BISC209: Agar Overlay

Agar Overlay Pour Plates
In this technique a small amount of melted agar (held in a hot water bath at ~42- 55°C) is inoculated with a dilution of a microorganism. Although the melting point of agar is ~85°C, once liquified at high temperature, agar doesn't gel when cooled until it reaches ~35-45°C, allowing you to add an inoculum of microorgansims (or other reagents) at a temperature that won't kill or inactivate them with heat, but allows even distribution throughout the medium before it solifies. The cooling, liquid agar is then poured evenly over surface of a solid agar plate. It is a useful technique when you want to evaluate surface and subsurface growth of a culture or create a lawn of microorganism for 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 42-55°C 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.  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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