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A spot test in lichenology is a
spot analysis Spot analysis, spot test analysis, or spot test is a chemical test, a simple and efficient technique where analytic assays are executed in only one, or a few drops, of a chemical solution, preferably in a great piece of filter paper, without using ...
used to help identify
lichen A lichen ( , ) is a composite organism that arises from algae or cyanobacteria living among filaments of multiple fungi species in a mutualistic relationship.dichotomous keys In phylogenetics, a single-access key (also called dichotomous key, sequential key, analytical key, or pathway key) is an identification key where the sequence and structure of identification steps is fixed by the author of the key. At each point i ...
for lichen species, and they take advantage of the wide array of lichen products 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 by the botanist 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 Calcium hypochlorite is an inorganic compound with formula Ca(OCl)2. It is the main active ingredient of commercial products called bleaching powder, chlorine powder, or chlorinated lime, used for water treatment and as a bleaching agent. Thi ...
(C test), or 5% alcoholic ''p''-phenylenediamine solution (P test). The colour changes occur due to presence of particular secondary metabolites in the lichen. 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.


Tests

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


K test

The reagent for the K test is an
aqueous solution An aqueous solution is a solution in which the solvent is water. It is mostly shown in chemical equations by appending (aq) to the relevant chemical formula. For example, a solution of table salt, or sodium chloride (NaCl), in water would be re ...
of
potassium hydroxide Potassium hydroxide is an inorganic compound with the formula K OH, and is commonly called caustic potash. Along with sodium hydroxide (NaOH), KOH is a prototypical strong base. It has many industrial and niche applications, most of which exp ...
(KOH) (10–25%), or, in the absence of KOH, a 10% aqueous solution of
sodium hydroxide Sodium hydroxide, also known as lye and caustic soda, is an inorganic compound with the formula NaOH. It is a white solid ionic compound consisting of sodium cations and hydroxide anions . Sodium hydroxide is a highly caustic base and alkali ...
(NaOH, lye), which provides nearly identical results. A 10% solution of KOH will retain its effectiveness for about 6 months to a year. The test depends on salt formation and required the presence of at least one acidic functional group in the molecule. Lichen compounds that contain a
quinone The quinones are a class of organic compounds that are formally "derived from aromatic compounds
uch as benzene or naphthalene Uch ( pa, ; ur, ), frequently referred to as Uch Sharīf ( pa, ; ur, ; ''"Noble Uch"''), is a historic city in the southern part of Pakistan's Punjab province. Uch may have been founded as Alexandria on the Indus, a town founded by Alexand ...
by conversion of an even number of –CH= groups into –C(=O)– groups with any necessary rearrangement of double ...
as part of their structure will produce a dark red to violet colour. Example compounds include the pigments that are anthraquinones, naphthoquinones, and
terphenylquinone Terphenylquinones are fungal dyes from the group of phenyl-substituted ''p''-benzoquinones having the following general structure. General chemical structure of terphenylquinones Also derivatives with a central ''o''-benzoquinone structure are k ...
s. Yellow to red colours are produced with the K test and some
depside A depside is a type of polyphenolic compound composed of two or more monocyclic aromatic units linked by an ester bond. Depsides are most often found in lichens, but have also been isolated from higher plants, including species of the Ericaceae, L ...
s (including atranorin and
thamnolic acid Thamnolic acid is a β-orcinol depside with the molecular formula C19H16O11. Thamnolic acid was first isolated from the lichen '' Thamnolia vermicularis'', but it also occur in ''Cladonia ''Cladonia'' is a genus of moss-like lichens in the fa ...
), and many β- orcinol
depsidone Depsidones (+ "depside" + "one") are chemical compounds that are sometimes found as secondary metabolites in lichens. They are esters that are both depsides and cyclic ethers. An example is norstictic acid Norstictic acid is a depsidone produced ...
s. In contrast,
xanthone Xanthone is an organic compound with the molecular formula O 6H4CO. It is a white solid. In 1939, xanthone was introduced as an insecticide and it currently finds uses as Insecticide, ovicide for codling moth eggs and as a larvicide. Xanthone ...
s,
pulvinic acid Pulvinic acids are natural chemical pigments found in some lichens, derived biosynthetically from the aromatic amino acids phenylalanine and tyrosine, via dimerization and oxidative ring-cleavage of aryl pyruvic acids, a process that also produ ...
derivatives, and
usnic acid Usnic acid is a naturally occurring dibenzofuran derivative found in several lichen species with the formula C18H16O7. It was first isolated by German scientist W. Knop in 1844 and first synthesized between 1933-1937 by Curd and Robertson. Usnic a ...
do not have any reaction.


