Agar is a gelatinous substance derived from red seaweed, a type of algae, which serves as the solidifying agent in culture media used across microbiology. This carbohydrate polymer remains solid at incubation temperatures, providing a stable, semi-solid surface for the cultivation of bacteria and fungi. The preparation process ensures the resulting material is both nutrient-rich to support microbial growth and completely sterile to prevent the introduction of unintended contaminants.
Essential Supplies and Materials
Preparing a basic nutrient agar requires a specific combination of dehydrated components and laboratory equipment to ensure consistency and sterility. The primary ingredients for a general-purpose medium include agar powder, a nutrient source such as peptone (an enzymatic digest of protein), and beef or yeast extract, which provides vitamins and carbohydrates. Distilled or deionized water is necessary to dissolve these components and serve as the transport medium.
The physical items needed include a large, heat-resistant glass beaker or flask for mixing and a stirring rod to facilitate dissolution of the powders into the water. Accurate measurement requires a precise balance and a graduated cylinder for the liquid volume. Finally, the resulting liquid medium must be housed in containers that can withstand the high temperatures of sterilization, often requiring specialized screw-cap bottles.
Mixing, Heating, and Sterilization
The preparation begins by carefully weighing the dehydrated powder and dissolving it completely in the measured water. The mixture must be heated, often in a hot plate or microwave, while stirring to achieve full dissolution. Agar does not fully dissolve until the temperature approaches 95°C. Failure to fully dissolve the agar at this stage will result in a soft, non-uniform medium after cooling.
Once the medium is fully dissolved, it is transferred to a vessel designed for sterilization, such as an autoclave or a pressure cooker, which uses saturated steam to kill all microbial life, including highly resistant bacterial spores. The standard sterilization cycle involves heating the media to a temperature of 121°C at a pressure of 15 pounds per square inch (psi) for a duration of 15 to 20 minutes. This combination of heat and pressure raises the boiling point of the liquid, preventing it from boiling over, and ensures the penetration of steam throughout the entire volume. The precise timing is adjusted based on the total volume of the liquid to guarantee that the center of the medium reaches the required temperature for the full duration.
Pouring Culture Plates Aseptically
Following sterilization, the liquid agar must be cooled before it can be poured into sterile petri dishes. The molten medium should be allowed to cool to a temperature between 50°C and 60°C. If it is too hot, it will create excessive condensation and potentially warp the plastic petri dishes. If the medium cools too much, however, it will begin to solidify, resulting in a lumpy or uneven surface.
The pouring process must be conducted using aseptic technique to prevent airborne contaminants from entering the sterile medium. This typically involves working near a flame, such as a Bunsen burner, which creates a cone of rising, sterile air, or working within a laminar flow hood. The lid of the petri dish should be lifted only slightly, exposing the base just enough to pour the agar, which minimizes the entry point for microbial spores.
An appropriate volume of liquid agar is poured to ensure the plate base is covered with a layer approximately 3 millimeters thick. This thickness is sufficient to allow for microbial growth and minimize dehydration during incubation. After pouring, the lid is quickly replaced, and the plate is gently swirled to ensure an even, bubble-free surface before the agar is allowed to solidify fully on a level benchtop.
Common Issues and Media Storage
One of the most frequent challenges encountered during preparation is the formation of air bubbles, which can obstruct the view of microbial colonies after incubation. These bubbles can often be removed immediately after pouring by briefly passing the flame of a Bunsen burner over the surface of the molten agar, which causes the bubbles to pop. Another common issue is post-pour contamination, which is usually a result of poor aseptic technique, such as lifting the petri dish lid too high or talking directly over the open dish.
To maximize the shelf life of the prepared plates, proper storage is necessary to prevent desiccation and contamination. Once the agar has solidified, the plates must be stored inverted (agar side up) to prevent condensation that forms on the lid from dripping onto the agar surface. The plates should be placed in sealed bags or containers and refrigerated at a temperature range of 2°C to 8°C. Storing plates in the dark and minimizing exposure to light also helps preserve any light-sensitive media components, such as certain dyes or blood.