Sterile Agar Plate Pouring Procedure

Overview

This lecture describes the sterile technique for pouring agar plates using a bottle of molten TSA agar and stacked sterile plates.

Materials and Preparation

• Sterile plates are provided in a stacked arrangement before beginning.
• A 100 milliliter bottle of sterile TSA agar is used for pouring.
• Agar in the bottle must be molten but not too hot to hold comfortably.
• A Bunsen burner is used to create an updraft and provide a sterile working area.

Sterile Pouring Technique

• Open the agar bottle carefully while holding it in one hand.
• Flame the neck of the bottle immediately after opening to maintain sterility.
• Place the bottle cap down with the open side facing up in the Bunsen flame updraft.
• Lift the lid of the bottom plate in the stack just enough to pour in agar.
• Pour approximately 20 to 25 milliliters of molten agar into the plate.
• Replace the plate lid promptly to minimize contamination.
• Flame the neck of the agar bottle again before pouring into the next plate.
• Lift the lid of the second plate and pour 20 to 25 milliliters of molten agar.
• Replace the lid and repeat this process until all plates are filled.

Volumes and Timing

Cooling and Solidification

• After pouring, plates must be left undisturbed to allow agar to solidify.
• Solidification generally takes about 30 minutes or longer depending on conditions.
• As agar cools and solidifies, its appearance changes from clear to opaque.
• Plates should remain stacked while cooling to reduce contamination risk.

Key Terms & Definitions

• TSA agar: Tryptic Soy Agar, a general-purpose growth medium for microorganisms.
• Molten agar: Agar that is liquefied by heating and remains in liquid form for pouring.
• Flame the neck: Passing the bottle opening through a flame to sterilize the glass surface.
• Updraft of the Bunsen flame: Rising warm air above the flame that carries dust and microbes away.