Observe the morphology and viability of cultures regularly and carefully. Examine the medium in the vessel for macroscopic evidence of microbial contamination. This includes unusual pH shifts (yellow or purple color from the phenol red), turbidity, or particles. Also, look for small fungal colonies that float at the medium-air interface. Specifically check around the edges of the vessel as these may not be readily visible
through the microscope.
With an inverted microscope at low power (40×), check the medium for evidence of microbial contamination and the morphology of the cells. Bacterial contamination will appear as small, shimmering black dots within the spaces between the cells. Yeast contamination will appear as rounded or budding particles, while fungi will have thin filamentous mycelia. For nonadherent cells grown in flasks, such as hybridomas,
this is a simple matter of viewing the flask directly on the microscope. For cells grown in spinner flasks or bioreactors, a sample of the cell suspension will need to be withdrawn and loaded into a microscope slide or hemocytometer for observation.
Most adherent cells should be attached firmly to the surface. In some cases, healthy cells will round up and detach somewhat during mitosis and appear very refractile. Following mitosis, they will reattach. Some of these will float free if the culture vessel is physically disturbed. In contrast, dead cells often round up and detach from the monolayer and appear smaller and darker (not refractile) than healthy cells. Cells in suspension culture grow either as single cells or as clusters of cells. Viable cells appear round and refractile whereas dead cells appear smaller and darker. Occasionally, a portion of the cells will attach and grow on the side of the culture vessel and appear round or flattened. The percentage of attached cells varies with the culture conditions and the cell density. Cellular debris may also be observed in healthy cell populations. Some cell lines grow as mixed adherent and suspension cultures.
As a reference, photomicrographs for some ATCC cell lines are available on the respective product details page on the website. Cells are shown at two different densities: just after subculturing (low) and just before they need to be subcultured (high).
In addition to daily examinations, periodically test a sample of the culture for the presence of fungi, bacteria, and mycoplasma. There are several methods that can be used to check for these contaminants. For additional information, refer to the section on microbial contamination.
ATCC® CCL-61™ at high density.