Agar plates are specialized tools used in microbiology to cultivate and isolate microorganisms. They consist of a Petri dish filled with a nutrient-rich medium solidified with agar, a gelatinous substance derived from seaweed. These plates provide a controlled environment where individual bacteria or fungi can grow into visible clusters called colonies, allowing for their isolation and study. This method is fundamental for observing microbial growth patterns and obtaining a pure culture from a mixed sample.
Preparing and Handling Agar Plates
Proper preparation and handling of agar plates are necessary to prevent outside contamination. Before use, plates should be stored inverted (agar-side up) in a refrigerator between 2°C and 8°C to prevent drying and minimize condensation. Check the plates for signs of contamination, such as mold or unwanted bacterial colonies, and discard any that appear compromised.
When setting up to inoculate a plate, work in a clean, sterile area, ideally near a heat source like a small flame, which creates a zone of sterile air. This practice, known as aseptic technique, prevents airborne microbes from settling on the exposed agar surface. Always label the bottom of the Petri dish, not the lid, with the date, sample source, and your initials. This ensures the label stays with the culture if the lid is accidentally removed.
Techniques for Inoculating the Plate
Inoculating the plate involves transferring the microbial sample onto the agar surface using sterile tools. A common method for collecting environmental samples, such as from a doorknob or a countertop, is the swab method. A sterile cotton swab is gently rubbed over the surface to be sampled and then lightly streaked across the agar plate in a zigzag pattern, depositing the collected microbes.
A more precise technique, used to isolate individual species from a dense, mixed culture, is basic streak plating, often called the quadrant streak. The goal is to progressively dilute the bacteria across the plate surface until individual cells are separated enough to grow into distinct colonies. Start by using a sterile inoculating loop or swab to place a small amount of the sample in Quadrant 1, near the edge of the plate.
After streaking the first quadrant, the tool must be sterilized again, typically by flaming it and allowing it to cool, or by using a fresh sterile swab. The plate is then rotated 90 degrees, and the sterile loop is dragged through a small section of the first quadrant before lightly streaking Quadrant 2. This action picks up a reduced number of bacteria from the first streak.
The process is repeated two more times, sterilizing the loop between each quadrant, ensuring the bacterial load is significantly reduced by the time Quadrant 4 is streaked. By the fourth streak, the cells are spaced far enough apart that they will grow into well-separated colonies, each originating from a single cell. The sterile technique is maintained by keeping the lid slightly ajar, using it as a shield to minimize airborne contaminants falling onto the agar during transfer.
Optimal Conditions for Incubation
After inoculation, the plates must be incubated under specific conditions to encourage microbial growth. The optimal temperature depends on the sample source; environmental samples from soil or air often grow well at room temperature (20°C to 25°C). Samples suspected of containing human-associated bacteria, such as those from skin or a clinical source, are often incubated near body temperature (35°C to 37°C).
Plates must always be incubated in the inverted position, meaning the lid is on the bottom and the agar-filled base is on top. This prevents condensation, which naturally forms on the lid when the warmer air inside meets the cooler exterior, from dripping onto the agar surface. If condensation falls, it can cause colonies to spread and merge, making it impossible to count or isolate them.
The duration of incubation is variable, but most fast-growing bacteria produce visible colonies within 24 to 48 hours. Fungi grow slower and may require several days to a week to form mature colonies. Check the plates periodically without removing them from the incubator, looking for the first signs of growth.
Reading Results and Safe Plate Disposal
Once the incubation period is complete, the results are examined by observing the colonies through the closed lid of the Petri dish. Morphological analysis involves noting the color, shape, size, and texture of the colonies, which provides initial clues about the type of microbe present. Fuzzy or filamentous growth often signals mold or fungal contamination, while unexpected colors or spreading colonies may indicate unwanted bacterial species.
Never open the Petri dish after microbial growth is visible, especially when dealing with environmental samples where the identity of the bacteria is unknown. Opening the plate risks releasing potentially harmful organisms into the air, making it a biohazard. All observations and measurements should be made with the lid securely closed.
The safe disposal of used agar plates is important, particularly for settings without access to an autoclave (a high-pressure sterilizing device). The most common decontamination method involves soaking the plates in a bleach solution. A 10% bleach solution can be poured directly onto the agar surface, ensuring the entire culture is saturated, and the plates should soak for a minimum of several hours. After decontamination, the plates can be placed into a sealed plastic bag and disposed of with regular household waste.