The use of antibiotics to improve phage detection and enumeration by the double-layer agar technique

Abstract

Background: The Double-Layer Agar (DLA) technique is extensively used in phage research to enumerate and identify phages and to isolate mutants and new phages. Many phages form large and well-defined plaques that are easily observed so that they can be enumerated when plated by the DLA technique. However, some give rise to small and turbid plaques that are very difficult to detect and count. To overcome these problems, some authors have suggested the use of dyes to improve the contrast between the plaques and the turbid host lawns. It has been reported that some antibiotics stimulate bacteria to produce phages, resulting in an increase in final titer. Thus, antibiotics might contribute to increasing plaque size in solid media.

Results: Antibiotics with different mechanisms of action were tested for their ability to enhance plaque morphology without suppressing phage development. Some antibiotics increased the phage plaque surface by up to 50-fold.

Conclusion: This work presents a modification of the DLA technique that can be used routinely in the laboratory, leading to a more accurate enumeration of phages that would be difficult or even impossible otherwise.

Figure 1

Plaques of phi PVP-SE1 obtained by classical DLA technique.

Figure 2

Heterogeneous phi PVP-SE1 plaque increase with 2 mg/l ampicillin added to the top layer. A and B – different plates with 2 mg/l ampicillin.

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.

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.

Figure 5

Optimized conditions 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.

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 0.06 mg/l cefotaxime and 5% glycerol; D – PAMA with 1.5 mg/l tetracycline and 5% glycerol.

Figure 8

Influence of PAMA on phi IBB-PF7A phage plaques. A – Classical DLA; B – PAMA with 0.5 mg/l ampicillin and 5% glycerol; C – PAMA with 0.06 mg/l cefotaxime and 5% glycerol; D – PAMA with 1.5 mg/l tetracycline and 5% glycerol.

Figure 9

Influence of PAMA on phi IBB-SL58B phage plaques. A – Classical DLA; B – PAMA with 0.5 mg/l ampicillin and 5% glycerol; C – PAMA with 100 mg/l ampicillin and 5% glycerol.

Figure 10

Microscopic observation of phage phi PVP-SE1 host (S1400/94). A – LB only; B – LB with 0.5 mg/l ampicillin; C – LB with 0.06 mg/l cefotaxime; D – LB with 1.5 mg/l tetracycline; E – LB and 5% glycerol; F – LB with 0.5 mg/l ampicillin and 5% lglycerol; G – LB with 0.06 mg/l cefotaxime and 5% l glycerol; H – LB with 1.5 mg/l tetracycline and 5% glycerol.