Spot test (lichen)

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A spot test in

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.[1]

Three common spot tests use either 10% aqueous

fluoresce
under UV, aiding in the differentiation of closely related species.

Tests

Spot tests on the foliose lichen Punctelia borreri showing thallus (top) and medulla (bottom). The pinkish-red colour change of the medulla in the C and KC tests indicate the presence of gyrophoric acid, a chemical feature that helps to distinguish it from similar species in the same genus.[2]

Four spot tests are used most commonly to help with lichen identification.[3]

K test

The reagent for the K test is an aqueous solution of potassium hydroxide (KOH) (10–25%), or, in the absence of KOH, a 10% aqueous solution of sodium hydroxide (NaOH, lye), which provides nearly identical results.[4] A 10% solution of KOH will retain its effectiveness for about 6 months to a year.[5] The test depends on salt formation and requires the presence of at least one acidic functional group in the molecule. Lichen compounds that contain a quinone as part of their structure will produce a dark red to violet colour. Example compounds include the pigments that are anthraquinones, naphthoquinones, and terphenylquinones. Yellow to red colours are produced with the K test and some depsides (including atranorin and thamnolic acid), and many β-orcinol depsidones. In contrast, xanthones, pulvinic acid derivatives, and usnic acid do not have any reaction.[4]

Some common and widely distributed lichens that have lichen products with a positive reaction to K include

didepside compound baeomycesic acid.[6]

C test

This test uses a saturated solution of

household bleach. These solutions are typically replaced daily since they break down within 24–48 hours; they break down even more rapidly when exposed to sunlight (less than an hour) and so are recommended to keep in a dark-coloured bottle. Other factors that accelerate the decomposition of these solutions are heat, humidity, and carbon dioxide.[7]

Colours typically observed with the C test are red and orange-rose. Chemicals causing a red reaction include anziaic acid, erythrin, and lecanoric acid, while those resulting in orange-red include gyrophoric acid.[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.[10]

Some common and widely distributed lichens that have lichen products with a positive reaction to C include Lecanora expallens, which is C+ (orange) because of the xanthone thiophanic acid, and Diploschistes muscorum, which is C+ (red) because of the didepside diploschistesic acid.[10]

PD test

p-phenylenediamine

This is also known as the P test. It uses a 1–5%

para-phenylenediamine (PD), made by placing a drop of ethanol (70–95%) over a few crystals of the chemical; this yields an unstable, light sensitive solution that lasts for about a day.[11] An alternative form of this solution, called Steiner's solution, is much longer lasting although it produces less intense colour reactions. It is typically prepared by dissolving 1 gram of PD, 10 grams of sodium sulfite, and 0.5 millilitres of detergent in 100 millilitres of water; initially pink in colour, the solution becomes purple with age. Steiner's solution will last for months.[5] The phenylenediamine reacts with aldehydes to yield Schiff bases according to the following reaction:[9]

R−CHO + H2N−C6H4−NH2 → R−CH=N−C6H4−NH2 + H2O

Products of this reaction are yellow to red in colour. Most β-orcinol depsidones and some β-orcinol depsides will react positively.[11] The PD test, known for its high specificity towards substances that yield K+ yellow or red reactions, has largely replaced the simpler yet less conclusive K test.[12] PD is poisonous both as a powder and a solution, and surfaces that come in contact with it (including skin) will discolour.[13]

Some common and widely distributed lichens that have lichen products with a positive reaction to P include

Parmelia subrudecta, which is PD+ (yellow) because of the didepside atranorin, and Hypogymnia physodes, which is PD+ (orange) because of the depsidone physodalic acid.[14]

KC test

This spot test may be performed by wetting the

ester bonds in depsides and depsidones. If a phenolic hydroxyl group is released that is meta to another hydroxyl, then a red to orange colour is produced as C is applied.[15] Alectoronic acid and physodic acid produce this colour, while a violet colour results when picrolichenic acid is present. The CK test is a less commonly used variation that reverses the order of the application of chemicals. It is used in special cases when testing for orange colour produced by barbatic acid or diffractaic acid, such as is present in Cladonia floerkeana.[8] Lugol's iodine is another reagent that may be useful in identifying certain species.[16]

Hypogymnia tubulosa is a lichen that is KC+ (orange-pink) because of the depsidone physodic acid; Cetrelia olivetorum is KC+ (pink-red) due to the depsidone alectoronic acid.[10]

Less common tests

There are several spot tests that are infrequently used due to their limited applicability, but may be useful in situations where particular lichen metabolites need to be detected, or to distinguish between certain species when other tests are negative.

