Cell culture is a fundamental technique widely used in biological research, enabling scientists to study cellular processes, develop new drugs, and produce biological compounds. Maintaining aseptic conditions during cell culture is important to ensure the reliability and validity of experimental results. Despite rigorous sterile practices, contamination remains a common challenge in cell culture laboratories. Early and accurate identification of contamination is important to mitigate its impact, prevent the loss of valuable cell lines, and avoid compromising research integrity.
Recognizing Contamination Through Visual Changes
Contamination in cell cultures often presents with macroscopic changes visible to the naked eye before microscopic examination. One common indicator is a change in the culture medium’s appearance. The medium may become turbid or cloudy, suggesting rapid proliferation of microorganisms like bacteria or yeast.
A noticeable shift in the medium’s pH, indicated by a change in the phenol red indicator, also signals contamination. Healthy cell cultures maintain a stable pH, appearing pink or red, but bacterial or fungal growth can produce acidic byproducts, causing the medium to turn yellow. Conversely, some contaminants or prolonged incubation may lead to an alkaline shift, turning the medium purple. Observing altered cell morphology or the presence of floating particles and films on the medium’s surface can also suggest contamination.
Identifying Contaminants Under the Microscope
Microscopic examination is a crucial step for identifying the specific type of contamination affecting a cell culture. Bacteria appear as small, distinct dots or rod-shaped structures. They often exhibit rapid, darting movements and can be observed freely suspended in the medium, adhering to cell surfaces, or even within infected cells. Their rapid proliferation can lead to a dense “snowstorm” effect in the medium.
Fungal contamination presents as two forms: yeast and mold. Yeast cells are larger than bacteria, appearing as oval or spherical particles often showing characteristic budding, where smaller daughter cells form from the parent cell. Mold contamination is distinguished by filamentous structures called hyphae, long, branching threads that can form a network in the medium. Spores, reproductive units of molds, might also be seen.
Cross-contamination, the introduction of another cell line, can also be identified microscopically. This type of contamination is suggested when the culture contains cells with morphologies or growth patterns different from the expected cell line. For example, a fibroblast culture might show patches of epithelial-like cells, or vice versa, indicating an unintended cell type. Observation of cell shape, size, and adherence properties helps distinguish these introduced cells from the primary culture.
Specialized Detection Techniques
Some contaminants are not visible through routine visual inspection or standard light microscopy, requiring specialized detection methods. Mycoplasma, a type of bacteria lacking a cell wall, is a common contaminant because it does not cause turbidity or pH changes in the medium.
Detection of mycoplasma often relies on molecular techniques like Polymerase Chain Reaction (PCR), amplifying specific mycoplasma DNA sequences, or DNA staining methods such as Hoechst staining, revealing characteristic fluorescent patterns. Enzyme-linked immunosorbent assays (ELISA) can also be used to detect mycoplasma antigens. Similarly, viral contamination often goes unnoticed as many viruses are non-cytopathic, meaning they do not cause visible cell damage. Advanced methods like PCR for viral nucleic acids, immunofluorescence to detect viral proteins, or electron microscopy for visualizing viral particles are employed for their identification.
Immediate Steps After Detecting Contamination
Upon suspecting or confirming contamination in a cell culture, immediate action is necessary to prevent its spread. The first step involves isolating contaminated cultures from healthy ones to contain microorganism spread. This means removing affected flasks or plates from common incubators and laminar flow hoods.
Thorough documentation of observations, including date, suspected contamination type, and reagent lot numbers, is important for tracking and identifying the source. Proper disposal of all contaminated materials, including cell cultures, media, and consumables, is then required, often by autoclaving for complete sterilization. Following disposal, all equipment that came into contact with contaminated cultures, such as incubators, water baths, and biosafety cabinets, must be thoroughly cleaned and disinfected. Reviewing aseptic techniques and examining reagent sterility are important follow-up steps to prevent future occurrences.