An agar plate is a foundational tool in microbiology, providing a contained environment for the study and cultivation of microorganisms. This specialized dish is filled with a nutrient medium solidified using agar, a gelatinous substance derived from red algae. The plate offers bacteria, molds, and yeasts a solid surface rich in food and moisture where they can grow into visible colonies. Preparing these plates is the first step in most basic microbiological investigations, allowing researchers to isolate, count, and identify various microbes. Precision, particularly during the sterilization phase, is required to ensure that only the intended organisms are grown.
Essential Equipment and Ingredients
Creating a microbial growth medium requires a nutrient base and a solidifying agent. The nutrient source is often a dehydrated powdered medium, such as Nutrient Agar or Lysogeny Broth (LB) powder, containing proteins, salts, and carbohydrates to support bacterial growth. The solidifying agent is agar powder, a complex polysaccharide that melts when heated but remains solid at temperatures conducive to growth. This combination is mixed with purified water, typically distilled or deionized, to ensure consistency. Necessary hardware includes sterile glass or plastic petri dishes, precise measuring tools, a heat-resistant container for preparation, and a reliable heat source capable of achieving high pressure for sterilization.
Step-by-Step Preparation and Sterilization
The initial steps involve accurately measuring the components of the growth medium. A typical recipe for Nutrient Agar requires suspending the powdered mixture, often around 28 grams, in one liter of water. The powder and water are combined in the heat-resistant vessel and heated gently while stirring to fully dissolve the agar and other components. Agar requires a temperature of approximately 95°C to enter a liquid state.
Following dissolution, the medium must undergo rigorous sterilization to eliminate all existing microorganisms, including highly resistant bacterial endospores. For home or classroom use, a pressure cooker serves as a functional substitute for an industrial autoclave. The container holding the liquid medium is placed inside the pressure cooker, elevated slightly off the bottom, along with a small amount of water.
The pressure cooker is heated until it reaches 15 pounds per square inch (psi), corresponding to approximately 121°C. This temperature must be maintained for 15 to 20 minutes to ensure complete sterilization. Once the sterilization period is complete, the heat source is turned off, and the pressure must be allowed to dissipate naturally. Manually releasing the pressure can cause the superheated liquid medium to boil over violently, risking injury and contamination.
Pouring, Setting, and Proper Storage
After the pressure returns to zero, the sterilized medium must be allowed to cool before pouring. The molten agar should be poured when its temperature drops to between 50°C and 55°C. This temperature is warm enough to remain liquid but cool enough to prevent excessive condensation inside the petri dish. The pouring process must minimize the risk of airborne contamination, often achieved by working quickly near the flame of a Bunsen burner or an alcohol lamp.
Each sterile petri dish is partially lifted, and the liquid medium is quickly poured to cover the bottom surface, usually filling the dish to a depth of four to five millimeters. The lid is immediately replaced to prevent contaminants from settling onto the exposed surface. The poured plates are left undisturbed on a level surface until the agar cools to room temperature and solidifies into a firm gel, a process that typically takes 30 to 60 minutes.
Once solidified, the plates are ready for use but require specific storage conditions if not inoculated immediately. Plates should be stored inverted to prevent water vapor from collecting on the lid and dripping onto the agar surface. This condensation could promote the spreading of microbial colonies. Un-inoculated plates are best kept in a refrigerator at approximately 4°C, where they can remain viable for several weeks.
Safe Handling and Disposal
Working with agar plates requires adherence to basic clean practices to prevent accidental contamination. All work surfaces should be wiped down with a 10% bleach solution or 70% isopropyl alcohol before and after handling the plates. Handwashing before and after the procedure is a highly effective measure to reduce the transfer of microbial contaminants.
Once an agar plate has been used for growth, it is considered a biohazard because it contains concentrated colonies of potentially harmful microorganisms. Used plates must never be discarded directly into the regular trash without first undergoing a final disinfection step.
The most secure disposal method is to sterilize the plates again using the pressure cooker for the same duration and temperature as the initial preparation. Alternatively, the plates can be flooded with a strong disinfectant, such as a bleach solution, and allowed to soak for several hours before the contents are scraped out and the plastic components are discarded. This final decontamination ensures that all cultured microbes are rendered inactive before the materials leave the contained work area.