maltophilia (30°C), Escherichia coli (37°C), Serratia marcescens (37°C), Enterobacter cloacae (37°C), Klebsiella pneumoniae (37°C), Proteus mirabilis (37°C), Pseudomonas aeruginosa (37°C), and Xanthomonas strains (28°C). Spot test, isolation of bacteriophage 3-Methyladenine supplier and plaque assay To detect the presence of phage in the culture supernatants and the phage sensitivity of a bacterium, spot tests were performed as described previously , except that LB broth and LB agar plates were used. The top agar containing the clearing zones was picked and soaked for 30 min in 100 μl of LB broth. Following appropriate dilution, the suspensions were plated for single plaque formation. Two more rounds of single-plaque isolation were performed
to obtain the pure phage culture. To AZD6738 research buy determine the phage titers, double-layered bioassays were performed on LB agar plates in which the top and bottom layers contained 0.75% and 1.5% agar, respectively. One-tenth of a milliliter each of a phage suspension after serial dilutions and cells of S. maltophilia strain from an overnight culture were mixed with 3 ml of molten soft agar and poured onto the bottom solidified agar (12 ml). Numbers of plaques were counted after the plates were incubated overnight. The same method was used to confirm phage susceptibility with the cells of different bacteria as the indicator hosts. Purification
of phage particles High-titer lysates of Smp131 (400 ml, approximately 1.0 × 1010 PFU/ml) were Alvespimycin datasheet centrifuged (10,000 × g,
20 min at 4°C). The supernatants were passed through a membrane filter (0.45 μm selleck chemicals llc pore size) and then centrifuged (15,000 × g at 4°C) for 2 hr. The phage pellets were suspended in 1.0 ml of the SM buffer (50 mM Tris–HCl, pH 7.5, containing 100 mM NaCl, 10 mM MgSO4, and 0.01% gelatin) and loaded on the block gradient of CsCl (1.2, 1.35, 1.45, 1.50, and 1.70 g/ml), followed by ultracentrifugation (28,000 rpm for 2 h at 4°C) with rotor TH641 (Sorvall OTD Combi) . The phage particles concentrated into a zone were recovered and dialyzed against the SM buffer. DNA techniques Phage particles purified following ultracentrifugation were treated with sodium dodecyl sulfate (SDS, 1%) and 20 U of proteinase K (Sigma P-2308) at 58°C for 1 h. An equal volume of phenol/chloroform (1:1) was then added to remove the proteinaceous materials. Phenol/chloroform extraction was repeated twice and the DNA was precipitated as described previously . Restriction enzyme digestion of the phage DNA was performed in accordance with supplier instructions. DNA fragments were separated in 0.7% agarose gels in a TAE buffer (40 mM Tris acetate, pH, 8.0, containing 2 mM EDTA). Isolation of DNA fragments from agarose gel was performed using commercial kits (Qiagen). Standard protocols were followed for blotting DNA fragments onto the membrane (NEN catalog number NEF988), preparation of probes by labeling with [α-32P] dCTP (Du Pont. NEN), and Southern hybridization.