Cell culture involves growing and maintaining cells outside of their natural environment in a laboratory setting. This technique is fundamental for research in medicine, drug development, and biology, providing a controlled system to study cell behavior and function. The artificial environment, which provides rich nutrients, warm temperature, and a specific pH, is also an ideal breeding ground for unwanted microorganisms. Maintaining absolute sterility is paramount; contamination introduces variables that skew experimental results and compromise data integrity. Identifying contamination swiftly is necessary to preserve the reliability of scientific findings.
Macroscopic Warning Signs in the Culture Media
The earliest indications of a problem can often be seen by observing the culture flask before microscopic examination. A common sign is cloudiness or turbidity in the culture medium, which should normally be clear and transparent. This suggests a large, rapidly growing population of suspended microorganisms, such as bacteria or yeast, is present.
Another noticeable change is the color of the medium, which is typically due to shifts in pH. Most culture media contain a pH indicator called phenol red, which is red-pink when healthy. Rapidly growing bacterial contaminants often produce acidic waste products, causing the medium to turn a distinct yellow color. Conversely, some fungi or prolonged contamination can lead to an alkaline shift, causing the medium to turn a dark pink or purple.
Sometimes, a thin film or pellicle may form on the surface of the liquid, or a sediment may settle at the bottom of the flask, separate from the cell monolayer. These physical signs, along with any unusual odor, warn that the culture is compromised. At this stage, a microscopic examination is necessary to identify the specific type of microbial invader.
Visual Identification of Microbial Contaminants
Microscopic examination reveals the morphology and behavior of the contaminants that cause the macroscopic changes. Bacterial contamination typically appears as numerous, tiny, dark dots or rod-shaped structures scattered throughout the media, often at a size range of around 1–5 micrometers. A distinguishing feature of bacteria is their rapid, darting, and vibrating motion across the field of view, which is distinct from the subtle, random jiggling of cellular debris caused by Brownian motion.
The density of bacteria can be overwhelming, sometimes coating the cell monolayer or appearing in concentrated clumps in the spaces between the cultured cells. A different presentation is seen with yeast contamination, which involves larger, oval or spherical organisms. These single-celled fungi may be observed as small clusters or chains of budding cells, a clear sign of their reproduction.
Fungal contamination, or mold, presents a unique visual signature, often starting as spores that sprout into filamentous structures. Under the microscope, this appears as long, branched hyphae, which are much larger than bacteria or yeast. Macroscopically, mold can sometimes be seen as furry, grey, white, or greenish patches floating on the surface of the medium, requiring careful inspection if they rest out of the microscope’s focal plane.
Recognizing Subtle and Non-Microbial Contamination
Not all contamination is obvious through simple observation, with some issues requiring specialized detection methods. Mycoplasma species are a frequent, hidden problem because they are among the smallest free-living bacteria, measuring only about 0.2 to 0.3 micrometers. Due to their minute size and lack of a cell wall, they do not cause the media to become turbid or cloudy like other bacteria, and they are generally invisible under a standard brightfield microscope.
Mycoplasma contamination is often detected by its subtle effects on the host cells, such as a reduced rate of cell proliferation or changes in cell morphology, like increased rounding or poor attachment. These organisms compete with the cultured cells for nutrients, and heavy infection can result in altered gene expression and metabolism. Definitive confirmation of Mycoplasma requires specific testing, such as DNA staining with a fluorescent dye or highly sensitive PCR-based assays.
Cell line cross-contamination is a different, non-microbial form of contamination where one type of cultured cell invades another. This is often visually suggested by the sudden appearance of mixed cell populations; for example, a culture expected to contain only elongated fibroblast cells may begin to show distinct, clustered epithelial-like cells. While changes in cell morphology can raise suspicion, definitive proof relies on species-specific testing, such as Short Tandem Repeat (STR) profiling.