Hanks' Balanced Salts is designed to equilibrate with air, hence does not require CO2 air mixture. All media used in tissue culture have a basis of a synthetic mixture of inorganic salts known as a physiological or balanced salt solution (BSS). All the physiological salt solutions have been derived from the salt solution originally described by Sydney Ringer (1885). The first balanced salt solution to be developed specifically for supporting the metabolism of mammalian cells was Tyrode's solution. Since then many modifications have been done to obtain better buffering salt solutions and to prevent calcium precipitation. 1X powdered Hanks' Balanced Salts mixture contains phenol red designed for use with cells maintained in less CO2 environment or CO2 free environment.