Phage work
Ten-fold dilutions of a stock are each mixed with excess log-phase host and molten soft agar, poured over hard agar, and incubated overnight so that every infectious particle leaves a countable hole in the lawn; the titre comes from the one plate that holds 30–300 plaques.
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Plaque assay
Ten-fold dilutions of a stock are each mixed with excess log-phase host and molten soft agar, poured over hard agar, and incubated overnight so that every infectious particle leaves a countable hole in the lawn; the titre comes from the one plate that holds 30–300 plaques.
How it differs from the standard method
- The whole series is plated, but only the plate in the countable window is quoted; the others are evidence that the dilutions were made properly.
- A fresh tip and tube at every step: a tip carries a few per cent of the previous, ten-fold stronger, tube, which biases every plate high.
- Top agar is held at 45–48 °C. Poured hotter it kills phage and host; poured onto a cold plate it sets in streaks before it spreads.
- A host-only overlay proves the lawn is a lawn and that the clearing comes from your phage rather than from a lysogen or a contaminating phage.
- Spot tests and soft-agar overlays with undiluted lysate show lysis but do not count particles: clearing by lysis from without can happen with no replication at all.
1One particle, one hole in the lawn
A plaque assay works because a bacteriophage cannot move far. Mix a dilution of a phage stock with excess host cells in a thin layer of soft agar and pour it over a hard agar plate: the cells grow into a continuous lawn, and wherever a single infectious particle landed, it infects a cell, bursts it, and its progeny infect the neighbours.
2Efficiency of plating, and why titres are host-specific
Not every infectious particle forms a plaque. Some never adsorb, some are inactivated by the warm top agar, some meet a cell that restricts the incoming genome or is in the wrong physiological state.
Soft agar matters
0.5–0.7% agar lets progeny diffuse a little: enough for a visible plaque, not so much that they merge. Harder agar gives tiny plaques, softer gives fuzzy ones.
Molten but not hot
Top agar is held just above its setting point. At 55–60 °C it kills both the phage and the indicator host, so the lawn goes patchy and the count collapses.
10–20 min first
Letting phage and host sit together before pouring means essentially every particle attaches, which is what makes the count a count of particles.
Divalent cations
Adsorption of most tailed phage needs them. Buffers and media for phage work are made with them for this reason.
3Making more: multiplicity is the whole trick
To propagate a phage you infect a growing culture and let it lyse. The counter-intuitive part is that adding more phage gives you less.
- Plaque-forming unit (PFU)
- Whatever gave rise to one plaque: usually one virion, but an aggregate of several counts once, which is why counts are slightly overdispersed relative to Poisson.
- Multiplicity of infection (MOI)
- Phage added divided by cells present. Because adsorption is random, the number per cell is Poisson: at MOI 1, 37% of cells are uninfected and 26% are hit twice or more.
- Burst size
- Progeny released per infected cell, 50–200 for many tailed phage. Measured in a one-step growth experiment, and reduced by multiple infection or a poorly growing host.
- Latent period
- Time from adsorption to lysis: tens of minutes for a lytic coliphage. It sets how long a propagation needs.
Key terms
Practise these as flashcards →The standard plaque assay: phage plus host in molten soft agar, poured over a set hard agar base so the lawn and the plaques form in a thin layer.
Plaques formed on a test host divided by plaques formed on a reference host from the same stock, a measure of host range as much as of technique.
The cleared culture after infection, containing the phage, host debris and medium; clarified and stored as the working stock.
Killing the surviving host with a lipid solvent. Effective and cheap, but it destroys lipid-containing phage, so it is not universal.
Picking a single plaque into buffer and re-plating it, repeated two or three times, to get a clonal phage stock.
A host carrying a temperate phage as a prophage. Lysogens give turbid plaques, and can release phage spontaneously, which shows up on a host-only control plate.
When to use it
- You need to know how many infectious particles are in a preparation, before an infection experiment, after a purification step, or to check a stock has not lost titre in storage.
- You are testing host range or resistance, where the ratio of titres on two hosts (the efficiency of plating) is the measurement.
- You want a clonal phage stock: plaque purification is the only way to be sure a preparation came from a single particle.
- You need a high-titre working stock to use as a reagent, for transduction, phage display, typing or a therapy study.
Compared with alternatives
The same counting statistics and the same 30–300 window, but for host cells; a plaque assay needs a CFU count behind it to know the MOI.
Animal-virus titration faces the same Poisson problem but counts transduced cells or focus-forming units instead of plaques in a bacterial lawn.
Pouring, drying and reading plates is plating technique; a patchy pour ruins a plaque assay in exactly the way it ruins a spread plate.
Following the turbidity of the infected culture is how you know when to harvest, and the uninfected control curve is what shows the clearing was lysis.
Display uses filamentous phage as a scaffold for selection rather than as a lytic agent; titres there are measured as colony-forming units of infected host, not plaques.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Ten-fold dilutions of a stock are each mixed with excess log-phase host and molten soft agar, poured over hard agar, and incubated overnight so that every infectious particle leaves a countable hole in the lawn; the titre comes from the one plate that holds 30–300 plaques.
- The whole series is plated, but only the plate in the countable window is quoted; the others are evidence that the dilutions were made properly.
- A fresh tip and tube at every step: a tip carries a few per cent of the previous, ten-fold stronger, tube, which biases every plate high.
- Top agar is held at 45–48 °C. Poured hotter it kills phage and host; poured onto a cold plate it sets in streaks before it spreads.
- A host-only overlay proves the lawn is a lawn and that the clearing comes from your phage rather than from a lysogen or a contaminating phage.
- Spot tests and soft-agar overlays with undiluted lysate show lysis but do not count particles: clearing by lysis from without can happen with no replication at all.