Spot tests are performed by placing a small amount of the desired reagent on the portion of the lichen to be tested. Often, both the cortex and medulla of the lichen are tested, and at times it is useful to test other structures such as soralia. One method is to draw up a small amount of the chemical into a glass capillary and touch it to the lichen thallus; a small paint brush is also used for this purpose. Reactions are best visualised with a hand lens or a stereo microscope. A razor blade may be used to remove the cortex and access the medulla. 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.
In a variation of this technique, suggested by the Swedish chemist Johan Santesson, a piece of filter paper is used to try to make the colour reaction more readily observable. The lichen fragment is pressed on the paper, and lichen substances are extracted with 10–20 drops of acetone. After evaporating the acetone, the lichen substances are left on the paper in a ring around the lichen fragment. The filter paper can then be spot tested in the usual way. 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. This technique is useful when testing lichens with dark pigments, such as Bryoria.
Spot tests may be used individually or in combination. The results of a spot tests are typically represented with a short code that includes, in order, (1) a letter indicating the reagent used, (2) a "+" or "−" sign indicating a colour change or lack of colour change, respectively, and (3) a letter or word indicating the colour observed. In addition, care should be taken to indicate which part of the lichen was tested. For example, "Cortex K+ orange, C−, P−" means the cortex of the test specimen turned orange with application of KOH and did not change under bleach or para-phenylenediamine. Similarly, "Medulla K−, KC+R" would indicate the medulla of the lichen was insensitive to application of KOH, but application of KOH followed immediately by bleach caused the medulla to turn red.
Occasionally, it takes some time for the colour reaction to develop. For example, in certain Cladonia species, the PD reaction with fumarprotocetraric acid can take up to half a minute. 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. There are several possible reasons that an anticipated test result does not occur. Causes include old and chemically inactive reagents, and low concentrations of lichen substances in the sample. If the colour of the thallus is dark, a colour change might be obscured, and other techniques are more appropriate, like the filter paper technique.