Figure 3 Determination of optimal glycerol concentration using

plaques of phi PVP-SE1. A – without glycerol (0%); B – with 5% glycerol; C – with 20% glycerol; D – with 10% glycerol. The combination of glycerol and antibiotics produced larger plaques and a dramatic increase in contrast compared with the use of antibiotics alone (Figure 4). In this way, glycerol appears to act synergistically with antibiotics in improving plaque observations. Figure 4 Influence of 5% glycerol in the top layer on phi PVP-SE1 phage plaques. A – classical DLA; B – PAMA with 0.2 mg/l cefotaxime; C – as in A but with 5% glycerol; D – as in B but with 5% glycerol. The optimum antibiotic concentration should be the highest possible to produce the maximum increase in plaque size but

not so high that it inhibits bacterial lawn formation. Therefore, the effects of different antibiotic concentrations Selleck VX-680 in both layers were analyzed, and the following optimal concentrations were determined: 0.5 mg/l ampicillin, PRI-724 0.06 mg/l cefotaxime and 1.5 mg/l tetracycline (Figure 5). Comparing these antibiotic concentrations with and without glycerol (Figure 6) we concluded that glycerol critically improves plaque observation, especially for tetracycline, for which both the plaque size and contrast were increased. Tetracycline was the antibiotic that induced the highest increment of phage plaque size and contrast (Table 2). Table 2 Comparison of phage phi PVP-SE1 plaque diameter with DLA and with PAMA using different antibiotics.   DLA AMP [0.5] CEF [0.06] TET [1.5] PLAQUE DIAMETER (mm) 0.47 ± 0.167 1.49 ± 0.433 1.91 ± 0.439 3.43 ± 0.398 AREA INCREASE 1 10 17 53 Values of plaque diameters are expressed in mm±standard deviation and area increase as the ratio between

the average values of each method and DLA. DLA: classical Double-Layer Agar technique; AMP [0.5]: PAMA with 0.5 mg/l ampicillin; CEF [0.06]: PAMA with 0.06 mg/l cefotaxime; TET [1.5]: PAMA with 1.5 mg/l tetracycline. Figure 5 Optimized conditions PJ34 HCl for improvement of phi PVP-SE1 plaques. Figure 6 Influence of glycerol in phage phi PVP-SE1 plaque improvement. A – with tetracycline alone at 1.5 mg/l; B – with 1.5 mg/l tetracycline and 5% glycerol. These optimized antibiotic concentrations plus glycerol (5%) were applied to three other phage-host systems to assess their ability to increase phage plaque. With phage phi PVP-SE2 only a slight increase in plaques was observed when cefotaxime and ampicillin were used, while the addition of tetracycline produced an enormous increase in phage plaque size (Figure 7). There was no significant effect on the plaquing behaviour of Pseudomonas fluorescens phage phi IBB-PF7A (Figure 8). In the case of Staphylococcus phage phi IBB-SL58B, ampicillin at 50–100 mg/l resulted in a very significant increase in plaque size (Figure 9). Figure 7 Influence of PAMA on phi PVP-SE2 phage plaques. A – Classical DLA; B – PAMA with 0.5 mg/l ampicillin and 5% glycerol; C – PAMA with 0.