Microorganisms are abundant in nature, but studying them individually requires separation from their complex native environments. Microbiology relies on growing these organisms in controlled settings, typically on a solid nutrient medium like agar poured into a petri dish. When a sample containing microbes is spread across this surface, the goal is to dilute the population so individual cells can grow far apart. This culturing process is necessary because most samples contain a mixture of different microbial species, requiring researchers to isolate a single type of microbe for accurate identification.
Understanding the Isolated Colony
An isolated colony is a visible mass of millions of microbial cells that originated from a single progenitor cell. This single cell, or sometimes a small cluster of cells, is referred to as a Colony Forming Unit (CFU). Over time, this CFU multiplies exponentially in a fixed location until the resulting mass becomes large enough to be seen with the naked eye. The very nature of an isolated colony means every cell within it is a direct descendant and, therefore, a genetic clone of the original cell.
Observing the visual characteristics, or morphology, of an isolated colony is the initial step in identifying the species. These characteristics include the colony’s size and its overall shape, often described as circular, irregular, or filamentous. Elevation describes how the colony rises above the agar surface, such as being flat, raised, or convex. Other features include the color, the texture (smooth, rough, or shiny), and the appearance of its edge (entire or lobate).
Why Pure Cultures Are Essential
The goal of obtaining an isolated colony is to establish a pure culture, which consists entirely of a single species of microorganism. A pure culture is fundamental to nearly all microbiological and clinical laboratory work because a mixed sample would produce unreliable or misleading data. If different species are growing together, any tests performed would reflect the combined characteristics of all organisms present. This makes accurate identification impossible.
Determining a microbe’s metabolic capabilities through biochemical testing requires the certainty that only one strain is being tested. Similarly, when a clinician needs to determine which antibiotics will successfully treat an infection, an antibiotic sensitivity test must be performed on a pure culture of the pathogen. Genetic sequencing and analysis also depend on having a pure, genetically uniform population to ensure the DNA being studied belongs to a single, specific organism.
Common Laboratory Isolation Methods
The most common technique used in laboratories to achieve isolated colonies is the Streak Plate Method, which relies on the principle of mechanical dilution. The procedure involves using a sterile inoculating loop to physically spread the microbial sample across the surface of the agar plate in a specific pattern, typically across four separate sections or quadrants. The initial section receives the densest concentration of the inoculum. After streaking the first section, the loop is sterilized by heating it in a flame and then cooled before touching the plate again.
The sterilized loop is then used to drag a small fraction of the microbes from the first section into the second, significantly reducing the number of cells being transferred. This sterilization and streaking process is repeated for the third and fourth sections. By the time the final section is streaked, the concentration of microbes has been diluted so extensively that individual cells are deposited far apart on the agar surface. Each of these single, separated cells then grows into a distinct, isolated colony after incubation.
While the streak plate is the standard for qualitative isolation, other methods also utilize the principle of physical dilution. The Pour Plate Method involves mixing a serially diluted sample directly into molten agar before pouring it into a plate. The Spread Plate Method involves plating a small volume of a highly diluted sample onto the surface and spreading it uniformly with a sterile instrument. Both methods aim to space out the individual CFUs, ensuring they grow into discrete colonies.
Interpreting Isolation Results
After the inoculated plate has been incubated, successful isolation is confirmed by visually inspecting the resulting pattern of growth. A successful streak plate will display heavy, dense growth in the initial quadrants and progressively fewer microbes in the later sections. True isolated colonies must appear as distinct, separate masses, surrounded by a significant margin of clear agar.
Failure to achieve isolation is indicated by confluent growth, where the entire plate is covered by a continuous, dense “lawn” of bacteria with no distinct colonies. A colony touching another colony is considered non-isolated and cannot be used to start a pure culture, as the two may have merged. Once a well-separated colony is identified, a scientist uses a sterile loop to carefully pick up a small amount of that single colony and transfer it to a fresh agar plate or broth tube. This final step is known as subculturing, and it permanently establishes the pure culture for future study.