C test

This test uses a saturated solution of
calcium hypochlorite Calcium hypochlorite is an inorganic compound with formula Ca(OCl)2. It is the main active ingredient of commercial products called bleaching powder, chlorine powder, or chlorinated lime, used for water treatment and as a bleaching agent. Thi ...
(bleaching powder), or alternatively a dilute solution (5.25% is typically used) of sodium hypochlorite, or undiluted
household bleach Bleach is the generic name for any chemical product that is used industrially or domestically to remove color (whitening) from a fabric or fiber or to clean or to remove stains in a process called bleaching. It often refers specifically, to ...
. 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 Carbon dioxide (chemical formula ) is a chemical compound made up of molecules that each have one carbon atom covalently double bonded to two oxygen atoms. It is found in the gas state at room temperature. In the air, carbon dioxide is transpar ...
. Colours typically observed with the C test are red and orange-rose. Chemicals causing a red reaction include
anziaic acid Anziaic acid is a depside found in lichens. It gives a red reaction in the C test. The two phenolic rings have a pentyl side chain. It is an ester dimer of olivetolic acid. Anziaic acid works as an antibacterial compound by inhibiting topoisomera ...
, erythrin, and
lecanoric acid Lecanoric acid is a chemical produced by several species of lichen.ubChem - Lecanoric acid"> Lecanoric acid is classified as a polyphenol and a didepside and it functions as an antioxidant. The acid is named after the lichen ''Lecanora''. The acid ...
, while those resulting in orange-red include gyrophoric acid. Rarely, an emerald-green colour is produced, caused by reaction with dihydroxy dibenzofurans, such as the chemical
strepsilin Strepsilin is a chemical found in Lichen, lichens. It produces an emerald green colour in the C test. It is a dibenzofuran dimer, with hydroxyl, hydroxy, ketone, oxy and methyl side groups. It is named after ''Cladonia strepsilis''. Strepsilin was ...
.


P test

This is also known as the PD test. It uses a 1–5%
ethanolic Ethanol (abbr. EtOH; also called ethyl alcohol, grain alcohol, drinking alcohol, or simply alcohol) is an organic compound. It is an alcohol with the chemical formula . Its formula can be also written as or (an ethyl group linked to a hyd ...
solution of
para-phenylenediamine ''p''-Phenylenediamine (PPD) is an organic compound with the formula C6H4(NH2)2. This derivative of aniline is a white solid, but samples can darken due to air oxidation. It is mainly used as a component of engineering polymers and composites lik ...
(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. 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 Sodium sulfite (sodium sulphite) is the inorganic compound with the chemical formula Na2 SO3. A white, water-soluble solid, it is used commercially as an antioxidant and preservative. A heptahydrate is also known but it is less useful because of ...
, and 0.5 millilitres of
detergent A detergent is a surfactant or a mixture of surfactants with cleansing properties when in dilute solutions. There are a large variety of detergents, a common family being the alkylbenzene sulfonates, which are soap-like compounds that are more ...
in 100 millilitres of water; initially pink in colour, the solution becomes purple with age. Steiner's solution will last for months. The phenylenediamine reacts with
aldehyde In organic chemistry, an aldehyde () is an organic compound containing a functional group with the structure . The functional group itself (without the "R" side chain) can be referred to as an aldehyde but can also be classified as a formyl grou ...
s to yield Schiff bases according to the following reaction: : Products of this reaction are yellow to red in colour. Most β-orcinol depsidones and some β-orcinol depsides will react positively. PD is poisonous both as a powder and a solution, and surfaces that come in contact with it (including skin) will discolour.


