Preparing for Plate Streaking
Meticulous preparation is crucial for successfully isolating bacterial colonies. An agar plate serves as a solid growth medium, providing nutrients and moisture for microorganisms to multiply and form visible colonies. These plates are typically prepared in a laboratory or purchased sterile, ready for inoculation.
Essential tools for this process include an inoculating loop, which can be a disposable plastic loop or a reusable wire loop made of nichrome or platinum. If using a reusable loop, a Bunsen burner or an alcohol lamp is necessary for flame sterilization. The microbial sample itself, often a broth culture or a swab from an environmental source, contains the mixed population of microorganisms to be separated.
Aseptic technique is fundamental to prevent contamination from unwanted microorganisms. This involves sterilizing the work area with a disinfectant like 70% ethanol before and after working. Sterilizing the inoculating loop by heating it in a flame until it glows red ensures no foreign bacteria are introduced. This maintains the purity of the sample.
Mastering Streaking Techniques
Quadrant streaking progressively dilutes a microbial sample across the agar surface, leading to isolated colonies. This technique systematically reduces the number of bacteria transferred from one section of the plate to the next.
The process begins by sterilizing the inoculating loop in a flame until it is red hot, then allowing it to cool for several seconds to avoid killing the microorganisms. Once cooled, the loop is gently touched to the microbial sample to pick up a small amount of inoculum. The first quadrant of the agar plate is then inoculated by making several parallel, closely spaced streaks across approximately one-quarter of the plate’s surface. This initial section will contain the highest density of bacterial growth.
After completing the first quadrant, the inoculating loop is re-sterilized and cooled. To inoculate the second quadrant, pass the loop through the edge of the first quadrant two or three times, picking up fewer bacteria. Then, extend into a new, unstreaked section of the plate with parallel lines. This further dilutes the bacterial concentration.
This pattern of re-sterilizing the loop, touching the previous quadrant, and streaking into a new section is repeated for the third and fourth quadrants. Each subsequent quadrant receives fewer bacteria, increasing the likelihood of individual cells forming distinct, isolated colonies. After the final quadrant is streaked, the plate is closed immediately and labeled on the bottom with details such as the date, sample name, and type of medium.
Incubation and Interpreting Results
After streaking, the agar plate needs appropriate incubation conditions for bacteria to grow and form visible colonies. Most cultures are incubated at 25°C to 37°C, depending on the microorganisms’ optimal growth temperature, for 24 to 48 hours. Plates are typically incubated inverted, with the agar side facing upwards, to prevent condensation from dripping onto the agar surface and spreading colonies.
Successful streaking is characterized by isolated colonies, particularly in the third and fourth quadrants. An isolated colony originates from a single bacterial cell or a cluster of identical cells that multiplied to form a visible mass. These distinct colonies appear as individual growth spots separated from others, indicating effective dilution.
Observing results helps identify common streaking issues. If the entire plate shows uniform, dense growth without isolated colonies, it suggests insufficient dilution or failure to re-sterilize the loop between quadrants. Conversely, no growth indicates the inoculating loop was too hot, killing bacteria, or the sample was not viable. Contamination, visible as unexpected colony types or fungal growth, indicates a breach in aseptic technique.