Agar plates are a foundational tool in microbiology, providing a solid, nutrient-rich surface upon which microorganisms can be grown and studied. The medium is a liquid broth solidified with agar, a polysaccharide derived from red algae, which serves as a stable, gelatinous matrix. The goal is to create a completely sterile environment, preventing unintended contamination from environmental bacteria or fungi. The presence of unwanted microbes invalidates experimental results, making sterilization a highly controlled and deliberate process.
Preparing the Agar Medium for Sterilization
The process begins with the precise measurement of the powdered medium and distilled water according to the specific formulation chosen. Accuracy is important to ensure the final medium has the correct nutritional balance and gelling consistency. The dry ingredients and water must be thoroughly mixed. The mixture is typically heated, often brought to a boil, to fully dissolve the agar component, which is insoluble in cold water.
The liquid medium must be placed into a heat-safe container, such as a borosilicate glass flask or a Pyrex bottle. The container should be large enough to hold the liquid and leave ample headspace, approximately twice the volume of the liquid, to prevent boiling over during heating. The lid is then capped loosely or covered with aluminum foil, which allows steam to escape and pressure to equalize during the heating cycle.
The Complete Sterilization Process
Sterilization is achieved using moist heat, a method effective at eliminating all forms of microbial life, including heat-resistant bacterial spores. The standard in a laboratory setting is the autoclave, which uses pressurized steam to reach 121°C (250°F) at 15 pounds per square inch (PSI). The exposure time for a typical batch of agar medium is 15 to 20 minutes once these conditions are met, though the duration depends on the total volume being sterilized.
For home use or in a low-resource setting, a standard kitchen pressure cooker can serve as an effective substitute. Many consumer pressure cookers operate at a slightly lower pressure, often between 10 and 13 PSI, meaning the required sterilization temperature of 121°C is not consistently achieved. To compensate for this lower pressure, the sterilization time must be significantly increased, sometimes ranging from 40 to 60 minutes, to ensure total spore eradication.
Regardless of the device used, safety protocols must be followed after the timed sterilization period is complete. The heat source must be turned off, and the cooker must be allowed to cool down gradually and depressurize naturally. Rapidly venting the pressure will cause the superheated liquid agar medium to violently boil over, which is a safety hazard and can compromise sterility. The container should only be removed once the pressure gauge reads zero and the device has cooled enough for safe handling.
Aseptic Pouring and Handling Techniques
Once sterilized, the liquid medium must be cooled to a pourable temperature, typically between 50°C and 55°C (120–130°F), before being transferred to sterile Petri dishes. Pouring at a higher temperature risks damaging the plastic dishes and causing excessive condensation, which can promote contamination. If the medium cools too much, the agar will begin to solidify, resulting in a lumpy or uneven plate surface.
The pouring process itself must be conducted using aseptic technique to prevent airborne contaminants from entering the sterile medium. This is most effectively accomplished by working near the flame of an alcohol lamp or Bunsen burner, which creates a cone of rising, sterile air through convection. The sterile Petri dishes are opened only slightly, just enough to pour the liquid agar, and the lid is replaced immediately to minimize exposure time.
The neck of the agar bottle should be passed quickly through the flame before and after each pour. This action heats the glass, creating an outward flow of air that prevents contaminants from settling on the lip of the bottle. After pouring the required amount of medium, the plate should be left undisturbed on a level surface to fully solidify. Once the medium has set, the plates should be inverted (lid on the bottom) to prevent residual condensation from dripping onto the agar surface.
Checking for Contamination
A quality control step is necessary to verify the success of the sterilization and aseptic pouring methods before the plates are used. This involves setting aside a small sample of the newly poured plates—typically three to five dishes—that will remain completely unopened. These control plates should be incubated at room temperature or a slightly warmer temperature, such as 25°C, for 24 to 48 hours.
A successful result is indicated by a plate that remains completely clear, showing no signs of microbial growth after this incubation period. If the plates are contaminated, a variety of signs may be visible, including fuzzy spots of mold, distinct bacterial colonies, or a general cloudiness within the agar itself. Plates that pass this sterility check can be safely stored, usually inverted in a refrigerator, and are ready for use in a controlled experiment.