The present invention relates to novel chromogenic substrates which are used to detect aminopeptidase activity in microorganisms or to determine whether at least one bacterium belongs to the Gram-positive group or to the Gram-negative group according to the color thereof. The invention also relates to culture media containing such substrates, to the use of the substrates or media for the detection of aminopeptidase activities and/or the differentiation of Gram-positive bacteria from Gram-negative bacteria and to methods of use.The aforementioned novel substrates have the formula below: ##STR00001## in which: R.sub.1 is nothing or an alkyl, allyl or aryl group, R.sub.2 consists of at least one amino acid, preferably alanine, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 consist, independently of one another, of H-- or --O-alkyl, preferably --O--CH.sub.3, R.sub.7 consists of H, O--CH.sub.3, alkyl or halogen, R.sub.8 consists of H or Cl, and n is an integer corresponding to 0 or 1. The invention is particularly suitable for use in the field of diagnostics.