KC test

This spot test may be performed by wetting the
thallus Thallus (plural: thalli), from Latinized Greek (), meaning "a green shoot" or "twig", is the vegetative tissue of some organisms in diverse groups such as algae, fungi, some liverworts, lichens, and the Myxogastria. Many of these organisms wer ...
with K followed immediately by C. The initial application of K breaks down (via
hydrolysis Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution reaction, substitution, elimination reaction, elimination, and solvation reactions in which water ...
)
ester bond In chemistry, an ester is a compound derived from an oxoacid (organic or inorganic) in which at least one hydroxyl group () is replaced by an alkoxy group (), as in the substitution reaction of a carboxylic acid and an alcohol. Glycerides ar ...
s in depsides and depsidones. If a
phenol Phenol (also called carbolic acid) is an aromatic organic compound with the molecular formula . It is a white crystalline solid that is volatile. The molecule consists of a phenyl group () bonded to a hydroxy group (). Mildly acidic, it req ...
ic hydroxyl group is released that is ''meta'' to another hydroxyl, then a red to orange colour is produced as C is applied. 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 Barbatic acid is an organic compound that is made by some lichens. It is in the structural class known as depsides. It is particularly common in the genera ''Usnea'' (the beard lichens) and ''Cladonia''. History The compound was first isolated ...
or diffractaic acid, such as is present in '' Cladonia floerkeana''.
Lugol's iodine Lugol's iodine, also known as aqueous iodine and strong iodine solution, is a solution of potassium iodide with iodine in water. It is a medication and disinfectant used for a number of purposes. Taken by mouth it is used to treat thyrotoxicosi ...
is another reagent that may be useful in identifying certain species.


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. * A 10% solution of barium hydroxide (Ba(OH)2) gives a violet colour when tested with diploschistesic acid, a chemical found in some ''
Diploschistes ''Diploschistes'' (crater lichen) is a genus of crustose lichens with a thick, cracked (areolate) body (thallus) with worldwide distribution.Field Guide to California Lichens, Stephen Sharnoff, Yale University Press, 2014, The fruiting part (ap ...
'' species. * A saturated solution of
barium peroxide Barium peroxide is the inorganic compound with the formula Ba O2. This white solid (gray when impure) is one of the most common inorganic peroxides, and it was the first peroxide compound discovered. Being an oxidizer and giving a vivid green c ...
(BaO2), when tested with olivetoric acid, will turn a yellow colour that becomes green after a few minutes. * A 1% (weight per volume) solution of
ferric chloride Iron(III) chloride is the inorganic compound with the formula . Also called ferric chloride, it is a common compound of iron in the +3 oxidation state. The anhydrous compound is a crystalline solid with a melting point of 307.6 °C. The colo ...
(FeCl3) in ethanol produces several possible colours when tested with compounds that have phenolic groups. * The N test uses a 35% solution of
nitric acid Nitric acid is the inorganic compound with the formula . It is a highly corrosive mineral acid. The compound is colorless, but older samples tend to be yellow cast due to decomposition into oxides of nitrogen. Most commercially available nitri ...
, which can be used to distinguish species of ''
Melanelia ''Melanelia'' is a genus of lichenized fungi in the family Parmeliaceae. The genus was circumscribed In geometry, the circumscribed circle or circumcircle of a polygon is a circle that passes through all the vertices of the polygon. The cent ...
'' from brown species of ''
Xanthoparmelia ''Xanthoparmelia'' (commonly known as green rock shields or rock-shield lichens) is a genus of foliose lichen in the family Parmeliaceae.Field Guide to California Lichens, Stephen Sharnoff, Yale University Press, 2014, ''Xanthoparmelia'' is syn ...
''. * The S test uses a
sulphuric acid Sulfuric acid (American spelling and the preferred IUPAC name) or sulphuric acid ( Commonwealth spelling), known in antiquity as oil of vitriol, is a mineral acid composed of the elements sulfur, oxygen and hydrogen, with the molecular formu ...
solution (0.5% to 10%) brushed over an
acetone Acetone (2-propanone or dimethyl ketone), is an organic compound with the formula . It is the simplest and smallest ketone (). It is a colorless, highly volatile and flammable liquid with a characteristic pungent odour. Acetone is miscib ...
-extracted, dried sample from a lichen thallus, followed by heating over a flame for 30 seconds or until colour develops. A persistent violet to bright pink colour indicates the presence of miriquidic acid and can be used to distinguish between the two morphologically similar
snow lichen ''Stereocaulon'' is a genus of lichens. Members of ''Stereocaulon'' are commonly called snow lichens. Species *'' Stereocaulon alpinum'' *'' Stereocaulon apocalypticum'' *'' Stereocaulon arcticum'' *'' Stereocaulon arenarium'' *'' Stereocau ...
s, ''
Stereocaulon alpinum ''Stereocaulon alpinum'' is a species of fungus belonging to the family Stereocaulaceae. It is similar to '' Stereocaulon paschale'' but differs from it in containing cyanobacteria of the genus ''Nostoc'' while ''S. paschale'' contains cyanobacter ...
'' and '' S. groenlandicum'' without having to resort to more laborious chemical analysis. *The Beilstein test involves heating a small sample of the substance to be tested on a copper wire; halogenated compounds cause a temporary deep green flame colour.


