Related Experiment Video
Updated: Jun 8, 2026
Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria
Published on: May 8, 2013
A single-layer spot assay for easy, fast, and high-throughput quantitation of phages against multidrug-resistant
Paschalis Paranos1, Spyros Pournaras1, Joseph Meletiadis1
1Clinical Microbiology Laboratory, Attikon University Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece.
Abstract:
Double-layer agar (DLA) overlay plaque assay is the gold standard for phage enumeration. However, it is cumbersome and time-consuming. Given the great interest in phage therapy, we explored alternative assays for phage quantitation. A total of 16 different phages belonging to Myoviridae, Siphoviridae, and Podoviridae families were quantitated with five K. pneumoniae, eight P. aeruginosa, and three A. baumannii host isolates. Phages were quantitated with the standard DLA assay (10 mL of LB soft agar 0.7% on LB hard agar 1.5%) and the new single-layer agar (SLA) assay (10 mL of LB soft agar 0.7%) with phages spread (spread) into or spotted (spot) onto soft agar. Phage concentrations with each assay were correlated with the standard assay, and the relative and absolute differences between each assay and the standard double-layer agar spread were calculated. Phage concentrations 1 × 104-8.3 x1012 PFU/mL with the standard DLA assay were quantitated with SLA-spread, SLA-spot, and DLA-spot assays, and the median (range) relative and absolute differences were <10% and <0.98 log10PFU/mL, respectively, for all phage/bacterial species (ANOVA P = 0.1-0.43), and they were highly correlated (r > 0.77, P < 0.01). Moreover, plaques could be quantified at 37°C after 4-h incubation for K. pneumoniae phages and 6-h incubation for P. aeruginosa and A. baumannii phages, and estimated concentrations remained the same over 24 hours. Compared to DLA assay, the SLA-spot assay required less media, it was 10 times faster, and generated same-day results. The SLA-spot assay was cheaper, faster, easier to perform, and generated similar phage concentrations as the standard DLA-spread assay.
Insights
A new single-layer agar spot assay offers a faster, cheaper, and easier method for phage enumeration compared to the traditional double-layer method. This rapid phage quantitation is crucial for advancing phage therapy research and applications.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- The double-layer agar (DLA) overlay plaque assay is the standard for bacteriophage enumeration but is labor-intensive and time-consuming.
- Phage therapy is gaining interest, necessitating efficient and reliable methods for phage quantitation.
- Existing methods present challenges in speed and resource requirements for widespread phage research and application.
Purpose of the Study:
- To explore and validate alternative, more efficient assays for bacteriophage quantitation.
- To compare the accuracy and efficiency of a novel single-layer agar (SLA) assay against the standard DLA assay.
- To assess the suitability of the SLA assay for enumerating diverse phages infecting key bacterial pathogens.
Main Methods:
- Quantitated 16 different phages infecting *K. pneumoniae*, *P. aeruginosa*, and *A. baumannii* using standard DLA and novel SLA assays (spread and spot methods).
- Correlated phage concentrations obtained from SLA assays with the standard DLA assay.
- Calculated relative and absolute differences and assessed correlation coefficients (r) and statistical significance (P-values).
Main Results:
- The SLA-spot assay demonstrated high correlation (r > 0.77, P < 0.01) with the standard DLA assay, with median relative differences <10%.
- Plaque quantification was achieved within 4-6 hours of incubation, with results remaining stable for 24 hours.
- The SLA-spot assay required less media, was 10 times faster, and provided same-day results compared to DLA.
Conclusions:
- The single-layer agar spot (SLA-spot) assay is a validated, rapid, cost-effective, and user-friendly alternative for phage enumeration.
- This assay generates comparable results to the gold-standard DLA assay, making it suitable for advancing phage therapy research.
- The SLA-spot assay significantly reduces time and resources, facilitating broader application of phage-based strategies.
