A spot test in lichenology is a spot analysis used to help identify lichens. It is performed by placing a drop of a chemical reagent on different parts of the lichen and noting the colour change (or lack thereof) associated with application of the chemical. The tests are routinely encountered in dichotomous keys for lichen species, and they take advantage of the wide array of lichen products (secondary metabolites) produced by lichens and their uniqueness among taxa. As such, spot tests reveal the presence or absence of chemicals in various parts of a lichen. They were first proposed as a method to help identify species by the Finnish lichenologist William Nylander in 1866.
Three common spot tests use either 10% aqueous KOH solution (K test), saturated aqueous solution of bleaching powder or calcium hypochlorite (C test), or 5% alcoholic p-phenylenediamine solution (P test). The colour changes occur due to presence of particular secondary metabolites in the lichen. In identification key reference literature, the outcome of chemical spot tests serves as a primary characteristic for determining the species of lichens. There are several other less frequently used spot tests of more limited use that are employed in specific situations, such as to distinguish between certain species. Variations of the technique, including using filter paper to enhance visibility of reactions or examining under a microscope, accommodate different lichen types and pigmentations, with results typically summarised by a short code indicating the substance and reaction observed. Other diagnostic methods like ultraviolet (UV) light exposure can help identify lichen metabolites and distinguish between species, as some substances fluoresce under UV, aiding in the differentiation of closely related species.