Streamlining phage enumeration: A glass drop plate approach for lytic Pseudomonas bacteriophage titration

Abstract

Due to its reliability, the double-layer agar (DLA) method is widely used for quantifying bacteriophages; however, it is time-consuming, labor-intensive, and requires careful optimization. This study developed a glass drop plate (GDP) method for rapid phage titration, capable of delivering results within 3 h while allowing nine spots per plate for testing multiple dilutions and triplicates. The GDP method produced phage titers comparable to those obtained using the DLA technique, with consistent results across three phages: SPA01 (2.20 × 10¹⁰ vs. 3.00 × 10¹⁰ PFU/mL), SPA05 (1.50 × 10⁹ vs. 1.93 × 10⁹ PFU/mL), and JJ01 (2.60 × 10¹⁰ vs. 7.67 × 10¹⁰ PFU/mL). Notably, the method replaces disposable plastic tips with reusable glass pipettes, offering an eco-friendly and sustainable alternative. This simple, cost-effective approach reduces material consumption and supports high-throughput screening, making it well-suited for rapid phage quantification in environmental, industrial, and clinical applications.