Bacterial plating is the fundamental technique in microbiology used to grow, isolate, and study microorganisms in a controlled environment. The process involves introducing a bacterial sample, known as the inoculum, onto a solid growth medium, typically nutrient-rich agar within a sterile petri dish. Successful plating allows scientists to observe colonies, which are macroscopic clusters of millions of bacteria originating from a single cell. Various plating methods exist, each designed to achieve a specific goal, such as obtaining a pure culture or quantifying microorganisms in a sample.
Preparing the Workspace and Growth Medium
Before inoculation, maintaining a sterile, or aseptic, working environment is required to prevent contamination from airborne microbes. The workspace should be thoroughly cleaned and disinfected with a sterilizing agent, such as 70% ethanol, both before and after the procedure.
The plating procedure should be conducted within a sterile field, often created by the updraft of heat from a Bunsen burner or within the filtered air environment of a laminar flow hood. Working near a flame creates a cone of sterile air that limits the settling of contaminants onto the exposed agar surface. All necessary materials, including pre-poured agar plates, sterile inoculation loops, and liquid cultures, should be organized and within easy reach to minimize unnecessary movements.
The growth medium provides a solid surface and the necessary nutrients for bacterial growth. Agar plates must remain sealed or with their lids closed until inoculation to preserve sterility. Tools like metal inoculation loops and spreaders must be sterilized, typically by heating them in a flame until they glow red-hot. The tool must be allowed to cool completely before touching the bacterial sample or the agar, as a hot loop will kill the bacteria.
The Isolation Method: Bacterial Streaking
The quadrant streak method is the primary technique employed to obtain a pure culture from a sample that may contain a mixture of different species. A pure culture is a population of cells derived from a single bacterium. The underlying principle is to physically dilute the bacterial concentration across the surface of the agar plate in a series of steps. This dilution gradually reduces the number of cells being spread until individual cells are deposited far enough apart to grow into isolated colonies.
To begin, the plate is mentally divided into four quadrants. A small amount of the mixed sample is introduced onto the first quadrant using a cooled, sterile inoculation loop, which is used to spread the inoculum in a tight zig-zag pattern over approximately one-quarter of the plate’s surface. This first area holds the highest concentration of bacteria.
After the first quadrant is complete, the loop must be sterilized again by flaming and allowed to cool. This step ensures that only a tiny fraction of cells from the previous section is carried over. The plate is then rotated 90 degrees, and the sterile loop is dragged through the edge of the first quadrant two or three times before spreading the material into the second quadrant. The loop is sterilized and cooled again, and the process is repeated, dragging from the second into the third quadrant, and then from the third into the fourth. By the time the fourth quadrant is streaked, the bacterial density is sufficiently low to yield well-separated, individual colonies after incubation.
The Quantification Method: Pour and Spread Plates
Quantification methods, specifically the spread plate and pour plate techniques, are used to determine the concentration of viable bacteria in a liquid sample, typically expressed as Colony Forming Units per milliliter (CFU/mL). Unlike streaking, which focuses on isolation, these methods require the preparation of serial dilutions of the original sample. This ensures that the final plate contains a countable number of colonies, ideally between 30 and 300.
In the spread plate method, a small, known volume of the diluted bacterial suspension, often 0.1 mL, is pipetted directly onto the surface of a solidified agar plate. A sterile, L-shaped glass or plastic spreader is then used to distribute the liquid evenly across the entire surface of the agar until the inoculum is absorbed. Since the bacteria grow only on the surface, this method is primarily suited for counting aerobic and facultative aerobic microorganisms.
The pour plate method involves combining a known volume of the bacterial sample with molten agar that has been cooled to approximately 45–50°C before it solidifies. The mixture is gently swirled to ensure even distribution and then poured into a sterile petri dish, where it is allowed to set. This technique allows for the growth of microaerophilic or anaerobic organisms that prefer a reduced oxygen environment, as colonies develop both on the surface and embedded within the agar. However, temporary exposure to the warm agar can sometimes stress or kill heat-sensitive bacteria, potentially resulting in a lower count compared to the spread plate method.
Post-Plating Handling and Storage
Once the bacteria are inoculated onto the agar, the plates must be immediately handled for successful growth and containment. Proper labeling is essential and should be done on the bottom of the plate, not the lid. The label should include the organism, the medium type, and the date of inoculation.
The inoculated plates are then placed in an incubator at a temperature suitable for the specific organism, typically 37°C for many common laboratory bacteria, and incubated for a set period, often 24 to 48 hours. A requirement for incubation is that the plates must be inverted, or placed lid-side down, to prevent condensation. Water vapor rising from the warm agar will condense on the cooler lid. If the plate is upright, these droplets would fall onto the agar surface, causing colonies to run together and making accurate counting or isolation impossible.
After incubation, the plates are examined for growth, and colonies are counted or selected for further study based on their size, shape, and color. When the experiment is complete, all used media and contaminated tools are considered biohazardous waste. They must be sterilized, usually by autoclaving, before final disposal to ensure all microorganisms are effectively killed, preventing their release into the environment.