Question

4. Plaques reach a certain size and stop enlarging. Why don’t they keep spreading until the whole plate is clear? (Hint: Think carefully about what phages need to replicate.)

Answer 1

4. Phages are too small (~ 200 nm) to be seen using light microscopy, but they can be detected if grown on a lawn of bacteria using an overlay plating technique. In this procedure, phages and their host cells are mixed in a small tube of soft agar and then poured (overlayed) on top of an agar base plate. Soft agar contains a lower concentration of agar and thus allows the phages to diffuse more freely. The plates are then incubated at the optimum growth temperature for the host bacteria.

During incubation, most of the bacteria multiply to produce a thick covering (a lawn). Some of the bacteria will become infected with a virulent phage. This phage will replicate to large numbers and ultimately kill, or lyse the host cell (lytic cycle). The phages released from the lysed cells will go on to infect neighboring cells, and so on. Cycles of infection and bacterial cell lysis continue until a clear area, called a plaque, is evident within the bacterial lawn. Once the bacteria stop growing due to crowding and lack of nutrients, the phages can no longer successfully infect the bacteria and the plaque will not increase in size.

The overlay plating technique is commonly used to determine the phage titer (the number of phage particles/mL). Theoretically, each isolated plaque originated from one phage. Therefore, the plaques can be counted to determine the number of phages in the original suspension (the titer). Each plaque can be designated as a plaque-forming unit or pfu. The titer is usually given in terms of pfus per mL.

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