Performing spot tests

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 Soredia are common reproductive structures of lichens. Lichens reproduce asexually by employing simple fragmentation and production of soredia and isidia. Soredia are powdery propagules composed of fungal hyphae wrapped around cyanobacteria o ...
. One method is to draw up a small amount of the chemical into a glass
capillary A capillary is a small blood vessel from 5 to 10 micrometres (μm) in diameter. Capillaries are composed of only the tunica intima, consisting of a thin wall of simple squamous endothelial cells. They are the smallest blood vessels in the body: ...
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 A magnifying glass is a convex lens that is used to produce a magnified image of an object. The lens is usually mounted in a frame with a handle. A magnifying glass can be used to focus light, such as to concentrate the sun's radiation to crea ...
or a
stereo microscope The stereo, stereoscopic or dissecting microscope is an optical microscope variant designed for low magnification observation of a sample, typically using light reflected from the surface of an object rather than transmitted through it. The instr ...
. 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 Pseudocyphellae (singular ''pseudocyphella'') are structures in lichens that appear as tiny pores on the outer surface (the cortex of the lichen. They are caused when there is a break in the cortex of the lichen, and the medullary hyphae extend ...
, or the underside of squamules. In a variation of this technique, suggested by 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 ''Bryoria'' is a genus of lichenized fungi in the family Parmeliaceae. Many members of this genus are known as horsehair lichens. The genus has a widespread distribution, especially in boreal and cool temperate areas. Taxonomy ''Bryoria'' was c ...
''. 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 ''Cladonia'' is a genus of moss-like lichens in the family Cladoniaceae. They are the primary food source for reindeer/caribou. ''Cladonia'' species are of economic importance to reindeer-herders, such as the Sami in Scandinavia or the Nenets ...
'' 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.


Other tests

It may sometimes be useful to perform other diagnostic measures in addition to spot tests. For example, some lichen metabolites fluoresce under 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 ''Cladonia deformis'', also known as the lesser sulphur cup or the lesser sulphur cup lichen, is a light-coloured, fruticose, cup lichen belonging to the family Cladoniaceae. This lichen was first described as ''Lichen deformis'' by Carl Linnaeus ...
'' (UV−) and ''
Cladonia sulphurina ''Cladonia sulphurina'' is a species of cup lichen belonging to the family Cladoniaceae. As of July 2021, its conservation status has not been estimated by the IUCN. In Iceland, it is classified as an endangered species An endangered sp ...
'' (UV+, due to presence of squamatic acid). Only long-wavelength UV is useful for observing lichens directly. More advanced analytical techniques, such as thin layer chromatography,
high performance liquid chromatography High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pa ...
, and
mass spectrometry Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a ''mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is use ...
may also be useful in initially characterizing the chemical composition of lichens or when spot tests are unrevealing.


History

Finnish lichenologist William Nylander is generally considered to have been the first to demonstrate the use of chemicals to help with lichen identification. In papers published in 1866, he suggested spot tests using KOH and bleaching powder to get characteristic colour reactions—typically yellow, red, or green. In these studies he showed, for example, that the lichens now known as ''
Cetrelia cetrarioides ''Cetrelia cetrarioides'' is a species of fungus belonging to the family Parmeliaceae The Parmeliaceae is a large and diverse family of Lecanoromycetes. With over 2700 species in 71 genera, it is the largest family of lichen-forming fungi. ...
'' and '' C. olivetorum'' could be distinguished as distinct species due to their different colour reactions: C+ red in the latter, contrasted with no reaction in the former. Nylander showed how KOH could be used to distinguish between the lookalikes '' 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. In the mid-1930s,
Yasuhiko Asahina Yasuhiko Asahina (朝比奈泰彦 ''Asahina Yasuhiko''; April 16, 1881 – June 30, 1975) was a Japanese chemist and lichenologist. Early life During his childhood, Asahina developed an interest in plants. In 1902, he enrolled in the School of ...
created the test with para-phenylendiamine, which gives yellow to red reactions with secondary metabolites that have a free aldehyde group. This spot test was later shown to be particularly useful in the taxonomy of the family Cladoniaceae.


References


Cited literature

* * *{{cite book , last1=Orange , first1=A. , last2=James , first2=P.W. , last3=White , first3=F.J. , year=2001 , title=Microchemical Methods for the Identification of Lichens , publisher=British Lichen Society , isbn=978-0-9540418-0-9 Chemical tests Lichenology