Columbia Agar is a modification of the original Columbia Agar formulation described by Ellner et al. from Columbia University and is tested in accordance with ISO 11133:2014. Columbia Agar provides a medium that is suitable for use with both defibrinated horse and sheep blood to support the growth and determining haemolytic reactions of a variety of microorganisms.

Columbia Agar with the addition of plain or chocolated blood is recommended by the UK Standards for Microbiology Investigations for various purposes, including as a standard, supplementary or primary isolation medium. Columbia agar with blood is a general-purpose, non-selective medium suitable for isolation and cultivation of most organisms including fastidious anaerobes from clinical specimens. Chocolating the blood provides a highly nutritious medium which supports the growth of a wide range of pathogens including the most fastidious organisms and is particularly useful for the cultivation of Haemophilus and Neisseria species.

Chocolate agar is the same as blood agar except that during preparation the red blood cells are lysed when added to molten agar base, which gives the medium a chocolate-brown colouration and from which the agar gets its name. As a result, cell lysis releases intracellular nutrients such as haemoglobin, haemin (X factor), and the coenzyme nicotinamide adenine dinucleotide (NAD or V factor) into the agar. These growth factors are available to fastidious organisms during growth.