Microcrystallization (or microcrystal test) is a method for identifying lichen metabolites that was predominantly used before the advent of more advanced techniques such as

thin-layer chromatography Thin-layer chromatography (TLC) is a chromatography technique that separates components in non-volatile mixtures. It is performed on a TLC plate made up of a non-reactive solid coated with a thin layer of adsorbent material. This is called the sta ...

and

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 specific components in mixtures. The mixtures can origin ...

. Developed primarily by Yasuhiko Asahina, this approach relies on the formation of distinctive

crystal A crystal or crystalline solid is a solid material whose constituents (such as atoms, molecules, or ions) are arranged in a highly ordered microscopic structure, forming a crystal lattice that extends in all directions. In addition, macros ...

s from lichen

extract An extract (essence) is a substance made by extracting a part of a raw material, often by using a solvent such as ethanol, oil or water. Extracts may be sold as tinctures or absolutes or dried and powdered. The aromatic principles of ma ...

s. Although now superseded by modern analytical methods, microcrystallization still holds importance for compound purification and analysis using

X-ray crystallography X-ray crystallography is the experimental science of determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to Diffraction, diffract in specific directions. By measuring th ...

History

Between 1936 and 1940, Japanese chemist and lichenologist Yasuhiko Asahina published a series of papers in the '' Journal of Japanese Botany'' detailing the microcrystallization technique. This simple and rapid method allowed for the identification of major metabolites in hundreds of lichen species, contributing significantly to taxonomic research. The technique was introduced to western lichenologists in a 1943 publication by Alexander Evans, and was used regularly until more advanced techniques such as

and

were introduced and integrated into laboratories. Decades of research on the

secondary metabolite Secondary metabolites, also called ''specialised metabolites'', ''secondary products'', or ''natural products'', are organic compounds produced by any lifeform, e.g. bacteria, archaea, fungi, animals, or plants, which are not directly involved ...

s of lichens culminated in the publication of ''Identification of Lichen Substances'', a 1996 work by Siegfried Huneck and Isao Yoshimura, that summarized analytical data for hundreds of lichen molecules, including images of microcrystals. Ultimately, the microcrystallization method had limitations, as it was unable to detect minor components or analyze complex mixtures of lichen substances. Despite these drawbacks, microcrystallization played a crucial role in the study of correlations between lichen chemistry,

morphology Morphology, from the Greek and meaning "study of shape", may refer to: Disciplines *Morphology (archaeology), study of the shapes or forms of artifacts *Morphology (astronomy), study of the shape of astronomical objects such as nebulae, galaxies, ...

, and geographic distribution.

Procedure

To perform microcrystallization, a small piece of lichen is extracted using

acetone Acetone (2-propanone or dimethyl ketone) is an organic compound with the chemical formula, formula . It is the simplest and smallest ketone (). It is a colorless, highly Volatile organic compound, volatile, and flammable liquid with a charact ...

or other

solvent A solvent (from the Latin language, Latin ''wikt:solvo#Latin, solvō'', "loosen, untie, solve") is a substance that dissolves a solute, resulting in a Solution (chemistry), solution. A solvent is usually a liquid but can also be a solid, a gas ...

s, filtered, and evaporated to yield a residue. The residue is transferred to a

microscope slide A microscope slide is a thin flat piece of glass, typically 75 by 26 mm (3 by 1 inches) and about 1 mm thick, used to hold objects for examination under a microscope. Typically the object is mounted (secured) on the slide, and then ...

, and a drop of microcrystallization reagent is added before capping with a

cover glass Cover or covers may refer to: Packaging * Another name for a lid * Cover (philately), generic term for envelope or package * Album cover, the front of the packaging * Book cover or magazine cover ** Book design ** Back cover copy, part o ...

. Commonly used

reagent In chemistry, a reagent ( ) or analytical reagent is a substance or compound added to a system to cause a chemical reaction, or test if one occurs. The terms ''reactant'' and ''reagent'' are often used interchangeably, but reactant specifies a ...

s include GAW (H₂O/

glycerol Glycerol () is a simple triol compound. It is a colorless, odorless, sweet-tasting, viscous liquid. The glycerol backbone is found in lipids known as glycerides. It is also widely used as a sweetener in the food industry and as a humectant in pha ...

ethanol Ethanol (also called ethyl alcohol, grain alcohol, drinking alcohol, or simply alcohol) is an organic compound with the chemical formula . It is an Alcohol (chemistry), alcohol, with its formula also written as , or EtOH, where Et is the ps ...

