Field of Science

About bacterial lawns and phage plaques

This was going to be a post where I do the planning to titer my new lysates today, but it turned into an explanation of how microbiologists use plaques in lawns of bacteria to study phages.

Wait, what's a 'lawn' and what's a 'plaque'?  A lawn is a thin layer of confluent bacterial growth, usually created by mixing a relatively large number of cells (≥10^6) with liquid agar solution ('top agar' or 'soft agar' and pouring the mixture onto the surface of a nutrient-containing agar plate. The top agar is usually at  0.5-0.75%, about half the concentration used for a normal solid plate.  The cells can't move around in the agar, and they grow to high density using the nutrients that diffuse upward from the bottom layer.


If a few of the initial cells were infected with a phage, the phage they release when they die will infect neighbouring cells and kill them, creating a cell-free zone called a 'plaque'.



Here is a detailed drawing of what's happening as a plaque forms:


Initially there's just one infected cell, and sparse uninfected cells in its neighbourhood.  When this cell lyses, the phages it releases can readily diffuse through the agar and infect nearby cells.  While this is happening, the uninfected cells are growing and dividing.  When the newly infected cells lyse, the phage they release add to the local population and infect more cells.  The phage continue to diffuse away, but soon the neighbouring cells become so dense that they stop growing and the phage can no longer replicate in them.  The cells are too big to diffuse through the agar like the phage, so lysis leaves a circular cell-free space called a plaque.  Typical plaques are 1-2 mm across so easy to see with the naked eye.

By counting the number of plaques that form in a lawn of bacteria, we know how many infectious phages were present in the mixture we poured on the plate.  This is the standard way to measure the number of phages (well, the number of 'plaque forming units', PFUs) in a preparation of phage (a 'lysate').

Regular poured-lawn method:  Cells and diluted phage are incubated together in a small volume of liquid (broth or phage-dilution solution) for long enough that most of the phage have attached to the cells.  Then hot liquid top agar is added to the tube and the contents are quickly mixed and poured onto an agar plate of whatever medium best supports lawn growth and plaque formation.  (Quickly so the mixture cools before the cells are damaged.)  The top agar quickly sets, and the plate is incubated overnight at an appropriate temperature for bacterial growth and phage plaque formation.

Spot-titer method:  Cells are quickly mixed with hot top agar (no phage) and the mixture is poured onto agar plates and left to set.  Sometimes the plates can be prepared days in advance, if the cells are happy sitting in the fridge. 10 ยตl dilutions of phage are then spotted onto the surface and the plates are incubated overnight as before.  If you're gentle you can even streak a drop of lysate across the lawn as you would streak cells on a normal plate, allowing you to grow well-isolated plaques without the nuisance of diluting your lysate.

Other ways we can use lawns and plaques:

Isolating phage from a single plaque:  Often you want to start an experiment with a genetically pure phage lysate that you grew up from a single plaque.  If plaques are well-separated (remember that the phage continue to diffuse out after the plaque forms and the lawn stops growing) you can use a Pasteur pipette to punch out the plaque away from the surrounding agar.  If this plaque is put into a small volume of phage-dilution solution, the many thousands of phages it contains will diffuse out over a few hours (or less) and the phage-containing liquid can be used in your experiment, or to prepare a new lysate whose phage all derive from the one that originated the plaque.

Plate lysates:  Lysates can be prepared in broth, by adding phage to growing cells, watching for the time when the culture clears because most of the cells have lysed, and pelleting out the cell debris.  This is a bit fussy to do, since clearing depends on having the right proportions of phage and cells.  A simpler method is to prepare a 'plate lysate', as follows.  Mix the liquid from a picked plaque or a small amount of a lysate with cells and top agar, and pour a lawn.  You want enough phage that the resulting plaques will be 'confluent' - will overlap just enough that very little intact lawn remains.  Once the plaques have formed, overlay the top agar with 5 ml of phage-dilution solution and leave for a few hours or overnight.  Half of the phage will diffuse into the liquid, and in the morning you just have to collect the liquid and add a few drops of chloroform to kill any cells.  These lysates usually have very high titers, because the cells in a lawn can grow to much higher density than those in a liquid culture.

Phage-resistant colonies:  
Sometimes, the area around an initially infected cell includes a cell that is genetically resistant to the phage due to a new mutation that blocks phage attachment or reproduction.  Such as cell (green in the diagram below) will be able to grow within the  area of spreading phage, and its descendants will form a visible colony within the plaque.


Turbid plaques:  One other phenomenon deserves mention, and that's the 'turbid' plaques formed when a 'temperate' phage infects a lawn.  Temperate phages are those that have a mechanism to enter a dormant state in host cells, where the phage genome is passively replicated by the cellular machinery, usually because it is integrated into the cell's chromosome.  Cells with such dormant phages ('lysogens', orange in the diagram below) are resistant to infection by external phages.  When a temperate phage forms a plaque, most infected cells lyse and produce infectious phage, but some form lysogens that grow and divide within the plaque.  Usually many such cells form causing the center of the plaque to appear cloudy ('turbid') rather than having visible colonies.





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