Subculturing is a fundamental technique in microbiology that involves transferring a small volume of microorganisms from an old growth medium to a fresh, sterile one. This process, often called passaging, is necessary because microbial populations cannot thrive indefinitely in their original container. Over time, available nutrients are consumed and toxic waste products accumulate, slowing growth and potentially leading to the culture’s death. A timely transfer ensures the continued viability and propagation of the desired microbial strain for ongoing study or industrial use. This practice underpins virtually all research, medical diagnostics, and biotechnological applications that rely on living cultures.
Fundamental Reasons for Transferring Cultures
The primary application of subculturing is to maintain the viability of a microbial strain. Transferring a culture to a new medium supplies a fresh source of nutrients, preventing the population from entering the stationary or decline phase due to starvation. This regular refreshment is necessary for keeping stock cultures alive and metabolically active over periods of weeks or months.
Subculturing is also performed to increase the overall biomass of a specific organism. Transferring a small inoculum into a larger volume of fresh liquid medium, such as broth, allows the population to multiply rapidly. This scaled-up growth provides the quantity of cells required for large-scale experiments, biochemical assays, or industrial production, such as fermentation.
A third reason for transfer is culture purification, often accomplished using the streak plate method. If a culture is contaminated or contains a mixture of different species, subculturing mechanically separates the cells. By isolating a single colony—which represents the descendants of a single cell—a pure strain can be established, ensuring experimental results are attributed only to the target microorganism.
Maintaining a Sterile Environment
The success of subculturing relies entirely on maintaining an environment free from external contamination, achieved through aseptic technique. This set of procedures prevents environmental microorganisms from entering the culture medium and also prevents the cultured organisms from escaping and contaminating the worker or laboratory area.
Specialized equipment provides a highly controlled workspace, most commonly a laminar flow hood or biosafety cabinet. These enclosed workstations draw air through a High-Efficiency Particulate Air (HEPA) filter, creating a continuous, unidirectional stream of particle-free air. This air stream sweeps across the work surface, preventing environmental contaminants from settling on exposed media or tools.
Tools that contact the culture, such as inoculation loops and needles, must be sterilized immediately before and after each transfer. Metal loops are rapidly heated in a Bunsen burner flame until they glow red-hot, incinerating any microorganisms present. This ensures the instrument is completely sterile before it touches a fresh culture medium. Tubes and flasks containing media are also briefly passed through the Bunsen burner flame after opening and before closing. This generates convection currents that prevent airborne particles from entering the container.
Methods of Culture Transfer
Once the sterile environment is established, the physical transfer of the culture uses several techniques tailored to the specific goal.
Quadrant Streaking
For achieving a pure culture from a mixed sample, the plate-to-plate method, specifically quadrant streaking, is the standard technique. This procedure involves dividing an agar plate into four sections and physically diluting the inoculum across the surface. A small sample is deposited in the first quadrant using a sterile loop. The loop is then sterilized, cooled, and used to drag inoculum from the first quadrant into the second, further diluting the concentration. This process is repeated into the third and fourth quadrants, with the loop sterilized before each new section. By the final quadrant, the bacterial density is low enough that individual cells are deposited far apart, allowing them to grow into isolated colonies. Each distinct colony can then be picked and transferred to a new medium to establish a pure stock.
Slants and Broth Transfers
When the objective is short-term maintenance, cultures are often transferred to agar slants or deeps. A slant is a test tube containing solid agar medium cooled at an angle, providing a large, sloped surface area for growth while minimizing drying. A broth-to-slant transfer involves drawing the loop in a light, zigzag pattern across the slant surface. Broth transfers, which move an inoculum into a liquid medium, are used for generating high-volume cultures.
Long-Term Culture Preservation
While regular subculturing maintains a strain for short periods, long-term preservation is necessary to prevent genetic drift and minimize labor.
Cryopreservation
The most common technique for preserving microbial strains for years or decades is cryopreservation, or freezing at ultra-low temperatures. This method requires mixing the culture with a cryoprotectant, such as glycerol or dimethyl sulfoxide (DMSO), before freezing. Glycerol prevents the formation of large, damaging ice crystals that would otherwise puncture the cell walls during freezing. The mixture is typically stored in an ultra-low freezer (e.g., -70°C to -80°C) or submerged in liquid nitrogen vapor phase. These extremely low temperatures halt metabolic activity, effectively pausing the life cycle of the microorganism.
Lyophilization
Another method for extended storage is lyophilization, commonly known as freeze-drying, which is useful for transport and storage at room temperature. In this process, the culture is first frozen and then subjected to a vacuum, which removes the water through sublimation, converting the ice directly into vapor. The resulting dry powder or pellet contains dormant, viable cells that can be rehydrated and cultured decades later. Lyophilization is frequently used by culture collection banks to maintain genetic stability.