cellulose nitrate membrane filters, 0.2 µm pore size, 47 mm, Sartorius, Göttingen, Germany). The filter was washed with 15 mL of 2.5% NaCl, matching the ionic strength of the medium, and was then transferred into a plastic cup, containing 2 mL of boiling deionized water [57 (link)]. The sample was incubated in a water bath at 100 °C for 15 min and then chilled on ice. The extract was transferred into a fresh vial and clarified from cell debris (5 min, 13,000× g, and 4 °C).
4
Quantification of Cell Growth and PHB
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Cell growth was monitored by the CDM, which was determined by filtering 5 mL of the culture broth through pre-weighed cellulose nitrate membrane filters (pore size 0.22 μm; Sartorius, Göttingen, Germany). The filters were dried at 80°C for 2 days and stored in desiccators. The net biomass was defined as the residual cell mass (RCM), which was calculated by subtracting the amount of PHB from the CDM. The PHB in dried cells was methyl-esterified using a mixture of chloroform and 3% (v/v) methanol-sulfuric acid (1:1 v/v) (Braunegg et al., 1978 (link)). The resulting monomeric methyl esters were quantified by a gas chromatograph (model CP3800, Varian Inc., Walnut Creek, CA, United States) using a Carbowax-PEG capillary column (0.25-μm df, 0.25-mm ID, 30 m length, Agilent Technologies, Inc., Santa Clara, CA, United States). The internal standard was benzoic acid, and the external standard was PHB (Sigma-Aldrich Corp., St. Louis, MO, United States). The total reducing sugar concentration was determined using a 3,5-dinitrosalicylic acid (DNS) assay (Miller, 1959 (link)). The NH4+ concentration in the culture medium was determined using a colorimetric assay (Kemper, 1974 (link)).
5
Isolation of Legionella spp. from Water
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Isolation of Legionella spp. was done as follows: Sterile water samples were used as negative control whereas sterile water samples inoculated with Legionella ATCC33152 (104–105 cfu/ml) were used as positive controls. Water samples of 1000 ml volume were passed through the cellulose nitrate membrane filters (Sartorius AG, Germany) with a pore size of 0.45 μm. Membranes were fragmented into small pieces while they were poured into 100 ml sterile plastic beaker with 50 ml original filtered water. This beaker was placed on a shaker at 37 °C for 30 min for releasing bacteria from the filter into the water. One ml was taken from each container and heat-treated at 50 °C for 30 min to inactivate the microorganisms other than Legionella spp. (16 (link)). Afterwards, 100 μl of each water sample was inoculated on a BCYE medium (Difco, USA) supplemented with glycine, vancomycin, cycloheximide and polymyxin B (GVPC). The plates were incubated under microaerophillic condition at 35 °C (90% humidity, 3% CO2) for 7 days (16 (link)).
6
Coking Wastewater Bioremediation Protocol
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Industrial wastewater from a coal coking factory in Egypt (sample A) was used in this study. The samples were collected from the biological wastewater treatment plant at the factory. Other water samples (13 samples, named as B–N) were also collected from a photo-bioreactor using an algal-bacterial system to treat coking wastewater.
The sufficient volume of the samples was calculated according to a predetermined relationship between the dry weight of biomass and the sample volume to reach a final biomass dry weight of 1.7 mg for the DNA extraction. These volumes were immediately filtered using a sterile filtration unit (Glassco®, India) and vacuum pump (Pro-set, CPS®, Germany). The samples were filtered using sterile 0.2 μm pore size cellulose nitrate membrane filters (47 mm diameter, Sartorius®, Germany). The membrane filters were then stored frozen at –20 °C till DNA extraction.
The sufficient volume of the samples was calculated according to a predetermined relationship between the dry weight of biomass and the sample volume to reach a final biomass dry weight of 1.7 mg for the DNA extraction. These volumes were immediately filtered using a sterile filtration unit (Glassco®, India) and vacuum pump (Pro-set, CPS®, Germany). The samples were filtered using sterile 0.2 μm pore size cellulose nitrate membrane filters (47 mm diameter, Sartorius®, Germany). The membrane filters were then stored frozen at –20 °C till DNA extraction.
