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Updated: Jun 21, 2026
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Published on: July 21, 2023
The use of antibiotics to improve phage detection and enumeration by the double-layer agar technique
Sílvio B Santos1, Carla M Carvalho, Sanna Sillankorva
1Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4700-057 Braga, Portugal. silviosantos@deb.uminho.pt
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.
Insights
This study enhances phage enumeration using the Double-Layer Agar (DLA) technique by incorporating antibiotics. Certain antibiotics significantly increase phage plaque size, improving detection and counting accuracy for difficult-to-observe plaques.
Area of Science:
- Microbiology
- Virology
- Bacteriology
Background:
- The Double-Layer Agar (DLA) technique is standard for phage research, but challenges arise with small, turbid plaques.
- Improving plaque visibility is crucial for accurate phage enumeration and characterization.
Purpose of the Study:
- To modify the DLA technique for enhanced phage plaque detection.
- To investigate the use of antibiotics to improve plaque morphology and enumeration.
Main Methods:
- Tested various antibiotics for their effect on phage plaque morphology.
- Assessed antibiotics' ability to enhance plaque size without inhibiting phage development.
Main Results:
- Some antibiotics significantly increased phage plaque surface area, up to 50-fold.
- The tested antibiotics did not suppress phage development.
Conclusions:
- A modified DLA technique using antibiotics improves phage enumeration accuracy.
- This method facilitates the detection and counting of phages with small or turbid plaques.
