Vibrio sp. V33 was grown to OD600 of 0.7, 0.8, or 0.9, measured using a UV-Vis spectrophotometer (Beckman, USA). Then, supernatants were collected by centrifuged at 12000 × g and 4 °C for 5 min and they were filtered through 0.22-μm polycarbonate membrane filters (Millipore) to prepare the cell-free supernatant. The same volumes of different cell-free supernatants were separately added into cell pellet of V. splendidus Vs to make cell suspensions of the same concentrations. V. splendidus Vs suspensions in fresh media were used as a control. After incubated for 12 h, OD600 of each culture was measured using a UV-Vis spectrophotometer.
0.22 μm polycarbonate membrane filters
Manufactured by Merck Group
Sourced in United States
The 0.22-μm polycarbonate membrane filters are laboratory equipment used for filtration purposes. These filters have a pore size of 0.22 micrometers, which allows for the efficient removal of particles and microorganisms from various liquids or solutions.
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4 protocols using 0.22 μm polycarbonate membrane filters
1
Vibrio sp. V33 Cell-free Supernatant Effect
2
Isolation and Characterization of Phage from Coastal Waters
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Host stain R. pomeroyi DSS-3 was grown in ½ YTSS medium (4 g yeast extract, 2.5 g trypone and 20 g Crystal Sea per liter) at 28 °C. Water samples, collected from Baltimore Inner Harbor Pier V in January 2012, were filtered through 0.22 μm polycarbonate membrane filters (Millipore, Bedford, MA, USA). Filtrate of 10 ml was added into 50 ml of exponentially growing bacterial cultures and incubated for two days. The DSS-3 culture was filtered through 0.22 μm polycarbonate membrane filter (Millipore, Bedford, MA, USA) to remove bacteria. 100 μl of this cell-free lysate was added to 500 μl of exponentially growing DSS-3 culture, and plated using plaque assay. Phage isolates were purified three times by plaque assay.
3
Analysis of Coastal Seawater Microbiome and Sulfur Compounds
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North Sea surface seawater was collected from Great Yarmouth coast, UK, (52° 35′ 27.5928″ N; 1°44′ 17.9268″ E) on 27th January 2018. To study the composition of the natural (T0) microbial community, 3 L of coastal seawater was filtered through 0.22-μm polycarbonate membrane filters (Millipore Corporation) using a vacuum pump. Filters were then stored at −80 °C for DNA extraction.
DMS concentrations in natural (T0) coastal seawater samples were quantified using a purge-and-trap gas chromatography (GC) system [75 (link)] using a flame photometric detector (Agilent 7890A GC fitted with a 7693 autosampler) and a HP-INNOWAX 30 m × 0.320 mm capillary column (Agilent Technologies J&W Scientific). DMSP content was quantified indirectly via alkaline lysis as previously described [76 (link)]. An eight-point calibration curve with DMS standards was made as in [76 (link)]. The detection limit for DMS in the headspace was 0.8 pmol.
DMS concentrations in natural (T0) coastal seawater samples were quantified using a purge-and-trap gas chromatography (GC) system [75 (link)] using a flame photometric detector (Agilent 7890A GC fitted with a 7693 autosampler) and a HP-INNOWAX 30 m × 0.320 mm capillary column (Agilent Technologies J&W Scientific). DMSP content was quantified indirectly via alkaline lysis as previously described [76 (link)]. An eight-point calibration curve with DMS standards was made as in [76 (link)]. The detection limit for DMS in the headspace was 0.8 pmol.
4
Seawater DNA Extraction Protocol
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Seawater samples were collected and filtered through 0.22-μm polycarbonate membrane filters (Millipore, MA, USA). The filters were stored in 5 mL of DNA storage buffer (500 mM NaCl, 50 mM Tris-HCl, 40 mM EDTA, and 50 mM glucose) at − 80 °C. The TIANamp Genomic DNA Kit (Tiangen Biotech, Beijing, China) was used to extract DNA from the seawater samples following the manufacturer’s protocol.
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