Agar is a gelatinous substance derived from red seaweed used as a solidifying agent in microbiology. When mixed with water and nutrients, it forms a solid, pliable growth medium for microorganisms. This mixture is poured into a Petri dish, a shallow, lidded container designed to provide a sterile environment. This prepared system, known as an agar plate, allows cultivation and observation of bacteria and fungi in a controlled setting.
Essential Materials and Safety Precautions
Creating a reliable agar plate requires specific supplies and cleanliness to prevent contamination. The base ingredients are agar powder, a source of nutrients like peptone or nutrient broth powder, and distilled water. Distilled water is necessary because tap water contaminants can interfere with microbial growth or medium consistency.
Essential equipment includes a heat-resistant glass container, such as an Erlenmeyer flask, for mixing and sterilization, and a precise measuring scale. You will need sterile Petri dishes (disposable or pre-sterilized reusable glass dishes). A heat source (stove or microwave) is needed to dissolve the agar, and a pressure cooker or autoclave is required for sterilization.
Safety involves preventing contamination and handling hot liquids. Working near a flame minimizes airborne contaminants settling into the media, following aseptic technique. Always wear heat-protective gloves when handling the flask after heating or sterilization. The work surface must be thoroughly cleaned with a disinfectant like 70% isopropyl alcohol before preparation.
Preparing and Sterilizing the Agar Medium
Preparation begins by calculating the required mass of dry ingredients, aiming for 1.5% to 2.0% agar concentration. For example, a common recipe uses 15 to 20 grams of agar powder per liter of distilled water. Combine the powdered nutrients and agar with water in a flask large enough to hold twice the liquid volume to prevent boiling over.
The mixture must be heated until the agar powder is completely dissolved, forming a clear, homogenous solution. Gentle heating and frequent swirling are necessary to ensure the agar dissolves fully and does not scorch. If the agar is not fully dissolved, the final product will be soft and uneven, making it unsuitable for culturing microbes.
Sterilization is achieved by exposing the medium to high-pressure steam, which kills all microbial life, including bacterial spores. The most common method uses an autoclave or a household pressure cooker set to 15 psi. This pressure allows the temperature to reach 121°C (250°F), which must be maintained for 15 to 20 minutes. This ensures the medium is completely sterile before pouring.
Pouring the Plates and Post-Preparation Handling
After sterilization, the flask must cool to the correct pouring temperature, generally 50°C to 60°C. Pouring too hot can damage plastic dishes or cause excessive condensation on the lid. Cooling too much causes the agar to solidify prematurely inside the flask, preventing uniform plates.
Pouring must be done quickly and precisely in an area with minimal airflow to maintain sterility. Lift the Petri dish lid only slightly and immediately replace it after pouring enough medium to cover the bottom (15 to 25 milliliters for a standard dish). This shallow layer creates the optimal surface for microbial growth.
The freshly poured plates must be left undisturbed at room temperature for 30 minutes to one hour until the agar solidifies into a firm gel. To prevent condensation from dripping onto the agar surface, the finished plates should be stored upside down in a refrigerator (2°C to 8°C). Stored this way, the plates remain sterile and usable for two to four weeks.