BISC209: Agar Overlay

Agar Overlay Pour Plates

In this technique a small amount of soft agar (held in a hot water bath at a temp. above the solidification temp of solid agar media) is added to the surface of an agar plate. It is useful when an even distribution of organisms 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 solid agar plates,
Tubes with about 3-5ml of melted agar held in hot water bath at 45-55C are called "Soft agar pours"

For Viral Plaque assays and other protocols (such as those for enumeration of viable bacteria) Serial Dilution of the inoculum, preceeds the inoculation of the "soft" agar. In assays where a lawn of growth is desired, the melted agar can be inoculated with a standard volume of log phase organism.

Once inoculated, the soft agar is poured over the surface of cooled, hard (solid) agar plate.

Once the soft agar hardens these plates can be used for several purposes, including:

Microbial senstivity testing: disks containing antibiotics, antiseptics/disinfectants, or other compounds are placed on the surface of soft agar inoculated for lawn growth.
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Plates are incubated for 24-48 hours.
After which:
Colony forming units can be counted or viral plaques (empty space created by bacteriphage lysis of areas in a bacterial lawn). 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 a zone of inhibition can be measured as the clear area around an antibiotic or other compound impregnated disk in mm or cm. The radius of the area where the inoculated microbe could not grow is related to the sensitivity of that organism to the compound.

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