Chemical Tests

Microscope work - Joe Hope, SNH Anacaun 2004

The best guide to the use of chemicals and to appropriate precautions and procedures is Microchemical Methods for the Identification of Lichens by Alan Orange et al. (reprinted 2010) available as a 102 page 8MB PDF download.

Small plastic dropper bottles in which to store your test chemicals are difficult to find on the high street, but can be found on the Internet. You might ask friends prescribed eye-drops or purchasing ear-wax softeners to recycle appropriate containers in the best possible sense and save them for you.

Commonly used test chemicals, which must be fresh/tested for freshness are:

K - potassium hydroxide
C – bleach
I – iodine
Pd - para-phenylenediamine
weak acid

These notes advise on suitable substitutes and cautions to be observed when using them. Wash any spills immediately, preferably under running water.

Some reactions are best observed under the microscope. A dissecting microscope is best for the thallus or medulla with the colour taken up onto absorbent paper. A compound microscope on x100 magnification is recommended when looking for diagnostic crystals which may form when certain chemicals are bled under the coverslip and react with the lichen.

K – potassium hydroxide

Many keys depend on the spot reactions given by potassium (K) in the form of potassium hydroxide. An easily obtained alternative may be found in a solution of caustic soda (sodium hydroxide), which can be purchased at any DIY store and a solution made up with water. The strength is not critical but about 10% caustic soda to 90% water by volume should work well. The solution should be tested on Xanthoria; if there is an almost instant crimson reaction, the strength is suitable. As it is caustic, strictly follow the warnings on the bottle; observe the usual precautions to prevent it getting on the skin, in the eyes, mouth etc.

C – bleach

Alan Orange recommends that the best source of bleach (sodium hypochlorite – C) is Milton Sterilising Fluid which you will find in the baby sections of shops; domestic bleaches may contain potassium or other ions which can give false colour reactions.

C must be fresh. Test it each time with a common lichen such as Ochrolechia androgyna; if the lichen does not give a really bright colour reaction the chemical should be discarded and fresh made up. One of the commonest errors in identification arises from false negative tests with C.

Some C reactions are fleeting and others are not very intense so it is essential to perform these tests carefully under a dissecting microscope.

Tests for C+ medulla in the field tend to be unreliable.

KC and CK.

These stains can be used sequentially, most commonly as K followed by C. First apply K and soak up the excess with absorbent paper; immediately thereafter apply C. Occasionally a diagnostic reaction requires them to be applied in the order C followed by K – different colour reactions occur with some lichen substances depending on the order of application!

I – Iodine

In addition to colour reactions, iodine in the form of Lugol’s solution is used to aid microscopical examination of asci; it is made by dissolving 0.5 g of iodine and 1.5 g of potassium iodide in 100 ml of distilled water.

Another form of iodine is Melzer’s solution which is made by dissolving 1.0 g of iodine, 1.5 g of potassium iodide in 50 ml of distilled water plus 50 g of chloral hydrate. Its advantages are that it clears the preparation, making complex structures more transparent; its high viscosity prevents flow movements of spores.

Pd - para-phenylenediamine

Pd is mutagenic, allergenic and may be carcinogenic. It is toxic by inhalation and skin contact; great care should be exercised when using it.

It is most safely used as Steiner’s solution, a stable form of this stain, made by dissolving 1 g of Pd, 10 g of sodium sulphite, and 0.5 ml of detergent in 100 ml of water.

Weak acid

Keys may ask you to decide whether a stone is siliceous or calcareous. If, when observed under a hand lens, it fizzes with lemon juice (from a plastic lemon), this should be sufficient to verify limestone.

Safe disposal

Material Safety Data Sheets (MSDS) are included with chemicals on delivery from official suppliers; if obtained privately this information should be passed on. Safety information may change with time as better understanding of hazards develops and should be kept up to date.

UV-fluorescence

A UV torch can be very useful in the field as well as in the lab. There are various UV torches on the market, some of higher quality than others, but you will need one of 365nm wavelength and 1W or 3W is usually the best choice. The lamp should be used in the dark and time allowed for the eyes to adapt to the low light levels emitted by the fluorescence. It is useful to have a fluorescent lichen (e.g. Cladonia portentosa) to hand for comparison.

All UV light, be it UVA or UVB, is hazardous, and exposure to UV light should be kept to a minimum. Be sure to use a protective screen or shield if advised, and do not look directly at the light. UV light can cause skin cancer and damage to eyes.

SUMMARY OF SIMPLE LICHEN TESTS
Many identification keys use the results of some very simple tests which are suitable for use by beginners to lichenology. These tests are summarized here.
Reagents/ tests	Formula	Positive reactions		Examples (use these species to test that the reagent is working)	Notes
K	10% solution of potassium hydroxide in water; replace when old or cloudy.	yellow, red (dirty yellow due merely to clearing effect is negative); some turn slowly red forming microscopic crystals		Phlyctis argena (red with crystals), Pertusaria corallina (bright yellow), Cladonia polydactyla (yellow) , Xanthoria parietina (red-purple)	May help to apply K, then after a while draw solution onto filter paper to help see pale reactions.
C	sodium hypochlorite solution (Milton's Sterilising Fluid recommended); replace frequently	pink, red, orange ( rarely green)		Ochrolechia androgyna, Melanelixia subaurifera (medulla pink or red)	Reactions often fleeting, only a second or two!
KC	K followed by C on the same fragment	pink, red, rarely violet		Hypogymnia physodes (medulla red), Pertusaria amara (soralia violet)	May help to apply K, then draw solution onto filter paper, add C to paper.
PD	a few crystals dissolved in alcohol (Industrial Methylated Spirit) in a watch glass	yellow, orange, red		Parmelia sulcata, Hypogymnia physodes (medulla orange in both), Cladonia pyxidata (orange-red)	PD is toxic and may cause cancer! Reactions may be slow (1-2 minutes).
I	0.5 g iodine, 1.5 g potassium iodide, dissolved in 100 ml water	blue, violet, rarely red		Porpidia tuberculosa, Lecidea lactea (medulla violet in both)	Pretreatment with K may help. I is not used in the field.
UV	long-wave ultra-violet (350 nm) from mains or portable lamp	fluorescence, usually white, bluish or orange		Cladonia portentosa, C. squamosa var. squamosa (whitish)	Cortex may mask fluorescence, try abrading the surface.
IN THE FIELD: Carry reagents K and C in stout brown glass dropper bottles. Do not use PD in the field as it is hazardous; I is not used in the field. Add a small drop of reagent to the lichen – use as little as possible as it will kill the parts it touches. Observe with a hand-lens if possible; reactions may be fleeting – observe while testing, or immediately afterwards! Try scraping very dark thalli to reveal paler tissues within. When using UV, shade the lichen from daylight.			IN THE LABORATORY: Add a small drop of reagent to a small piece removed from the rest of the lichen (to avoid ruining the whole specimen). Watch the test with a dissecting microscope if possible. Reactions may be fleeting – observe while testing! Try scraping or sectioning very dark dark thalli to reveal paler tissues. Add I to sections or squashes – it is usually necessary to use a high power microscope. Use UV lamp in a darkened room.