Agar, a gelling agent derived from seaweed, provides the solid surface necessary for growing microorganisms in a laboratory setting. This polysaccharide forms a gel-like substance when combined with a liquid growth medium, creating an environment where individual microbes can multiply to form visible colonies. This guide explores the standard agar concentrations, factors that influence the necessary amount, and proper preparation techniques for quality plates.
Standard Agar Concentration
For general microbiology applications, a standard agar concentration ranges from 1.5% to 2% weight per volume (w/v). This means that for every 100 milliliters of liquid medium, 1.5 to 2 grams of agar powder are added. For instance, preparing one liter (1000 mL) of a standard nutrient agar medium involves using about 15 grams of agar powder. A common 90-100mm diameter petri dish usually holds between 15 and 25 milliliters of agar medium to cover the bottom. To fill approximately 40 standard 100mm plates from one liter of prepared medium, each plate receives about 25 mL.
Factors Influencing Agar Quantity
The amount of agar varies depending on intended use and microbial growth medium type. Different microbial growth media, such as nutrient agar, potato dextrose agar, or specialized selective and differential media, have varying agar requirements. The desired firmness of the gel also influences the agar quantity; a softer gel might be preferred for motility assays, while a firmer gel is suitable for general plating where a stable surface is needed. Some media might also require specific additions that affect the final gel properties, requiring slight adjustments to the agar content.
Preparing Agar for Plates
Preparing agar medium for petri dishes involves measurement and sterilization. Begin by weighing the appropriate amount of agar powder and dehydrated growth medium per recipe or manufacturer’s instructions. Combine these dry ingredients with the specified volume of distilled or deionized water in a heat-resistant flask. Stir the mixture thoroughly to ensure the powders are well-dispersed before heating. The medium must then be heated, often to boiling, to fully dissolve the agar, appearing as a clear solution.
Sterilization is an important step, typically performed using an autoclave at 121°C (250°F) for 15-20 minutes to eliminate contaminants. After autoclaving, cool the hot agar medium to a pouring temperature (45-50°C) before dispensing into petri dishes. Pouring at too high a temperature can lead to excessive condensation, while pouring when too cool can result in premature solidification and an uneven surface.
Work in a sterile environment (e.g., near a Bunsen burner flame or in a laminar flow hood) to minimize contamination during pouring. Pour 15-25 mL of medium evenly into each standard petri dish, quickly replacing the lid to prevent airborne contamination.
Ensuring Quality Agar Plates
Quality agar plates require addressing common preparation issues. If the agar is too soft or too hard, it indicates incorrect agar concentration or insufficient mixing. Accurate measurement of ingredients and thorough dissolution are important for consistent gel strength. Contamination from unwanted microbial growth can result from inadequate sterilization, improper handling, or non-aseptic pouring techniques. Sterile equipment and a clean pouring environment prevent this.
Uneven plate surfaces occur if dishes are not level during solidification or if agar is poured too cool. Lid condensation is common, caused by pouring hot agar or incorrect storage. Cooling plates fully before stacking and storing them inverted reduces condensation. Drying plates before use (e.g., in a laminar flow hood) also prevents excess moisture.