Cell Culture Contamination Prevention and Removal Protocols

This unit describes how to detect and recognize bacterial, fungal, or mycoplasma contamination and how to verify the presence of mycoplasma. Strategies are described for dealing with culture contamination, if necessary. - [Read Assessing and Controlling Microbial Contamination in Cell Cultures Protocol]

Protocol describes a method for mycoplasma detection using Hoechst 33258, a fluorescent dye that stains DNA. Staining of particulate DNA matter around the cell nucleus (on the cell surface or in the cytoplasm) is indicative of mycoplasma contamination. - [Read Detection of Mycoplasma in Mammalian Cell Cultures Using Fluorescence Microscopy Protocol]

One of the most important, but frequently overlooked, cell culture procedures is testing cultures for microbial contamination, especially mycoplasma. It is critical for every cell culture laboratory to only use cell lines that have been carefully screened for mycoplasma. Fortunately, there is a simple fluorochrome DNA staining test that can detect both mycoplasma and virtually any other prokaryote contaminants. - [Read Mycoplasma Detection Using DNA Staining Protocol]

For both biological and economical reasons, it is important to eliminate mycoplasmas from cell cultures being used for basic research, diagnosis, and biotechnological production. The most commonly used method for elimination, inactivation, or suppression of mycoplasmas in cell cultures is treatment with antibiotics. In general, antibiotic therapies do not result in long-lasting, successful elimination. Also, the cytotoxic properties of antibiotics can cause undesirable side effects on cells. - [Read Mycoplasma Elimination Reagent Protocol]

Quality is important in all aspects of tissue culture since the quality of materials used i.e. media and other reagents) will affect the quality of the cultures and products derived from them. The main areas of quality control that are of concern for tissue culture are: The quality of the reagents and materials; The provenance and integrity of the cell lines; The avoidance of microbial contamination. - [Read Quality Control Considerations for Cell Culture]

This method is advantageous for saving the occasional cultures that become contaminated. Yeast contaminated cultures will appear cloudy when slightly shaken and lymphocytes will not cluster together as much as normal. If cultures are suspect, a drop of culture can be streaked on a YPD media plate to check for growth of yeast colonies, or a 5 ml sample can be taken to Barnes Diagnostic Center for identification of yeast strain. - [Read Removal of Yeast Contamination from Lymphoblast Cultures Protocol]

No cell culture problem is as universal as that of culture loss due to contamination. All cell culture laboratories and cell culture workers have experienced it. Culture contaminants may be biological or chemical, seen or unseen, destructive or seemingly benign, but in all cases they adversely affect both the use of your cell cultures and the quality of your research. Contamination problems can be divided into three classes: Minor annoyances, Serious problems, Major catastrophes. - [Read Understanding and Managing Cell Culture Contamination Protocol]