BISC209: Agar Overlay

Agar Overlay Pour Plates

In this technique a small amount of soft agar is added to the surface of an agar plate. It is useful when an even distribution of organisms inoculated into the soft agar is desired. Most often this technique is used when a lawn of growth is needed as in viral plaques assays or in some antibiotic sensitivity assays.

materials: pre-poured agar plates, Soft agar pours in hot water bath (tubes with about 3-5ml of melted agar)

For Viral Plaque assays and other protocols such as those for enumeration of viable bacteria Serial Dilution technique preceeds the inoculation of soft agar pours. In assays where a lawn of growth is desired the soft agar can be inoculated with a standard volume of log phase organisms.

Once inoculated the soft agar is poured over the surface of an agar plate.

Once the soft agar hardens:

Lawn growth plates can have disks containing antibiotics, or other antiseptics/disinfectants, or other compounds you wish to test on the lawn growth placed on the surface of the agar.
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Otherwise the plates are incubated:

24-48 hours later:
Plaques of viruses or colonies to be enumerated can be counted. The number of plaque forming units (PFU) (or enumerated colonies (CFU) are calculated as number/ml of original using the formula:

PFU or CFU/volume plated*dilution factor

OR the effects of the tested substances on the growth of organisms can be evaluated.

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