1:1:1, v/v/v) and GE (

acetic acid Acetic acid , systematically named ethanoic acid , is an acidic, colourless liquid and organic compound with the chemical formula (also written as , , or ). Vinegar is at least 4% acetic acid by volume, making acetic acid the main compone ...

/glycerol 1:3). Slides using GE or GAW are gently heated and then allowed to cool, promoting the crystallization process. Once formed, crystals are best observed under

polarized light , or , is a property of transverse waves which specifies the geometrical orientation of the oscillations. In a transverse wave, the direction of the oscillation is perpendicular to the direction of motion of the wave. One example of a polarize ...

with a 200–1,000-fold magnification. This method requires basic laboratory equipment, including a

microscope A microscope () is a laboratory equipment, laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic ...

equipped for polarized light,

test tube A test tube, also known as a culture tube or sample tube, is a common piece of laboratory glassware consisting of a finger-like length of glass or clear plastic tubing, open at the top and closed at the bottom. Test tubes are usually placed in s ...

pipette A pipette (sometimes spelled as pipet) is a type of laboratory tool commonly used in chemistry and biology to transport a measured volume of liquid, often as a media dispenser. Pipettes come in several designs for various purposes with differing ...

s, a micro

spirit-lamp A portable stove is a cooking stove specially designed to be portable and lightweight, used in camping, picnicking, backpacking, or other use in remote locations where an easily transportable means of cooking or heating is needed. Portable stove ...

or micro

Bunsen burner A Bunsen burner, named after Robert Bunsen, is a kind of ambient air gas burner used as laboratory equipment; it produces a single open gas flame, and is used for heating, sterilization, and combustion. The gas can be natural gas (which is main ...

spatula A spatula is a broad, flat, flexible blade used to mix, spread and lift material including foods, drugs, plaster and paints. In medical applications, "spatula" may also be used synonymously with tongue depressor. The word ''spatula'' derives ...

scalpel A scalpel or bistoury is a small and extremely sharp bladed instrument used for surgery, anatomical dissection, podiatry and various handicrafts. A lancet is a double-edged scalpel. Scalpel blades are usually made of hardened and tempered ...

, and microscope slides and cover glasses. Lichen substances can be identified based on the distinctive shape and color of their crystals.

Identification and interpretation

The process of crystal identification involves comparing them to images of crystals in different solvents found in published sources. Although the shape of the crystals depends on the solvent and, to a certain degree, the substance concentration, it is usually possible to recognize the fundamental crystalline forms. Care should be taken to differentiate between undissolved substances, which might be crystalline but lack a characteristic shape, and recrystallized substances. Microcrystal samples cannot be preserved for long, as they start to degrade within hours or days. Distinguishing between gyrophoric acid and lecanoric acid using thin-layer chromatography can be challenging. However, if one of these substances is known to be present, a microcrystal test can help differentiate them. In the GAW solvent system, lecanoric acid forms long, curved crystal clusters, although the results can be inconsistent, especially in the presence of other substances. Gyrophoric acid, when present in the GE solvent system, may manifest as small, fine crystal clusters or rounded aggregations of tiny crystals. Lecanoric acid in the GE solvent system produces needle-like crystal clusters, but these are not as well-formed as in GAW. These tests can help distinguish '' Punctelia borreri'' (which contains gyrophoric acid) from '' Punctelia subrudecta'' (which contains lecanoric acid). When two substances generate similar-looking crystals, their optical properties can be used to differentiate between them. Certain crystals alter the polarization plane of transmitted light, and when rotated between crossed polarizers, they alternate between bright and dark every 90°. The

extinction angle Extinction is a term used in optical mineralogy and petrology, which describes when cross-polarized light dims, as viewed through a thin section of a mineral in a petrographic microscope. Isotropic minerals, opaque (metallic) minerals, and amo ...

is the angle between a specific crystal axis and the filter's polarization plane when the crystal appears dark (in extinction). For instance, this method can be employed to distinguish between perlatolic acid and imbricaric acid, which both form long, straight crystals in the GE solvent system but exhibit extinction angles of 0° and 45°, respectively, in relation to their long axis.

