Agar plates are fundamental tools in microbiology, providing a solid surface for cultivating and studying various microorganisms in a controlled environment. Preparation involves gathering materials, sterilization, and storage, all aimed at creating an optimal, contamination-free growth medium.
Gathering Your Supplies
Specific ingredients and equipment are required for preparing agar plates. Ingredients include agar powder, a gelatinous substance derived from red algae that solidifies the medium. Distilled or deionized water is essential for dissolving components. For general-purpose media like nutrient agar, additional ingredients such as peptone (providing organic nitrogen) and beef extract (offering carbon, vitamins, and salts) supply microbial sustenance.
Equipment includes:
A heat-resistant flask or bottle to mix and heat the solution.
A stirring rod or magnetic stirrer for uniform dissolution.
A hot plate or microwave to boil the mixture.
A pressure cooker or autoclave for sterilization.
Sterile petri dishes for pouring the cooled agar.
Measuring cups or cylinders for accurate ingredient measurement.
The Preparation Process
Begin by accurately measuring and combining ingredients. For nutrient agar, suspend about 28 grams of dehydrated nutrient agar powder in 1000 milliliters of distilled or deionized water. Add the measured agar powder and nutrient sources to a heat-resistant flask. Stir the mixture thoroughly for proper dissolution.
Once combined, the solution requires heating to fully dissolve the agar and other components. Heat the flask on a hot plate or in a microwave, bringing it to a boil while stirring. This ensures the agar completely dissolves, forming a homogeneous solution. Prevent scorching during this step.
Sterilization is a crucial phase to eliminate contaminating microorganisms from the agar medium and glassware. Place the dissolved agar solution, in a loosely capped heat-resistant bottle, in an autoclave or pressure cooker. Sterilization is typically performed at 121°C (250°F) under 15 pounds per square inch (psi) of pressure for 15 to 20 minutes. This high-temperature, high-pressure steam effectively kills bacteria, fungi, and their spores.
After sterilization, cool the molten agar to a pourable temperature, typically between 45-55°C (113-131°F). Cooling to this temperature range prevents condensation inside petri dish lids and avoids thermal shock to heat-sensitive additives, such as antibiotics. Pouring requires aseptic technique. In a clean workspace, lift the lid of a sterile petri dish minimally and carefully pour the warm agar into the dish, filling it to about 1/8 inch thick (15-25 mL for a standard 90-100 mm plate). Promptly replace the lid to prevent airborne contamination.
Finally, allow the poured plates to solidify and cure. The agar will transition from liquid to a solid gel within 20 minutes to an hour at room temperature. After solidification, allow plates to cure for a period, often by leaving them undisturbed for several hours or overnight. This curing process allows any remaining condensation to evaporate from the agar surface, which helps prevent contamination and ensures a drier surface for microbial growth.
Storing Your Agar Plates
Proper storage of prepared agar plates maintains their sterility and extends their shelf life. After the agar has fully solidified, plates should be stored upside down in a cool, dark environment, such as a refrigerator, at around 4°C. Storing them inverted helps prevent condensation, which can form on the lid, from dripping onto the agar surface and potentially spreading contaminants or creating an overly moist environment.
To prevent dehydration, plates should be sealed. They can be placed back into their original plastic sleeves or sealed in airtight plastic bags. This barrier minimizes moisture loss, which can otherwise lead to the agar drying out and becoming unusable. While plain agar plates can last for 30 to 90 days when properly sealed and refrigerated, plates containing additional nutrients or sensitive additives like antibiotics may have a shorter shelf life, sometimes as little as 7 days for blood agar. Signs of spoilage or contamination include visible mold growth, bacterial colonies, or signs of dehydration such as cracking or shrinking of the agar.
Safety Considerations
Safety precautions are important when preparing and handling agar plates. Personal protective equipment (PPE), including gloves and eye protection, should always be worn to prevent contact with hot liquids and potential microbial contaminants. Handling hot glassware and the agar solution requires caution to avoid burns; using heat-resistant gloves or mitts is recommended. Work in a clean, disinfected area to minimize the risk of introducing unwanted microorganisms.
Maintain sterile technique throughout preparation. This involves minimizing exposure of the sterile agar and petri dishes to the air and surrounding environment, as airborne microbes can easily contaminate the plates. Proper disposal of used agar plates is also necessary. Plates that have been used for microbial growth should be sterilized, typically by autoclaving, before being discarded. After autoclaving, the solidified agar can then be disposed of as regular waste.