Agar plates are a foundational tool in microbiology and mycology, providing a stable, nutrient-rich surface for cultivating various microorganisms. The medium is created by combining a nutrient broth, which supplies elements for growth, with agar, a gelatinous polysaccharide derived from red seaweed. This mixture solidifies into a gel, offering a stable environment where a single microbe can grow into a visible colony. Creating successful plates requires precise measurements to determine the medium’s firmness and the quantity used per dish.
Understanding Agar Concentration
The firmness of the final culture medium is determined by the concentration of agar powder added to the liquid nutrient base. The typical concentration for a firm gel is between 1.5% and 2.0% weight per volume (w/v) of the total medium, translating to 15 to 20 grams of agar powder per liter of water or broth. Using 15 grams per liter is a common standard for general-purpose media like Nutrient Agar or Luria-Bertani (LB) agar. This concentration ensures the medium is firm enough to withstand inoculation techniques like streaking or spreading without tearing the surface. A softer agar, such as 0.5%, is sometimes intentionally used in specialized studies to observe microbial motility. When preparing specific media like Potato Dextrose Agar (PDA) or Tryptic Soy Agar (TSA), follow the manufacturer’s instructions for the nutrient component, ensuring the final agar addition meets the 15-20 grams per liter threshold for proper solidification.
Calculating Volume per Dish
Determining the volume of liquid media needed follows establishing the correct concentration of ingredients. For a standard 100-millimeter diameter Petri dish, the ideal volume of molten agar media ranges from 15 milliliters to 25 milliliters. This volume creates a layer approximately 3 millimeters thick, sufficient for most applications. Pouring less than this results in a thin layer that dries out quickly, shortening the plate’s shelf life and potentially cracking during storage. Exceeding 25 milliliters wastes media and increases the chance of condensation forming on the lid, which can contaminate the culture surface. To scale a recipe efficiently, multiply the number of desired plates by the chosen volume per plate (e.g., 20 dishes $\times$ 20 mL/dish = 400 mL total media).
Preparation and Pouring Technique
The preparation process involves several distinct phases to transform the measured ingredients into a sterile, pourable medium. The agar powder and nutrient components must first be completely dissolved in the liquid. This requires heating the mixture, often to boiling, while stirring to prevent scorching. A clear solution indicates the agar has fully integrated into the liquid base.
Sterilization is achieved by placing the media in a pressure cooker or autoclave at 121°C and 15 pounds per square inch (psi) for 15 to 20 minutes. This high-heat, high-pressure process eliminates all living microorganisms and spores that could contaminate the final plates.
After sterilization, the media must cool to a temperature between 50°C and 55°C before pouring, as this range is just above the agar’s solidification point.
Pouring must use aseptic technique to prevent airborne contamination, often near a flame or in a clean, draft-free area. The cap of the media container is removed, and the neck is briefly flamed to create an updraft of sterile air before the molten agar is poured into the sterile Petri dish. The lid of the Petri dish is lifted only slightly and immediately replaced after pouring, filling the bottom just enough to cover the surface. The plate is then gently swirled to ensure a smooth, even layer free of bubbles.
Optimal Storage and Shelf Life
After pouring, plates must be left undisturbed at room temperature for at least an hour to allow the medium to fully solidify. Once solidified, the plates are commonly stored inverted (agar surface facing down) to prevent condensation from dripping onto the culture surface. Excess moisture can lead to a running culture or contamination.
Prepared agar plates are typically stored in a refrigerator between 2°C and 8°C to slow water evaporation and degradation. Shelf life varies based on the medium’s composition and sealing method. General-purpose plates often last between 30 and 90 days, especially when stored in sealed plastic bags or wrapped with film to minimize drying. Media containing sensitive additives, such as certain antibiotics, may have a much shorter shelf life of only a few weeks, as these components degrade over time.