Agar, a gelatinous substance derived from red seaweed, is the foundational material in microbiology for growing and studying microorganisms in a controlled setting. It functions as a solidifying agent, transforming a liquid nutrient solution into a firm, gel-like surface within a petri dish. This solid surface, known as a culture medium, provides a stable environment for bacteria, fungi, and other microbes to form distinct, observable colonies. The preparation of this medium, often called nutrient agar, is the first step in conducting any experiment involving microbial growth.
Gathering Supplies and Ingredients
The preparation of this culture medium requires specialized ingredients and basic laboratory equipment. The medium is typically composed of dehydrated nutrient powder, which contains a blend of components like peptone, beef or yeast extract, and agar powder. Peptone and the extracts serve as the primary food source, supplying organic nitrogen, amino acids, vitamins, and trace minerals necessary for broad microbial growth.
Agar powder, making up about 1.5% of the final mixture, is a complex carbohydrate that provides the rigid structure, as most microbes cannot digest it. Other ingredients, such as sodium chloride, are included to maintain the osmotic balance, ensuring microbial cells remain healthy. To prepare the medium, you will need a precise digital scale, a heat-resistant glass container, a stirring rod, and sterile petri dishes.
Mixing and Sterilizing the Medium
Preparation begins by accurately weighing the dehydrated powder and mixing it with distilled or deionized water according to the manufacturer’s instructions, typically aiming for about 28 grams per liter. The mixture must be heated to the boiling point while stirring gently to ensure all components are dissolved. The solution will appear clear once the agar is dissolved.
After dissolving, the medium requires sterilization to eliminate any contaminating microbes, including bacterial spores, which are highly resistant to heat. While professional laboratories use an autoclave, a common household pressure cooker can serve as an effective substitute by utilizing high-pressure steam. The medium, loosely capped in the heat-resistant container, is placed inside the cooker with an adequate amount of water.
To achieve sterility, the pressure cooker must reach 121°C (250°F), corresponding to 15 pounds per square inch (PSI). Before reaching this pressure, the cooker must vent steam for about ten minutes to displace trapped air, as air pockets prevent even heat distribution. Once 15 PSI is reached, the medium should be maintained at this level for 15 to 20 minutes to kill contaminants. After the required time, turn off the heat source. The pressure must drop naturally without opening the vent, which prevents the liquid from boiling over.
Pouring and Solidifying the Agar Plates
After sterilization, the molten medium must cool to between 50°C and 60°C before pouring. If the medium is too hot, excessive steam creates condensation inside the petri dish lids; if it is too cool, it will solidify prematurely. Pouring the medium requires an aseptic technique to prevent airborne contaminants from entering the dishes.
Work in a clean area, ideally near a flame source (like a Bunsen burner or alcohol lamp), which creates a small zone of sterile air through upward convection. The sterile petri dish lid should only be opened minimally, using a slight “clamshell” motion to pour the medium into the bottom half of the plate. A volume sufficient to cover the entire base (usually about 15–20 milliliters) is poured to create a layer approximately 3 to 4 millimeters thick.
Immediately replace the lid, and gently swirl the plate to ensure the medium covers the base evenly. Any bubbles that form can be popped by briefly passing a burner flame over the molten agar, but this must be done quickly to avoid melting the plastic dish. The plates are then left undisturbed on a level surface until the agar sets, which typically takes 30 minutes to a few hours at room temperature.
Storing and Incubating the Plates
Once the agar has solidified, the plates are ready for storage or immediate use. To prevent moisture loss and minimize airborne contaminants, the plates should be stored upside down. Storing them inverted prevents condensation that forms on the lid from dripping onto the agar surface, which could interfere with microbial growth.
Unused plates are preserved by sealing them in plastic bags and refrigerating them at about 4°C, where they remain viable for four to six weeks. Before inoculation, refrigerated plates should warm up to room temperature to prevent temperature shock. After inoculation, they are placed into an incubator, again upside down, to promote growth at a specific temperature, such as 37°C for bacteria or 20–25°C for environmental samples.