The Bacteriophage Grower's Playbook

The ATCC four-day phage propagation protocol

At ATCC, broth-based phage propagation is the standard due to its scalability and efficiency. This adaptable four-day protocol offers a reliable framework for diverse host-phage systems across research and industrial applications. 

Day 1: Host cultivation
Start by growing the bacterial host under correct atmospheric conditions using suitable media. Host health is crucial—temperature, agitation, and nutrients must be optimized to ensure robust growth.

Day 2: Amplification
For optimal bacteriophage propagation, ensure your host cells are in the early log phase of growth. This stage provides actively dividing cells, which are most susceptible to bacteriophage infection and yield the highest viral output.

Day 3: Lysate prep and titration
After incubation, remove bacterial debris via vacuum filtration and perform a spot titer assay to estimate plaque-forming units (PFUs) for downstream use. Tip: Let titered plates dry before incubation to prevent spots from merging.

Day 4: Plaque check
Examine plates for individual plaques at higher dilutions. Calculate PFUs to confirm successful propagation and readiness for downstream applications. 

Decoding bacteriophage problems: From prep to plaques to purification

To optimize plaque development, evaluate key factors affecting bacteriophage infectivity, replication, and purification. Once the filtrate is concentrated, a purification step may be necessary to remove contaminants that could interfere with plaque formation. Refer to the following factors to improve plaque development.

• Host growth phase: Bacteriophages need actively dividing cells. Use a spectrophotometer to confirm OD₆₀₀ between 0.1–0.3 for early log phase. Starting with healthy, early log-phase cultures sets the foundation for consistent and efficient bacteriophage amplification. 
• Multiplicity of infection (MOI) optimization: Optimize host-to-bacteriophage ratios. Extreme MOIs can disrupt infection—high ratios overwhelm the host; low ratios may not initiate replication. Use a microplate reader to optimize MOI conditions. 
• Media composition: If plaques remain absent after determining appropriate MOI, supplement media with MgCl₂ and CaCl₂ (1–10 mM) to boost bacteriophage adsorption.
• Anaerobic host viability: To keep anaerobic bacteriophages healthy, the host bacteria must thrive in low-oxygen conditions. Use an anaerobic chamber or a sealed system and always work with pre-reduced media to maintain a stable, oxygen-free environment.
• Improving nucleic acid recovery: Polyethylene glycol (PEG) is often used as a preliminary step to concentrate bacteriophages by promoting aggregation and precipitation, enhancing lysate quality and yield before extraction.

ATCC: Supporting bacteriophage research excellence

ATCC provides reliable materials and expert guidance for bacteriophage research and development. When issues arise—like low yields or inconsistent plaques—revisit host health, media composition, and MOI. For deeper insights, watch the webinar ATCC’s Best Practices for Optimal Growth and Propagation of Bacteria and Bacteriophages, which covers techniques to improve plaque clarity and consistency. 

Explore the ATCC Genome Portal

The ATCC Genome Portal provides customers with high-quality bacteriophage genomic data for research in microbial ecology, therapeutics, and synthetic biology. Access sequences and metadata to enhance reproducibility and innovation. Notable entries include:

• Escherichia coli phage Phi X174 (ATCC®13706-B1™): The first viral genome ever sequenced, a key reference for evolutionary studies.
• Pseudomonas fluorescens phage phi-S1 (ATCC®27663-B1™): Known for strong biofilm-lytic activity and potential in antimicrobial research.

Did you know?

The ATCC collection includes over 340 bacteriophages and their host strains.

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