Background

Phage quality testing and analysis are essential steps in ensuring the safety, efficacy, and reliability of bacteriophages for various applications, including phage therapy, research, and industrial use. High-quality phage preparations must be free from contaminants, possess consistent biological activity, and exhibit well-characterized genetic and physical properties.

Unlike traditional biologics, phages require specialized analytical approaches to confirm purity, identity, and potency. However, many labs lack the experience or infrastructure to perform this rigorously—leading to inconsistent results, regulatory setbacks, or ineffective formulations. Key aspects of phage quality testing include:

Purity Assessment

• Plaque Assay: This method quantifies phage titers by counting plaque-forming units (PFU) on bacterial lawns, providing a measure of phage concentration and infectivity.
• Endotoxin Testing: Ensuring low levels of endotoxins is crucial, as these bacterial contaminants can cause adverse reactions. Endotoxin levels are typically measured using the Limulus Amebocyte Lysate (LAL) assay.
• Protein Analysis: SDS-PAGE and Western blotting are used to verify the presence of phage structural proteins and assess the purity of phage preparations.

Potency Evaluation

• Host Range Testing: Determining the spectrum of bacterial strains that a phage can infect is essential for therapeutic applications. This involves testing phage activity against a panel of bacterial isolates.
• Lytic Activity: The ability of phages to lyse bacterial cells is evaluated through assays that measure bacterial lysis kinetics and efficiency.

Morphological and Structural Analysis

• Transmission Electron Microscopy (TEM): This technique provides detailed images of phage particles, confirming their morphology and structural integrity.
• Genomic Analysis: Sequencing of phage genomes ensures the absence of unwanted genetic elements and confirms the identity of the phage.

Creative BioMart Microbe offers a broad suite of analytical assays that assess key quality attributes of your phage preparation, whether for in vitro use, in vivo testing, or downstream commercialization. Contact us today to discuss your phage quality analysis project!

Service Procedure

Service Details

Sample Output

• Comprehensive Quality Certificate Report
• Morphology images (TEM or AFM, if applicable)
• Sequencing files and annotation summary
• Purity and endotoxin levels

Our Advantages

• Comprehensive Testing Portfolio: Full suite of physical, microbiological, and molecular assays to characterize all key attributes of phage quality.
• Customizable and Scalable Services: Modular testing options tailored to early-stage research, preclinical development, or regulatory submission requirements.
• Regulatory-Ready Reporting Format: Test reports are structured for compatibility with academic, clinical, and regulatory documentation standards.
• High Sensitivity and Specificity: Utilization of qPCR/ddPCR and LAL-based methods ensures sensitive detection of contaminants and precise quantification of phage components.
• Experienced Team and Technical Expertise: Led by virologists and microbiologists with extensive experience in phage characterization, ensuring high analytical reliability and scientific rigor.

Case Study

Case Study 1: Rapid single-layer spot assay for phage quantitation.

The study evaluated alternative methods to the traditional double-layer agar (DLA) plaque assay for quantifying bacteriophages, which is accurate but time-consuming. Researchers tested a simpler single-layer agar (SLA) assay—using both spot and spread techniques—on 16 phages across multiple bacterial hosts. Results showed that the SLA methods produced comparable phage counts to the DLA assay, with high correlation and minimal variation. The SLA-spot assay, in particular, was faster, cheaper, and easier, delivering accurate same-day results with significantly less labor and materials.

Figure 1. Pearson's correlation analysis comparing DLA-spread assay as a reference method with other assays tested in the present study for K. pneumoniae, P. aeruginosa, and A. baumannii isolates. For K. pneumoniae (upper left), all assays were highly correlated with the standard DLA-spread assay. Similarly, for P. aeruginosa (upper right) and A. baumannii (middle), all assays were highly correlated with the standard DLA-spread assay. The blue dots represent the SLA-spread method, the green dots are DLA-spot, and the red dots are the SLA-spot assay. (Paranos et al., 2024)

Case Study 2: Metagenomic analysis reveals temperate phage and bacterial DNA contamination.

Widespread antibiotic resistance has renewed interest in bacteriophage therapy, but concerns remain about the quality of commercial phage cocktails. This study reanalyzed published metagenomic data using updated viromics tools and found evidence of temperate phages and bacterial DNA contamination in all tested cocktails. These findings raise safety concerns, particularly regarding horizontal gene transfer, and highlight the urgent need for stricter quality control before therapeutic use of phage products.

Figure 2. Comparative genomic map of four temperate contigs identified in cocktail sequences with the most similar sequences 143 found in the NT database. The minimum identity of connections between genes is 0.6. Lysogeny-related genes are labeled. (Peng et al., 2024)

FAQs

References:

1. Paranos P, Pournaras S, Meletiadis J. A single-layer spot assay for easy, fast, and high-throughput quantitation of phages against multidrug-resistant Gram-negative pathogens. Simner PJ, ed. J Clin Microbiol. 2024;62(8):e00743-24. doi:10.1128/jcm.00743-24
2. Peng X, Smith SE, Huang W, Ru J, Mirzaei MK, Deng L. Metagenomic analyses of single phages and phage cocktails show instances of contamination with temperate phages and bacterial DNA. Published online September 12, 2024. doi:10.1101/2024.09.12.612727