Contamination of cell cultures can easily become the most common problem in cell culture laboratories, sometimes causing very serious consequences. Cell culture contaminants can be divided into two categories, chemical contaminants such as medium, serum and water impurities, endotoxins, plasticizers and detergents, and biological contaminants such as bacteria, molds, yeasts, viruses, mycoplasmas cross infection. Contaminated by other cell lines. Although it is impossible to completely eliminate contamination, its frequency and severity can be reduced by thoroughly understanding its source and following good aseptic techniques
1.This section outlines the main types of biological contamination:
Bacterial contamination
Mold and virus contamination
Mycoplasma contamination
Yeast contamination
1.1Bacterial contamination
Bacteria are a large group of ubiquitous single-celled microorganisms. They are usually just a few microns in diameter and can come in a variety of shapes, from spheres to rods and spirals. Because of their ubiquity, size, and rapid growth rate, bacteria, along with yeasts and molds, are the most common biological contaminants in cell culture.
1.1.1 Detection of Bacterial Contamination
Bacterial contamination is easily detected by visual inspection of the culture within a few days of it becoming infected;
Infected cultures usually appear cloudy (i.e., turbid), sometimes with a thin film on the surface.
Sudden drops in the pH of the culture medium is also frequently encountered.
Under a low-power microscope, the bacteria appear as tiny, moving granules between the cells, and observation under a high-power microscope can resolve the shapes of individual bacteria.
1.2Mold & Virus contamination
1.2.1 Mold Contamination
Molds are eukaryotic microorganisms of the fungal kingdom that grow in the form of multicellular filaments called hyphae. The connective networks of these multicellular filaments contain genetically identical nuclei called colonies or mycelium.
Similar to yeast contamination, the pH of the culture remains stable during the initial phase of contamination and then increases rapidly as the culture becomes more severely infected and becomes cloudy. Under the microscope, mycelium is usually filamentous, sometimes as dense clusters of spores. The spores of many molds can survive in extremely harsh and inhospitable environments during their dormant phase and are activated only when the right growth conditions are encountered.
1.2.2 Virus Contamination
Viruses are microscopic infectious agents that take over the host cell’s machinery for reproduction. Their extremely small size makes them difficult to detect in culture and to remove from reagents used in cell culture laboratories. Since most viruses have very strict requirements for their hosts, they usually do not adversely affect cell cultures of species other than the host.
However, the use of virus-infected cell cultures can pose a serious risk to the health of laboratory personnel, especially if human or primate cells are grown in the laboratory.
Viral infection in cell cultures can be detected by electron microscopy, immunostaining with a set of antibodies, ELISA, or PCR with appropriate viral primers.
1.3Mycoplasma contamination
Mycoplasmas are simple bacteria without cell walls, and they are thought to be the smallest self-replicating organisms. Due to their extremely small size (usually less than 1 micron), mycoplasma is difficult to detect until they reach extremely high densities and cause cell cultures to deteriorate; Until then, there is usually no obvious sign of infection.
1.3.1 Detection of mycoplasma contamination
Some slow-growing mycoplasmas may persist in cultures without causing cell death, but they alter the behavior and metabolism of host cells in cultures.
Chronic mycoplasma infection may be characterized by reduced cell proliferation rate, decreased saturation density and agglutination in suspension culture.
However, the only reliable way to detect mycoplasma contamination is to test the culture regularly using fluorescent staining (e.g., Hoechst 33258), ELISA, PCR, immunostaining, autoradiography, or microbial testing.
1.4Yeast contamination
Yeasts are single-celled eukaryotes of the fungal kingdom, ranging in size from a few microns (usually) to 40 microns (rarely).
1.4.1Detection of yeast contamination
As with bacterial contamination, cultures contaminated with yeast can become cloudy, especially if the contamination is in an advanced stage. The pH of cultures contaminated with yeast changes very little until the contamination becomes more severe, at which stage the pH usually increases. Under the microscope, yeast appears as individual ovoid or spherical particles and may produce smaller particles.
2.Cross infection
Although not as common as microbial contamination, extensive cross-contamination of many cell lines with HeLa and other fast-growing cell lines is a clearly defined problem with serious consequences. Obtain cell lines from reputable cell banks, regularly check the characteristics of the cell lines, and use good aseptic techniques. These practices will help you avoid cross-contamination. DNA fingerprinting, karyotyping and isotyping can confirm whether there is cross-contamination in your cell culture.
Although not as common as microbial contamination, extensive cross-contamination of many cell lines with HeLa and other fast-growing cell lines is a clearly defined problem with serious consequences. Obtain cell lines from reputable cell banks, regularly check the characteristics of the cell lines, and use good aseptic techniques. These practices will help you avoid cross-contamination. DNA fingerprinting, karyotyping and isotyping can confirm whether there is cross-contamination in your cell culture.
Post time: Feb-01-2023