TMPD dihydrochloride, a readily oxidizable compound, is an enzymatically convert redox active substrate molecule. TMPD dihydrochloride is also an electron donor and serves as a reducing cosubstrate for heme peroxidases[2]. TMPD dihydrochloride is also a complex IV substrate.In Vitro: TMPD (N,N,N′,N′-tetramethyl-para-phenylene-diamine) is used in cell culture and microbiology to differentiate organisms that exhibit cytochrome c oxidase activity and to distinguish between Gram-negative and Gram-positive pathogenic and non-pathogenic bacteria. The concept of TMPD oxidation by microorganisms is widely used and is known as a successful colorimetric indicator for bacterial oxidases, as the radical cation TMPD+˙, formed by oxidation, shows a characteristic deep blue colour. The electrochemical recognition of the TMPD oxidation by bacterial oxidases can be applied to a variety of pathogens.