The bacteriophages used in this study and their sources are listed in S12 Table . All phages except P2 phage and N4 phage were propagated on E. coli BW25113 strain. To propagate P2 phage and N4 phage, we used E. coli C and E. coli W3350 strains, respectively. We used a host-range mutant of T3 (from our in-house phage stock that can grow on both E. coli K-12 and BL21 strains), mutant λ phage (temperature-sensitive mutant allele cI857) [9 (link),249 ], and a strictly virulent strain of P1 phage (P1vir) [250 (link)] that favors a lytic phage growth cycle. We followed standard protocols for propagating phages [231 ]. Phage titer was estimated by spotting 2 μl of a 10-fold serial dilution of each phage in SM buffer (Teknova) on a lawn of E. coli BW25113 via top agar overlay method using 0.7% LB agar. SM buffer was supplemented with 10 mM calcium chloride and magnesium sulphate (Sigma). We routinely stored phages as filter-sterilized (0.22 μm) lysates at 4 °C.
Sm buffer
Manufactured by Merck Group
Sourced in United States
SM buffer is a laboratory reagent used to maintain the stability and integrity of biological samples during analytical procedures. It provides a suitable pH environment and ionic conditions to preserve the natural state of the sample. The core function of SM buffer is to facilitate consistent and reliable sample preparation for various analytical applications.
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Lab products found in correlation
5 protocols using sm buffer
1
Bacteriophage Propagation and Titration
2
Immunogold Electron Microscopy of Bacteriophages
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Immunogold electron microscopy was performed as described previously with polyclonal antibodies directed against Phi15-NPS, TpeX or phage p2 MTP48 (link) developed by Davids Biotechnologie (Regensburg, Germany) using their standard protocol. Purified bacteriophage suspensions obtained from CsCl were dialysed against SM buffer (50 mM Tris-HCL, 100 mM NaCl and 8 mM MgSO4, pH 6.5, Sigma). Staining was performed with 2% (w/v) uranyl acetate on freshly prepared carbon films. Grids were analysed in a Tecnai 10 transmission electron microscope (TEM) (FEI Company, Eindhoven, The Netherlands, OR, USA) at an acceleration voltage of 80 kV. Micrographs were taken with a MegaView II charge-coupled device camera at the Max Rubner-Institut, Kiel, Germany48 (link)49 (link)50 (link)51 (link)52 . Phage proteins, from CsCl-purified phage particles, were separated on a 12% SDS-PAGE gel. Western hybridisation was performed as outlined previously45 (link)46 (link) using polyclonal antibodies raised against Phi15-NPS, TpeX or phage p2 MTP.
3
Phage Stability Under pH and Temperature
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To determine phage stability under various pH conditions, the sodium chloride-magnesium sulfate (SM) buffer (100 mM NaCl, 10 mM MgSO4 7H2O, and 50 mM Tris HCl, pH 7.5; Sigma, St. Louis, MO) pH was adjusted with 4 N HCl or 2 N NaOH over a pH range of 1 to 12, and the phage HY01 was added at a final concentration of 109 PFU/ml. After incubation at 37°C for 12 h, the phage suspensions were neutralized, and phage titers were determined using plaque assays with E. coli O157:H7 ATCC 43890 as a reference strain. To determine phage stability under various temperature conditions, the phage HY01 (final concentration, 109 PFU/ml) was added to SM buffer and incubated at -20, 20, 30, 37, 40, 50, 60, 65, and 70°C for 12 h. After incubation, phage titers were determined with plaque assays using the same reference strain [13 (link)].
4
Phage KN4-1 Infection Kinetics
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A total of 5 × 103 pfu of phage KN4‐1 was mixed with 5 × 106 cfu of exponential phase culture of 1461 or 4565 strain individually. The culture was continuously incubated at 37°C. Samples were taken at 0.5, 1, 3 and 22 h. After adding CHCl3 (Sigma‐Aldrich, St Louis, MO) to a final concentration of 2% and shaking for 5 min (treated with CHCl3, both intracellular and free phages were determined), the culture was appropriately diluted (or undiluted) with SM buffer, containing 100 mM NaCl, 8 mM MgSO4·7H2O and 50 mM Tris (pH 7.5) (Sigma‐Aldrich, St Louis, MO). For plating, 100 μl of phage suspension was mixed with 100 μl of exponential phase culture of 4565 strain (we use 4565 but not 1461 because phage numbers can be estimated by calculating the visible plaques on 4565). After incubation at 37°C for 10 min, 4 ml 0.7% top agar was added. The mixture was lightly vortexed, poured on a LB agar and incubated at 37°C for 18 h. Phage titres at each time point were determined by plaque counting.
5
Isolation and Characterization of KPC-producing K. pneumoniae
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A total of nine K. pneumoniae strains were isolated from two elderly patients (>60 years) with urinary tract infections (UTI) at Shanghai Public Health Clinical Center, Shanghai, China. Phage 117 and phage 31 used in this study were kindly provided by the Chen lab (Hualien Hospital, Taiwan). All of the strains were stored at –80 °C in Luria Broth (LB) medium (10 g tryptone, 5 g yeast extract, and 10 g NaCl per liter) (Sigma-Aldrich, St. Louis, MO, USA) with sterile glycerol (25%, vol/vol). The blaKPC gene was detected by PCR and sequencing with K. pneumoniae carbapenemase (KPC) primers KPC-F (5′-TGTAAGTTACCGCGCTGAGG-3′) and KPC-R (5′-CCAGACGACGGCATAGTCAT-3′) [14 (link)].
Proliferation of phage 117 and phage 31 was performed by using the double-layer agar method [15 ]. To obtain high-titer phage stocks, 5 mL SM buffer (50 mM Tris-Cl, pH 7.5, 99 mM NaCl, 8 mM MgSO4, 0.01% gelatin, Sigma-Aldrich, St. Louis, MO, USA) was added to a fully lysed plate (90 × 15 mm), and the top agar (5 g/L) overlay was shredded with a sterilized inoculation loop. In order to release phage particles, the mixture was collected in a conical tube, stirred gently for at least 2 h at room temperature (RT) and then centrifuged at 12,000× g for 10 min at 4 °C. The supernatant was filtrated through 0.22 μm syringe filter (Millipore, Billerica, MA, USA), and the phage lysate was titrated and stored at 4 °C.
Proliferation of phage 117 and phage 31 was performed by using the double-layer agar method [15 ]. To obtain high-titer phage stocks, 5 mL SM buffer (50 mM Tris-Cl, pH 7.5, 99 mM NaCl, 8 mM MgSO4, 0.01% gelatin, Sigma-Aldrich, St. Louis, MO, USA) was added to a fully lysed plate (90 × 15 mm), and the top agar (5 g/L) overlay was shredded with a sterilized inoculation loop. In order to release phage particles, the mixture was collected in a conical tube, stirred gently for at least 2 h at room temperature (RT) and then centrifuged at 12,000× g for 10 min at 4 °C. The supernatant was filtrated through 0.22 μm syringe filter (Millipore, Billerica, MA, USA), and the phage lysate was titrated and stored at 4 °C.
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