A liquid culture is a broth or suspension where microorganisms, such as bacteria or yeast, are grown in a nutrient-rich liquid medium for rapid propagation. Agar is a gelatinous substance derived from seaweed, mixed with nutrients, and solidified in Petri dishes to create a solid growth surface. Transferring a microbial suspension from a liquid culture to agar transitions the organisms from bulk growth to surface growth. This process separates individual cells, allowing them to divide and form distinct, visible clusters called colonies, which confirms viability and allows for purity checks.
Essential Preparation and Sterile Setup
Transferring a liquid culture requires aseptic technique, a stringent methodology designed to prevent environmental contamination. Maintaining a sterile environment is necessary because microbes exist everywhere, ensuring that only the target organism grows on the agar plate. The working surface must first be thoroughly cleaned with a disinfectant, such as 70% ethanol solution, and allowed to air dry completely.
A flame source, such as a Bunsen burner, is positioned near the work area to create a sterile zone of heated air. This convection current helps keep airborne contaminants away from the open media and tools. Necessary materials include pre-poured agar plates, the liquid culture, and sterile implements like inoculation loops, spreaders, or pipettes. All tools must be sterilized, typically by flaming metal loops until they glow red-hot or by using pre-sterilized, disposable plastic tools.
Core Techniques for Plating the Culture
Once the sterile environment is established, the liquid culture is transferred to the agar using two primary methods, each serving a different analytical purpose.
Streak Plating
The streak plating method is primarily used to achieve colony isolation and check for culture purity. This technique involves drawing a small amount of the liquid culture onto the agar surface with a sterile inoculation loop. The loop creates a series of directional lines across the first section of the plate. The loop is re-sterilized, and a fresh set of lines is drawn that only briefly touches the previous section. This process is repeated across quadrants, with the loop sterilized between each section to progressively dilute the microbes and reduce microbial density. The goal is for the final section to contain individual cells spaced far enough apart to grow into discrete colonies.
Spread Plating
The spread plating method is often used to count the number of viable organisms in the original liquid culture. This technique requires pipetting a measured, small volume (typically 0.1 milliliters) of the liquid culture onto the center of the agar plate. A sterile, L-shaped glass or metal spreader is used to distribute the liquid sample evenly across the entire surface. The spreader must be allowed to cool completely after sterilization to avoid killing the microorganisms upon contact. For accurate counting, the liquid culture is often subjected to serial dilutions before plating, aiming for a final plate count between 30 and 300 colonies.
Once the liquid is evenly spread, the plate is left undisturbed for five to ten minutes. This allows the inoculum to fully absorb into the agar before incubation.
Incubation and Interpreting Growth Results
After the culture is transferred to the agar, the plates are prepared for incubation, which provides optimal conditions for microbial growth. The plates must be incubated in an inverted position (agar medium on top, lid on bottom). This orientation prevents condensation from dripping onto the agar surface, which could cause colonies to run together or introduce contamination.
Incubation temperature and time are determined by the specific organism being grown; a common bacterial incubation is 37°C for 24 to 48 hours. Following incubation, the resulting growth is examined to assess both viability and purity. Viability is confirmed by the simple presence of growth on the plate.
Purity is assessed by checking if all colonies on the plate look identical. The presence of colonies with different shapes, colors, or textures suggests the culture is mixed or contaminated. The visual characteristics of the colonies, collectively known as colony morphology, provide clues for identification. These characteristics include the colony’s size, form (e.g., circular or irregular), margin appearance, and elevation (e.g., flat, raised, or convex).