Phage Titer Calculators

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Phage titer measures the concentration of infectious bacteriophage particles in a sample, expressed as plaque-forming units per milliliter (PFU/mL). It is determined by plaque assay — the viral equivalent of a bacterial plate count. Serial dilutions of the phage stock are mixed with host bacteria in soft agar, poured over solid agar plates, and incubated. Each phage particle infects one bacterium, initiates the lytic cycle, and spreads to neighboring cells, creating a clear zone (plaque) in the bacterial lawn. PFU/mL = plaques / (dilution factor × volume plated in mL).

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PFU/mL Calculation

PFU/mL = plaques counted / (dilution factor × volume plated mL)

Example: 38 plaques on a 10⁻⁷ dilution plate, 0.1 mL plated: PFU/mL = 38 / (10⁻⁷ × 0.1) = 38 / 10⁻⁸ = 3.8 × 10⁹ PFU/mL.

Plaque Assay Protocol

  1. Prepare serial 10-fold dilutions of phage stock (10⁻⁵ through 10⁻⁹ or as appropriate)
  2. Mix 0.1 mL phage dilution with 0.2 mL mid-log host bacteria (e.g., E. coli)
  3. Allow 10 min adsorption at 37°C
  4. Add 3 mL of soft agar (0.7%) at 45°C; pour immediately onto solid agar plate
  5. Allow to solidify 10 min; incubate overnight at 37°C
  6. Count plaques (clear zones); use plates with 20–200 plaques

Plaque Morphology

Clear plaques: lytic phages (virulent); all bacteria in the clearing zone are lysed. Turbid plaques: temperate phages; center appears cloudy due to lysogenic bacteria surviving in the plaque. Plaque size, shape, and turbidity are phage-specific characteristics.

High-Titer Stock Preparation

Phage stocks typically 10⁹–10¹² PFU/mL. Store at 4°C in SM buffer (saline-magnesium buffer) + trace chloroform. Repeated freeze-thaw cycles reduce titer.

Glossary

PFU/mL (Plaque-Forming Units per mL)
The measure of phage titer; PFU/mL = plaques / (dilution × volume); counts only infectious particles capable of forming visible plaques in a bacterial lawn; analogous to CFU/mL for bacteria.
Plaque Assay
Method for measuring phage titer: phage dilutions mixed with host bacteria in soft agar overlay; each phage forms a visible clear zone (plaque) in the bacterial lawn after overnight incubation.
SM Buffer
Standard storage medium for bacteriophages: 100 mM NaCl, 8 mM MgSO₄, 50 mM Tris-HCl pH 7.5; maintains phage infectivity at 4°C for months to years.

Frequently Asked Questions

Phage titer = concentration of infectious phage particles, expressed as PFU/mL (plaque-forming units per milliliter). Measured by plaque assay: dilute phage serially; mix with log-phase host bacteria; embed in soft agar overlay on solid agar plates; incubate overnight; count clear plaques (zones of bacterial lysis). Each plaque represents one infectious phage particle. PFU/mL = plaques / (dilution × volume plated). Unlike bacterial CFU, PFU measures only phages capable of completing a lytic cycle — defective or non-infective phage particles are not counted.

PFU/mL = number of plaques / (dilution factor × volume plated in mL). Step 1: Identify the plate with 20–200 plaques (statistically reliable range). Step 2: Determine the dilution factor (cumulative product of all dilution steps). Step 3: Apply the formula. Example: 75 plaques on 10⁻⁸ dilution, 0.1 mL plated: PFU/mL = 75 / (10⁻⁸ × 0.1) = 75 / 10⁻⁹ = 7.5 × 10¹⁰ PFU/mL. If multiple plates fall in the 20–200 range, average the PFU/mL estimates from each.

Clear plaque: produced by a virulent (obligately lytic) phage that kills all bacteria in the zone — no lysogenic bacteria survive; example: T4 phage on E. coli. Turbid plaque: produced by a temperate phage (e.g., lambda phage) — the center of the plaque is turbid/cloudy because some bacteria survive by becoming lysogenic (integrating the phage as a prophage). The turbid appearance is diagnostic for temperate phages with the ability to undergo lysogeny. Turbidity can be used to select for lysogenic vs. non-lysogenic variants: in lambda genetics, clear plaque mutations (cI repressor mutations) produce clear plaques instead of turbid ones.

Typical phage titer ranges: fresh phage stock: 10⁹–10¹² PFU/mL. Crude lysate (first harvest): 10⁸–10¹⁰ PFU/mL. Purified and concentrated: up to 10¹² PFU/mL. Storage: SM buffer (100 mM NaCl, 8 mM MgSO₄, 50 mM Tris-HCl pH 7.5) at 4°C → stable for months to years. Trace chloroform (0.3%): added to kill residual bacteria and prevent bacterial regrowth; not used for phages sensitive to chloroform (enveloped phages). Avoid freeze-thaw cycles — reduces titer significantly. Some phages can be frozen at −80°C in 15–25% glycerol for long-term storage.