7
Formulation and Characterization of Azithromycin Nanocarriers
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Soy lecithin (Lipoid S75) and dipalmitoylphosphatidylcholine (DPPC) were gifts from Lipoid GmbH (Germany). Azithromycin (AZT) in the form of dihydrate was generously donated by PLIVA Croatia Ltd. (Croatia). Sodium deoxycholate (SDCh), dimethyldioctadecylammonium bromide (DODAB), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (USA). All organic solvents (methanol, ethanol, and acetonitrile) were of HPLC grade, purchased from BDH Prolabo (UK). Müller--Hinton broth (MHB), Müller-Hinton agar (MHA), and tryptic soy broth (TSB) were obtained from Merck (Germany). HMW chitosan was a product of Fluka Chemie GmbH (Switzerland). All other chemicals or solvents used in this study were of analytical grade and purchased from Kemika (Croatia) or Sigma-Aldrich (USA). Phosphate buffered saline (PBS, 0.01 mol mL -1 ) was prepared by dissolving KH 2 PO 4 (1.3609 g) in 1000 mL of purified water, whereas an appropriate pH of 7.5 was adjusted by adding 10 mol L -1 KOH. PBS was filtered through cellulose nitrate membrane filters (0.45 µm pore size) purchased from Sartorius AG (Germany). Commercially available phosphate buffer with pH 7.4 (Gibco, Thermo Fisher Scientific, UK) was used for in vitro antimicrobial assays.
8
HPLC Quantification of AZT
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Quantification of AZT was performed by HPLC. The chromatographic system was composed of a Shimadzu (Milan, Italy) LC-10ATVP chromatographic pump and a Shimadzu SPD-10AVP UV-vis detector set at 215 nm. Separation was obtained on a Phenomenex (Torrance, CA, USA) Kinetex (150 mm × 4.6 mm I.D., 5 mm) coupled to a Phenomenex (Torrance, CA, USA) Security Guard C18 guard cartridge (4 mm x 3.0 mm I.D., 5 mm). A phosphate-buffered saline was prepared by dissolving KH2PO4 in purified water (0.01 M), and the pH was adjusted to 7.5 by adding 10M KOH. The buffer was then filtered through cellulose nitrate membrane filters (0.45 μm pore size; Sartorius AG, Göttingen, Germany). The mobile phase was composed of a mixture of phosphate-buffered saline at pH 7.5, methanol and acetonitrile (10/50/40, v/v). The flow rate was 0.8 mL/min. Solutions of AZT in methanol at drug concentrations ranging from 13 μg/mL to 400 μg/mL were used to construct a standard curve (R2 = 0.9964). For the evaluation of drug diffused through the skin, another calibration curve of AZT in PBS pH 7.4/ ethanol (80:20 v/v) was obtained, with drug concentrations ranging from 0.5 μg/mL to 50 μg/mL (R2 = 0.9943).
9
Enumeration of ESBL E. coli in Water Samples
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A standard plate count method on CHROMagar ESBL (CHROMagar, Paris, France) plates was used for the enumeration of ESBL E. coli in all water samples. Depending on the expected bacterial concentration, serial decimal dilutions were prepared in sterile 0.2% buffered peptone water (BPW, Scharlab, Barcelona, Spain) and, subsequently, spread plating (0.1 mL), pour plating (1 mL) or membrane filtration (10 and 100 mL) methods were used. Samples were filtered through 0.45 μm cellulose nitrate membrane filters (Sartorius, Madrid, Spain) using a filter holder manifold (Millipore, Madrid, Spain). Plates were then incubated for 24 h at 37 °C, and dark pink-reddish colonies were counted. The analysis was performed in duplicate, and the results were expressed as cfu/100 mL. The detection limit (LOD) for ESBL E. coli counts in the raw water samples was 3.0 log cfu/100 mL (100 cfu/100 mL), while in the tertiary effluents, the LOD was 0 log cfu/100 mL (1 cfu/100 mL). When possible, five dark pink-reddish colonies on E. coli ESBL agar were picked from each positive sample and sub-cultured in brain infusion (BHI) at 37 °C for 24 h. After incubation, 1 mL of each culture was supplemented with 30% glycerol and kept at −20 °C until further analysis.
10
Artificial Urine Medium for Proteus mirabilis
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Owing to urease activity, P. mirabilis produces crystalline biofilm in the presence of urinary components such as urea, chloride, sodium, potassium, and other dissolved ions, and organic and inorganic compounds. For this study, artificial urine (AU) media was prepared in accordance with the procedure described by Durgadevi et al. (2019) (link) with minor changes. Briefly, 0.05% calcium chloride, 0.07% magnesium chloride, 0.2% sodium disulfate, 0.5% sodium chloride, 0.002% sodium oxalate, 0.06% trisodium citrate, 0.1% ammonium chloride, 0.3% potassium dihydrogen orthophosphate, 0.2% potassium chloride, and 2.5% urea were dissolved in sterile distilled water. Then, the pH of the prepared AU media was adjusted to 6.2 ± 0.2 and sterilized using a cellulose nitrate membrane filter (0.2 μm; Sartorius, United Kingdom). Finally, the filtered AU medium was aseptically pooled with equal volume of sterile 2 × LB medium for experimental purposes.
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