The agar plate is a foundational tool in microbiology, providing a solid, jelly-like growth medium for cultivating microorganisms. These plates allow scientists to isolate and grow colonies of bacteria or fungi in a controlled environment. The integrity of any experiment depends entirely on preventing contamination from unwanted microbes in the air, on surfaces, or from hands. Maintaining an aseptic technique is paramount to ensure only the desired organism grows, as contaminants can easily ruin an experiment. This necessity requires a strict, standardized procedure for opening and working with agar plates.
Preparing the Work Area
The preparation of the work environment begins with thorough disinfection of the workbench surface. This involves wiping the bench with a disinfectant solution, such as 70% ethanol or a diluted bleach solution, and allowing sufficient contact time.
A localized sterile zone is established using a heat source, usually a Bunsen burner or a micro-incinerator. This creates convection currents of heated air, which move upward and carry airborne dust and contaminants away from the immediate work area. The protective zone around a standard Bunsen burner flame can range from approximately 10 to 30 centimeters in radius.
Working within this protective zone minimizes the exposure of the open plate and sterile tools to falling microorganisms. Before beginning the procedure, all tools that will contact the culture, such as inoculating loops or spreaders, must be sterilized. This is typically done by heating them to a red-hot temperature in the burner flame. Tools must then be allowed to cool completely within the sterile zone before touching the agar or the sample.
The Clamshell Technique for Opening Plates
The correct method for opening an agar plate centers on the “clamshell technique,” which minimizes the exposure time and surface area vulnerable to contamination. Throughout the entire process, the plate must remain right-side up, with the lid resting on top of the base. This orientation uses the lid as a physical shield against falling airborne particles.
When the plate needs to be accessed, the lid should only be lifted slightly, creating a small, protective opening. This angle should be kept as shallow as possible, ideally less than 45 degrees, which creates a barrier against the immediate environment. The lid should never be fully removed from the base or placed down on the workbench.
This slight opening allows the sterile tool, such as an inoculating loop or a sterile pipette tip, to be inserted quickly to either collect a sample or apply an inoculum to the agar surface. The entire action of opening the lid, performing the task, and closing the lid must be done swiftly and deliberately within the boundaries of the aseptic zone. The lid must be returned to its fully closed position immediately after the tool is withdrawn.
The clamshell lid acts as a mechanical barrier, ensuring that any dust or spores that might settle from the air are deposited onto the lid’s inner surface rather than the exposed agar. This technique, combined with the updraft from the Bunsen burner, provides the highest degree of protection for the culture during the inoculation process. The agar surface should never be gouged or pierced by the inoculating tool, as this damages the medium and impedes the isolation of individual microbial colonies.
Post-Procedure Handling
Once the inoculation or sampling procedure is complete, the plate should be quickly and firmly closed. The next immediate step is to label the plate clearly by writing directly onto the bottom of the base, not the lid. Labeling the base ensures the identifying information, such as the date, organism name, and technician’s initials, remains with the culture even if the lid is accidentally separated.
The inoculated plates are typically placed into the incubator in an inverted position (base on top, lid underneath). This inversion serves a mechanical purpose by preventing condensation from forming on the lid and dripping down onto the agar surface. If water droplets were to fall, they would spread the colonies, making it impossible to count or isolate individual microbial growths.
Inverting the plates also slows the rate at which the agar medium dries out, preserving the moisture necessary for microbial growth. This inverted incubation position is a standardized practice that maintains the purity and clarity of the cultures. If intended for long-term storage or transport, plates may be sealed with a flexible film, such as Parafilm, to prevent desiccation and secure the lid.