How to Pour Agar Plates: A Step-by-Step Process

Agar plates provide a stable, nutrient-rich surface for cultivating microorganisms like bacteria and fungi in a controlled environment. These specialized dishes are widely used in scientific research, educational settings, and by hobbyists exploring microbiology. Creating your own agar plates at home is a practical way to engage with fundamental biological principles.

Gathering Your Supplies

Assemble all necessary materials. You will need sterile petri dishes. The primary ingredient for the growth medium is agar powder, such as nutrient agar or potato dextrose agar, chosen based on the type of microorganisms you intend to grow. Distilled or deionized water is essential for preparing the solution.

A heat-safe container is needed for mixing and heating the agar solution. A stirring rod or magnetic stirrer will ensure the powder dissolves completely. For heating and sterilization, a hot plate for dissolving and a pressure cooker or autoclave are typically used to achieve the high temperatures required to eliminate contaminants. Safety equipment, including heat-protective gloves and eye protection, is important for handling hot materials.

Preparing the Agar Mixture

Creating the agar solution begins with accurately measuring the agar powder and distilled water. A common ratio for nutrient agar is approximately 15 grams of agar powder per liter of distilled water, though specific product instructions should always be followed for precise measurements. Some nutrient agar formulations may require 28 grams per liter. Thoroughly mix the agar powder with the water in your heat-safe container to prevent clumping.

The mixture then needs to be heated to dissolve the agar completely, until the solution appears clear. Constant stirring during this phase helps ensure even dissolution and prevents the agar from sticking to the bottom of the container. After dissolving, the agar solution requires sterilization to eliminate any unwanted microorganisms. A pressure cooker works similarly to an autoclave for this purpose, using high pressure and saturated steam.

Sterilization in a pressure cooker involves heating the mixture at 15 pounds per square inch (psi) and 121°C (250°F) for 15 to 20 minutes. Ensure the pressure cooker has enough water at the bottom, and place the agar container on a rack to prevent direct contact with the heat source. After the sterilization cycle is complete, allow the pressure cooker to cool naturally before carefully opening it and removing the hot agar solution.

Pouring and Setting the Plates

Once the agar mixture has been sterilized, it needs to cool to a pourable temperature, between 50 and 60°C (122 to 140°F), before pouring. Pouring at this temperature allows the agar to solidify evenly without premature gelling or excessive condensation. If the agar cools too much, it can start to solidify and become lumpy.

Maintaining sterility during pouring is important to prevent contamination. Working in a clean, draft-free area is advisable, and some prefer working near a flame, like a Bunsen burner, as the rising heat helps create an upward air current that deters contaminants. Quickly opening and closing the petri dish lids, minimizing the time the agar is exposed to the air, helps maintain a sterile environment.

For standard 100 mm petri dishes, 20 to 25 milliliters of agar solution is poured into each dish. This volume provides a sufficient layer for microbial growth without overfilling the plate. Pour the agar onto a clean, level surface to ensure an even thickness across the plate as it solidifies. Once poured, allow the plates to sit undisturbed at room temperature until the agar has fully solidified, which usually takes about 30 minutes to an hour.

Storing and Using Your Plates

Proper storage is important for extending their shelf life and preventing contamination or dehydration. After the agar has fully solidified, store the plates inverted (agar side up). This practice helps minimize condensation from collecting on the lid, which could drip onto the agar surface and spread contaminants or create an uneven growth environment.

For optimal longevity, store the sealed plates in a refrigerator at 4°C. Refrigeration slows dehydration and inhibits the growth of any stray contaminants that might have settled on the plates. Plain, poured agar plates can generally last anywhere from 30 to 90 days when stored properly. Nutrient-rich agar plates, or those with additives, may have a shorter shelf life, sometimes lasting up to two to four weeks.

Before using your stored plates, visually inspect them for any signs of contamination, such as unexpected colored spots or fuzzy growth. Plates showing contamination should be discarded to avoid compromising experimental results. After use, agar plates containing microbial growth should be properly disposed of, typically by autoclaving to sterilize them before discarding in regular waste, or by treating with a bleach solution to kill any microorganisms.