References

{{Reflist, colwidth=30em, refs= {{cite journal , last1=Asahina , first1=Y. , year=1936 , title=Mikrochemischer nachweiss der Flechtenstoffe (I) , journal=Journal of Japanese Botany , volume=12 , pages=516–525 , language=de {{cite journal , last1=Evans , first1=Alexander W. , year=1943 , title=Asahina's microchemical studies on the Cladoniae , journal=Bulletin of the Torrey Botanical Club , volume=70 , issue=2 , pages=139–151 , doi=10.2307/2481365 , jstor=2481365 {{cite book , editor-last1=Upreti , editor-first1=Dalip Jumar , editor-last2=Divakar , editor-first2=Pradepp K. , editor-last3=Shukla , editor-first3=Vertika , editor-last4=Bajpal , editor-first4=Rajesh , first1=Pierre , last1=Le Pogam , first2=Gaëtan , last2=Herbette , first3=Joël , last3=Boustie , year=2015 , chapter=Analysis of Lichen Metabolites, a Variety of Approaches , title=Modern Methods and Approaches in Biomonitoring and Bioprospection , series=Recent Advances in Lichenology , pages=229–261 , publisher=Springer India , isbn=978-81-322-2180-7 {{cite book , last1=Elix , first1=J.A. , last2=Stocker-Wörgötter , first2=E. , chapter=Biochemistry and secondary metabolites , editor-first=Thomas H. , editor-last=Nash III , title=Lichen Biology , edition=2nd , publisher=Cambridge University Press , location=Cambridge, UK , year=2008 , pages=118–119, isbn=978-0-521-69216-8 {{cite book , last=Huneck , first=Siegfried , last2=Yoshimura , first2=Isao , title=Identification of Lichen Substances , publisher=Springer Berlin Heidelberg , publication-place=Berlin, Heidelberg , year=1996 , isbn=978-3-642-85245-9 , oclc=851387266 , page=47 {{cite journal , last=Mitchell , first=M.E. , year=2014 , title=De Bary's legacy: the emergence of differing perspectives on lichen symbiosis , journal=Huntia , volume=15 , issue=1 , pages=5–22 36–137, url=https://www.huntbotanical.org/admin/uploads/02hibd-huntia-15-1-pp05-22.pdf {{cite book , editor-last1=Galun , editor-first1=Margalith , last1=Galun , first1=Margalith , last2=Shomer-Ilan , first2=Adiva , chapter=Secondary Metabolic Products , volume=III , year=1988 , title=CRC Handbook of Lichenology , publisher=CRC Press , location=Boca Raton , page=134 , isbn=978-0-8493-3583-9 {{cite journal , last1=Olivier-Jimenez , first1=Damien , last2=Chollet-Krugler , first2=Marylène , last3=Rondeau , first3=David , last4=Beniddir , first4=Mehdi A. , last5=Ferron , first5=Solenn , last6=Delhaye , first6=Thomas , last7=Allard , first7=Pierre-Marie , last8=Wolfender , first8=Jean-Luc , last9=Sipman , first9=Harrie J.M. , last10=Lücking , first10=Robert , last11=Boustie , first11=Joël , last12=Le Pogam , first12=Pierre , title=A database of high-resolution MS/MS spectra for lichen metabolites , journal=Scientific Data , volume=6 , issue=1 , year=2019 , pmid=31780665 , pmc=6882832 , doi=10.1038/s41597-019-0305-1 , page=e294 , bibcode=2019NatSD...6..294O {{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 , pages=40–43 {{cite journal , last=Shibata , first=Shoji , title=Yasuhiko Asahina (1880–1975) and his studies on lichenology and chemistry of lichen metabolites , journal=The Bryologist , volume=103 , issue=4 , year=2000 , doi=10.1639/0007-2745(2000)103 710:yaahso.0.co;2 , pages=710–719

External links

Microcrystal Tests for Lichen Substances
YouTube tutorial presented by Yoshihito Ohmura,

National Museum of Nature and Science The is in the northeast corner of Ueno Park in Tokyo. The museum has exhibitions on pre-Meiji period, Meiji science in Japan. It is the venue of the taxidermied bodies of the legendary dogs Hachikō and Taro and Jiro. A life-size blue whale mod ...

Lichenology Crystallography

History

Procedure

Identification and interpretation

See also

References

External links