5, 100 mM NaCl, 10 mM MgSO4 and 0.01% gelatin solution). Contaminated bacterial DNA was eliminated by DNase I. Phage nucleic
acids were extracted with the phenol : chloroform method and dissolved in TE buffer (Sambrook & Russell, 2001). The types of nucleic acids were identified by agarose gel electrophoresis and treated with DNase or RNase enzymes. For double-stranded DNA, 1 μg of DNA was digested with BamHI, HindIII, EcoRI, PstI or XhoI restriction endonucleases using the manufacturer’s recommended conditions HCS assay (Promega, Wisconsin) and the patterns were observed by agarose gel electrophoresis. The MOI that gave the highest phage titer was determined with some modification (Lu et al., 2003). Burkholderia pseudomallei P37 at mid-log phase was transfected with a selected phage at three different MOIs, 0.01, 0.1 and 1 PFU CFU−1. Phage titers were determined by the drop plate method. Phage-free cultures containing only bacteria and bacterial-cell-free cultures containing only phages were used as controls in all experiments. All assays were performed
in duplicate. The bacterial challenge test was performed by growing B. pseudomallei Ibrutinib clinical trial P37 in 300 mL nutrient broth in the presence of 3.6 mM CaCl2 at 37 °C and the phage solution was added at 5 h after inoculation with 0.1 MOI at the mid-log phase (O’Flynn et al., 2004). The numbers of bacteria were measured every hour for 16 h using OD550 nm and the plate count technique in triplicate. The phage that could lyse a broader range of B. pseudomallei, but not other related bacteria except B. mallei, and also provide a high titer was selected to study using the one-step growth method to obtain the latent period,
the eclipse period and the burst size (Pajunen et al., 2000). Ten milliliters of a mid-log phase of B. pseudomallei P37 was centrifuged and resuspended in a 0.25 volume of fresh nutrient broth/CaCl2 (c. 109 CFU mL−1). The phage at an MOI of 0.0005 was added and allowed to adsorb for 5 min at 37 °C and then centrifuged at 10 000 g Isoconazole for 5 min. The pellet was resuspended in 10 mL nutrient broth and incubated at 37 °C for 2 h. Three hundred microliters of culture were taken at 10-min intervals, half of which was treated with 1% (v/v) chloroform to release the intracellular phage and plated on the bacterial lawn for the latent period determination. The other half was immediately diluted and plated to measure the phage titer. The experiment was repeated three times. Twenty-one soil samples from 140 tested that yielded positive plaques on B. pseudomallei P37 lawn (plaque sizes were approximately 0.1–1.0 mm in diameter) were chosen for repropagation. After repropagation, only six isolates, named ST2, ST7, ST70, ST79, ST88 and ST96, clearly lysed the bacterium in liquid culture.