Performing spot tests

Chemical spot tests on the crustose and saxicolous lichen Aspicilia epiglypta

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

pseudocyphellae, or the underside of squamules.[19]

In a variation of this technique, suggested by the Swedish chemist Johan Santesson,[20] 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.[21] 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.[8] This technique is useful when testing lichens with dark pigments, such as Bryoria.[5]

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.[12]

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.[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. 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.[8]

Other tests

apothecia of the crustose lichen Ochrolechia africana; the yellowish colour results from the fluorescence of lichexanthone.[22]

It may sometimes be useful to perform other diagnostic measures in addition to spot tests. For example, some lichen metabolites

ultraviolet radiation such that exposing certain parts of the lichen to a UV light source can reveal the presence or absence of those metabolites similarly to spot tests. Examples of lichen substances that give a bright fluorescence in UV are alectoronic, lobaric, and divaricatic acids, and lichexanthone. In some cases, the UV light test can be used to help distinguish between closely related species, such as Cladonia deformis (UV−) and Cladonia sulphurina (UV+, due to presence of squamatic acid).[19] Only long-wavelength UV is useful for observing lichens directly.[5]

More advanced analytical techniques, such as thin-layer chromatography, high-performance liquid chromatography, and mass spectrometry may also be useful in initially characterising the chemical composition of lichens or when spot tests are unrevealing.[23]

History

Finnish lichenologist

Xanthoria candelaria and Candelaria concolor because the presence of parietin in the former species results in a strong colour reaction. 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.[24] 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.[27][28] This spot test was later shown to be particularly useful in the taxonomy of the family Cladoniaceae.[29][24]

See also

References

  1. ^
    doi:10.1111/j.1095-8339.1866.tb01301.x
    .
  2. ^ Truong, Camille; Clerc, Philippe (2003). "The Parmelia borreri group (lichenized ascomycetes) in Switzerland". Botanica Helvetica. 113 (1): 49–61.
  3. ISBN 0-87071-394-9
    .
  4. ^ a b Ahmadjian & Hale 1973, p. 636.
  5. ^
    OCLC 862053107
    .
  6. ^ Orange, James & White 2001, p. 15.
  7. ^ Ahmadjian & Hale 1973, p. 635.
  8. ^ a b c d e f Walker, F.J.; James, P.W. (May 1980). "A revised guide to microchemical techniques for the identification of lichen products". Bulletin of the British Lichen Society. 46 (Supplement): 13–29.
  9. ^
    ISBN 978-81-322-2180-7
    .
  10. ^ a b c Orange, James & White 2001, p. 16.
  11. ^ a b Ahmadjian & Hale 1973, pp. 636–637.
  12. ^
    ISBN 978-0-7131-2456-9
    .
  13. ^ a b Dahl & Krog 1973, p. 23.
  14. ^ Orange, James & White 2001, p. 17.
  15. ^ Ahmadjian & Hale 1973, p. 637.
  16. ISBN 978-0300082494
    .
  17. ^ Orange, James & White 2001, p. 9.
  18. ^
    doi:10.1017/s0024282913000418
    .
  19. ^ a b Dahl & Krog 1973, p. 24.
  20. doi:10.3891/acta.chem.scand.21-1162
    .
  21. ^ Ahmadjian & Hale 1973, p. 634.
  22. doi:10.1139/b91-099
    .
  23. ^ "Arizona State University Lichen Herbarium: Lichen TLC". nhc.asu.edu. Retrieved 18 September 2016.
  24. ^
    JSTOR 3244891
    .
  25. ^ Nylander, W. (1866). "Circa novum in studio lichenum criterium chemicum" [A new chemical criterion in the study of lichen]. Flora (in Latin). 49: 198–201.
  26. ^ Nylander, W. (1866). "Quaedam addenda ad nova criteria chemica in studio lichenum" [New criteria to be added to the chemical study of lichens]. Flora (in Latin). 49: 233–234.
  27. ^ Asahina, Y. (1934). "Über die Reaktion vom Flechten-Thallus" [About the response from the lichen thallus]. Acta Phytochimica (in German). 8: 47–64.
  28. ^ Asahina, Y. (1936). "Mikrochemischer Nachweis der Flechtenstoffe (I)" [Microchemical detection of lichen substances (I)]. The Journal of Japanese Botany (in German). 12: 516–525.
  29. JSTOR 40597010
    .

Cited literature
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