Media and buffers

Most media in use today are chemically defined, and are supplemented with 5–20% serum (which contains stimulants and other components necessary for cell division), although serum-free medium with added stimulants is used for an increasing number of cell types. Media contain an isotonic balanced salts solution with amino acids, vitamins, and glucose. In addition to serum, media can also be supplemented with antibiotics (usually penicillin and streptomycin) to help prevent bacterial contamination. Generally, mammalian cells divide well between pH 7.0 and 7.4 but cell metabolic processes affect this narrow range rapidly – exposure to oxygen causes the pH to rise and metabolic production of lactic acid causes the pH to fall. A buffer is thus incorporated into the culture medium, commonly sodium bicarbonate, in conjunction with an incubator atmosphere containing 5% CO₂. These compounds work together to absorb or release hydrogen (H⁺) ions, maintaining the pH of the medium within a narrow range. It is critical to monitor the changing pH of a culture, and most media contain phenol red as a pH indicator. The compound allows for a visual measurement of pH and it is red at pH 7.4. As the  cells divide  over several days the  production of lactic  acid  outstrips the  buffering capacity of the  bicarbonate/CO2  system, and  the  indicator becomes orange at pH 7.0 and  yellow at pH 6.5. A failure of cell division within the culture would mean that the medium would become more alkaline; hence the indicator would become pink at pH 7.6 and then purple at pH 7.8.