Determination of multiplicity of infection (MOI) Serial dilutions of bacteriophage stock solution were mixed with the same amount of A. baumannii cells. After 15 minutes adsorption,
free bacteriophages were removed by centrifugation at 5,000 g for 10 min, pellets were resuspended with LB medium, and samples were taken for bacteriophage titer analysis after 4 hours incubation at 35°C. Adsorption rate, latent period, and phage burst size As described previously [20, 21], 10 mM CaCl2 was added to the infected culture to measure divalent metal ions effects on adsorption rate of phage AB1, samples were taken at different time intervals to analyze the free phage particles in the solutions with and without addition of calcium ions. One-step growth experiment was carried out according to the previous descriptions [45, 46] to determine the latent period GF120918 clinical trial and phage burst size. In brief, 50 ml bacterial cells of A. baumannii KD311 were incubated to mid-exponential-phase (OD600 = 0.4-0.6) and harvested by centrifugation. The pellet was resuspended in 0.5 ml fresh LB medium and mixed with 0.5 ml phage AB1 solution (1 × 108 PFU/ml). Phage AB1 was allowed to adsorb for 1 min and the mixture was subjected to centrifugation immediately
at 13,000 rpm for 30 seconds to remove free phage particles. The pellet was resuspended in 100 ml fresh LB medium and the culture was continuously incubated at 35°C. Samples were taken at 3 min intervals and phage titre was determined by the double-layer-agar plate method. The results were analyzed and the constant phage titer, which represented the many number of infective centres, selleck compound along the latent stage was deduced. The burst size of phage AB1 was calculated by dividing the phage titers at plateau phase
by the number of infective centres. pH stability and thermal stability test pH stability and thermal stability tests were carried out as previously described[47, 48]. Briefly, certain amount of phage particles were treated under specified conditions. Samples were taken at different time intervals and supernatants from centrifugation were used directly in the assays. Initial phage concentration was about 3.5 × 1010 PFU/ml in LB medium. Host range determination 108 bacterial cells were mixed with melted 0.6% agar (50°C) and this mixture was poured on a 2% solid agar to make double layer agar plates. After solidification, we spotted the isolated bacteriophage stock solution on each plate with different bacterium strain and observed whether lysis plaques emerged. The susceptibility test BioMerieux Vitek 32 system (BioMerieux, Inc., USA) was used in clinical samples diagnosis for bacterial identifications and antibiotics susceptibility tests. Acknowledgements The authors thank Dr Jingfu Huang (Tianjin Children Hospital, Tianjin, China) for generously providing the bacterial strains used in this study. This study was supported by a grant (No.