At time 0, 5 and 15 min, 300 μl of the culture was withdrawn and immediately filtered on a 0.2
μm 96-well filter plate (Pall, East Hills, NY). The CBL-0137 purchase number of free phages in each sample was then determined by plating. Six replicates were performed for each phage strain. An exponential function of y = be -at , where a and b are the parameters to be estimated, and t the time, was used to fit the data from individual experiments. The adsorption rate was obtained by dividing each of the estimated parameter a with its corresponding cell concentration. For more detail on how the adsorption rates were calculated, please see Additional file 3. Determination of plaque size For each phage strain, images of four to five plates with phage plaques were taken with Qcount (Spiral Biotech, Inc.; Norwood, MA) and then analyzed using
the ImageJ software (NIH). To convert the pixel count to surface area, we arbitrarily generated a computer printout with a known surface area and used it as the size standard. In this study, we found that 1 pixel = 0.01588 mm2. Besides the phage traits, many other factors may also influence the plaque size. Several precautions were taken to minimize potential unintended effects. For example, to minimize plaque variation due to plating conditions [12], the plating conditions were standardized and only freshly prepared plates were used (see above). To reduce variation due learn more to the timing of the formation of the initial attachments of phage particles, adequate amount of pre-adsorption time and high host concentrations (see above) were used to synchronize the timing of the formation of the initial infection centers before plating. This practice is especially critical for phages with low adsorption rates. To reduce the incidence of fusion of two nearby plaques, thus being measured as one large plaque, the Amino acid number of phages on each plate was kept below 100. However, other factors, such as the edge effect
(plaques on the edge of the plate were usually smaller), were unable to be controlled. Therefore, to further minimize potential skewing effects, plaque size distributions obtained from the four to five replicated plates were pooled, and the mode, rather than the mean, was used as the descriptive measure of these distributions. The determination of plaque size was performed nine times independently. Determination of plaque productivity In order to estimate phage numbers in plaques (productivity), three random plaques from each of the four plates (used to estimate plaque size – see above) were obtained by taking agar plugs containing the plaques [17]. The 12 plaques were pooled STAT inhibitor together and then homogenized in 6 mL TB medium using a glass homogenizer with a Teflon plunger [17]. The homogenate was centrifuged for 10 min at 3000 × g (Eppendorf centrifuge 5702) at room temperature and the supernatant was then plated in triplicates at appropriate dilutions on a lawn of E.
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