Agar is a polysaccharide derived from red seaweed that forms a stable gel at room temperature, making it the preferred solidifying agent for microbial growth media. This firm, nutrient-rich surface provides a stable environment where microorganisms can be isolated and cultured for study. Preparing this medium requires precise, quantitative measurements to ensure the final product supports optimal microbial growth, and achieving the correct firmness and volume is essential.
Calculating the Agar Concentration Ratio
Determining the required amount of agar involves calculating the correct concentration ratio to achieve the desired gel rigidity. For most standard microbiological applications, the concentration of agar powder must fall between 1.5% and 2.0% weight-per-volume (w/v). This means 1.5 to 2.0 grams of agar powder are needed for every 100 milliliters (ml) of liquid medium.
A 1.5% concentration is the standard, translating to 15 grams of agar powder per 1-liter (1,000 ml) batch of medium. Concentrations below 1.0% result in a soft, semi-solid medium unsuitable for standard plating techniques. Conversely, concentrations significantly higher than 2.0% create a medium that is too firm or brittle, hindering nutrient diffusion and potentially affecting microbial motility.
To prepare a smaller batch, the calculation must be scaled directly to the desired volume. For example, making 500 ml of a 1.5% agar medium requires 7.5 grams of agar powder. This calculation ensures the final gel strength is consistent, regardless of the total batch size. The agar powder is simply one component of the medium, which also includes various nutrients, salts, and water that must be measured precisely according to the specific medium’s formula.
Determining the Pour Volume Per Dish
Once the medium concentration is established, the next consideration is the volume of liquid medium required for each petri dish. The most common sizes used are the standard 90-millimeter (mm) or 100-mm diameter dishes. The goal is to create a uniform layer that provides sufficient depth for culturing without being wasteful.
For a standard 90-mm petri dish, the recommended pour volume is between 15 ml and 20 ml of the prepared liquid medium. For the 100-mm dish, a volume of 20 ml to 25 ml is often used. This range achieves a depth of about 3 to 5 millimeters across the entire base of the dish.
Maintaining this specific depth is important for several practical reasons. A sufficiently deep layer ensures the medium lasts longer without drying out during incubation or storage, which can inhibit growth or alter results. Furthermore, proper depth provides a stable surface area for inoculation and allows for better diffusion of nutrients or antimicrobial agents. If the layer is too thin, the medium quickly dehydrates, rendering the plate unusable.
Essential Steps for Preparation and Pouring
After the correct amounts of agar and medium components are weighed and mixed with water, the mixture must first be heated. Heating, typically to the boiling point, fully dissolves the agar particles, which are highly resistant to dissolution at room temperature. Consistent stirring or swirling prevents the agar powder from scorching on the container bottom.
Following dissolution, the medium requires sterilization to eliminate contaminating microorganisms that would interfere with the intended culture. This is accomplished using an autoclave or pressure cooker, subjecting the medium to high-pressure steam at 121°C for at least 15 minutes. The container cap must be loosened during this process to allow for pressure equalization.
After sterilization, the molten medium must be cooled to a specific temperature before pouring. Cooling prevents excessive condensation on the lid and damage to heat-sensitive components. The ideal pouring temperature range is between 45°C and 50°C, just above the solidification point (around 40°C to 45°C). Cooling can be done in a temperature-controlled water bath to maintain consistency.
The final step involves the sterile pouring technique, performed in a draft-free area, such as near a flame or in a laminar flow hood, to minimize contamination risk. The lid of the sterile petri dish is lifted slightly, and the correct volume of cooled medium is gently poured into the bottom half. Once dispensed, the lid is immediately replaced. The plates are left undisturbed on a level surface to solidify completely, then stored upside down to prevent condensation from dripping onto the agar surface.