The Wellcome strain of FCoV,13 at a titre of 1.5 × 106/ml was used and 3 ml put onto 1 g of each cat litter in a tube. The tubes were rotated at room temperature for 2 h. After spinning at 8316 g for 10 mins, the supernatant was spun in a microfuge at 18,078 g for 5 mins to remove any remaining cat litter. The supernatant was filtered through a 0.45 µm filter (Pall Life Sciences) and 10-fold dilutions were made. Two-hundred µl of each dilution was applied to a well of feline embryo A cells14 (link) plated the previous day at 2 × 105 cells per well of a 12 well plate and cultured overnight at 37°C. Inoculae were left in contact for 4 h then removed and replaced with Dulbecco’s cell culture medium (Gibco Thermo Fisher Scientific) with 10% fetal bovine serum (PAA Laboratories). The cells were then placed at 37ºC and examined after 48 h for evidence of cytopathic effect. Supernatant from each cat litter was titrated in 10-fold dilutions in triplicate. Three millilitres of medium without FCoV were put onto 1 g of cat litter and treated as described above to control for a possible cytotoxic effect of the cat litter. Virus titres were assessed by the 50% end-point calculated according to the method of Ramakrishnan.15 (link)
0.45 m filter
Manufactured by Pall Corporation
Sourced in United States
The 0.45 µm filter is a laboratory filtration product manufactured by Pall Corporation. It is designed to remove particles larger than 0.45 micrometers (µm) from liquid samples. The filter is made of materials suitable for laboratory use and can be used in various filtration applications where precise particle removal is required.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
0.22 m filter, by Sarstedt (1 mentions)
Amicon centrifugal filter, by Merck Group (1 mentions)
Tryptic soy broth (tsb), by BD (1 mentions)
Toc analyzer, by Shimadzu (1 mentions)
Ph meter, by Hanna Instruments (1 mentions)
Icp ms, by PerkinElmer (1 mentions)
5 protocols using 0.45 m filter
1
Feline Coronavirus Titration in Cat Litter
2
Neem Leaf Powder Extraction Protocol
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Dried Neem leaf powder (Lot# PRM03042104PD) was obtained from Premier Herbal Inc. (Toronto, ON, Canada). The leaf powder was extracted in either boiled double distilled water (ddH 2 O) or 100% anhydrous ethanol in a 1:10 ratio (1 g leaf powder to 10 mL anhydrous ethanol or ddH 2 O). The ethanol extraction was carried out on a shaker at room temperature overnight, while the water extraction was completed in three hours on low heat. The water extract then undergoes gravity filtration with a P8 coarse filter, followed by vacuum filtration with a 0.45 µm filter (PALL Life Sciences, VWR, Mississauga, ON. CA. Cat. No. 28148-028). The solvent from the ethanolic extract was evaporated using a RotorVap at 40°C and reconstituted in dimethylsulfoxide (Me 2 SO) at a final stock concentration of 500 mg/ml. The extract was then passed through an Acrodisc® 0.2µm DMSO-safe syringe filter (PALL Life Sciences, VWR, Mississauga ON, CA. Cat No. 28144-050) in a biosafety cabinet. For the water extract, the extract is frozen at -80˚C, freeze dried using a lyophilizer and then reconstituted in ddH 2 O for a final stock concentration of 100 mg/mL. Prior to use, the water extract is passed through a 0.22 µm filter (Sarstedt, Montreal, QC, CA Cat No. 83.1826.001) in a biosafety cabinet.
3
Isolation of Extracellular Proteome from Trophozoites
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Both the attenuated and virulent cultures were harvested upon reaching 70% confluency (approximately two weeks of culturing). Trophozoites were mechanically scraped from several MYA plates and pooled into 50 mL falcon tubes. The trophozoites were centrifuged at 1000× g for 10 min at 4 °C. The supernatant of the cultures was aspirated into fresh 50 mL falcon tubes followed by centrifugation at 10,000× g for 10 min at 4 °C to further remove bacterial and amoebae debris. After centrifugation, the supernatant was passed through a 0.45 µm filter (Pall Corporation, Port Washington, NY, USA) and subsequently a 0.25 µm filter (Pall Corporation) to obtain a cell free filtrate containing the putative extracellular proteome or exoproteome. The filtrate was concentrated 10-fold using Amicon centrifugal filters with a 3 kDa molecular-weight cut off (Millipore Corp, Watford, UK). Protein quantification was measured with the BCA assay (Pierce™Thermofisher Scientific, Waltham, MA, USA).
4
Sewage Water Phage Isolation
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In total, 3 mL of sewage water was mixed with 500 µL of bacterial culture (108 colony forming units (CFU)/mL) and 2 mL of trypticase soy broth (TSB, Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The whole mixture was incubated for 24 h, and the next day, the mixture was centrifuged at 7800× g for 15 min. The supernatant was collected and filtered with a 0.45 µm filter (Pall Corporation, Cortland, NY, USA). To assess phage activity (qualitatively) against the host strain, a spot assay followed by a plaque assay was used [11 (link),12 (link)].
5
Wastewater Characterization and Analysis
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Chemical oxygen demand (COD), total suspended solids (TSS), and volatile suspended solids (VSS) were determined according to Standard Methods [12] . pH was measured by a pH meter (Hanna, USA). After centrifugating sample (for feed and effluent samples) at 4,000×g for 10 min, the supernatant of the samples were taken for soluble COD (sCOD) and extracellular polymeric substances (EPS, mainly includes polysaccharides and protein) measurement. The polysaccharides and protein were analyzed according to Dubois [13] and Lowry [14] method, respectively. The sample was filtered with a 0.45 µm filter (Pall Corporation, USA), and the dissolved total organic carbon (TOC) were measured by a TOC analyzer (Shimadzu, Japan). Inorganic elements were measured using ICP-MS (Perkin-Elmer, USA). It is noted that the membrane used in this study was 0.1 µm, thus, sCOD and sTOC in the permeate were the soluble organics with sizes less than 0.1 µm.
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