Spot test (lichen)
In identification key reference literature, the outcome of chemical spot tests serves as a primary characteristic for determining the species of lichens.
Lichen compounds that contain a quinone as part of their structure will produce a dark red to violet colour.
Yellow to red colours are produced with the K test and some depsides (including atranorin and thamnolic acid), and many β-orcinol depsidones.
[8] Rarely, an emerald-green colour is produced, caused by reaction with dihydroxy dibenzofurans,[9] such as the chemical strepsilin.
[8] Another rare colour produced by this test is yellow, which is observed with Cladonia portentosa as a result of the dibenzofuran usnic acid.
[14] This spot test may be performed by wetting the thallus with K followed immediately by C. The initial application of K breaks down (via hydrolysis) ester bonds in depsides and depsidones.
Alternatively, the solution can be applied to lichen features that lack a cortex or that leave the medulla exposed, such as soralia, pseudocyphellae, or the underside of squamules.
[20] In a variation of this technique, suggested by the Swedish chemist Johan Santesson,[21] a piece of filter paper is used to try to make the colour reaction more readily observable.
[22] In cases where the results of a spot test on the thallus are uncertain, it is possible to squash a thin section of the tissue on a microscope slide in a minimal amount of water and reagent under a cover slip.
A colour change is visible under a low-power microscope objective, or when the slide placed against a white background.
For example, in certain Cladonia species, the PD reaction with fumarprotocetraric acid can take up to half a minute.
[13] In contrast, the reactions with C and KC are usually fleeting and occur within a second of applying the reagent, so a colour change can easily be missed.
Examples of lichen substances that give a bright fluorescence in UV are alectoronic, lobaric, and divaricatic acids, and lichexanthone.
[25] In papers published in 1866, he suggested spot tests using KOH and bleaching powder to get characteristic colour reactions—typically yellow, red, or green.
He also knew that in some cases the lichen chemicals were not evenly distributed throughout the cortex and the medulla due to the differing colour reactions on these areas.
[25] In the mid-1930s, Yasuhiko Asahina created the test with para-phenylendiamine, which gives yellow to red reactions with secondary metabolites that have a free aldehyde group.
