Agar plates are fundamental tools in microbiology, serving as a solid growth medium for various microorganisms. These transparent dishes, containing a gel-like substance, enable scientists and students to cultivate and observe bacteria, fungi, and other microbes in a controlled environment. By providing essential nutrients and a stable surface, agar plates allow for the isolation, identification, and study of microbial life, which is crucial for understanding infectious diseases, developing new medicines, and exploring microbial ecosystems.
Gathering Your Supplies
Gathering all necessary materials ensures a smooth and sterile process. Key ingredients include agar powder, which solidifies the growth medium, and a nutrient source like nutrient broth or peptone to support microbial growth. Distilled water is essential for creating the solution, as its purity prevents contaminants.
Heat-resistant glassware (beakers or flasks) is necessary for mixing and heating, and a stirring rod for dissolution. A heat source (hot plate or Bunsen burner) is used to boil the mixture. For sterilization, a pressure cooker or autoclave is needed to eliminate microorganisms using high heat and pressure. Sterile Petri dishes are for the solidified medium, and personal protective equipment (gloves, eye protection) for safety.
The Step-by-Step Preparation
Begin by accurately measuring and combining ingredients. For a common preparation, mix approximately 20-25 grams of agar powder with about 5 grams of nutrient broth or peptone powder into 1 liter of distilled water. Add the agar powder to cold water to prevent clumping.
Heat the combined mixture to dissolve the agar. Stir gently while heating on a hot plate or with a Bunsen burner until it boils for about one minute. This ensures the agar completely dissolves and the medium becomes clear. Incompletely dissolved agar can result in a squishy or fragile medium.
Sterilization prevents contamination. The heated agar solution, in a heat-resistant bottle with a loosened lid or foil cover, is placed into a pressure cooker or autoclave. Sterilize at 121°C (250°F) and 15 pounds per square inch (psi) for 15 to 20 minutes. This high-temperature, high-pressure environment kills any spores or microbes present.
After sterilization, cool the agar solution to 50-55°C (122-131°F) before pouring into Petri dishes. Pouring too hot can damage plastic dishes or lead to excessive condensation. Pouring too cool will cause it to solidify prematurely, resulting in lumpy plates. A good indicator is when you can comfortably hold the flask with gloves.
Pour the cooled agar into sterile Petri dishes in a clean environment (e.g., near a flame or in a laminar flow hood) to minimize airborne contamination. Quickly lift the lid of each Petri dish just enough to pour approximately 25 milliliters of agar, or enough to just cover the bottom, then immediately replace the lid. Allow the plates to sit undisturbed on a level surface at room temperature until the agar solidifies into a firm gel, which takes a few hours or overnight.
Ensuring Success: Proper Handling and Storage
Proper handling and storage maintain sterility and usability after solidification. Always keep the lids on Petri dishes to prevent airborne contaminants. When handling, minimize exposure to air by quickly opening and closing lids only when necessary.
Store prepared agar plates inverted (upside down) in a refrigerator at 4°C (39°F). Storing them upside down prevents condensation from dripping onto the agar surface, which could create a moist environment conducive to contamination or interfere with microbial growth. Refrigeration extends shelf life by slowing drying and inhibiting microorganism growth.
The shelf life of prepared agar plates can vary, but plain agar plates generally last between 30 to 90 days when stored correctly. Factors like the type of agar, packaging, and adherence to sterile storage conditions influence how long they remain viable.
Important Considerations for Safe Practice
Safety is important when preparing and handling agar plates, due to heat, pressure, and microorganisms. Always wear personal protective equipment, including heat-resistant gloves, eye protection, and a lab coat, to shield against splashes, burns, or accidental exposure to microbes. Handle hot glassware with caution.
Proper disposal of used or contaminated materials prevents microorganism spread. Agar plates used for culturing, or those showing signs of contamination, should be sterilized before disposal. Autoclave plates at 121°C (250°F) and 15 psi for at least 20-30 minutes to kill all microbial life. Place plates in autoclavable bags or specialized baskets. After sterilization, the melted agar and plastic can be disposed of with general waste.
Common issues have straightforward solutions. If contamination occurs, it often points to a break in sterile technique during preparation or pouring. Meticulous sterilization of all equipment and working in a clean environment can prevent this. If the agar does not solidify, it could be due to insufficient agar powder or inadequate heating. Bubbles in the plates, which can obscure observation, result from pouring too hot or shaking excessively; pouring slowly and allowing the agar to cool can minimize bubble formation.