Contents
| Congo Red Clemencon’s Reagent Cresyl Blue | Ferrous Sulphate Ferric Alum Crystal Immersion Oil | Melzer’s Reagent Methylene Blue Potassium Hydroxide (KOH) | Water References |
Congo Red
1% aqueous solution; saturated solution in NH40H.
Formula 1
Dissolve 1 (2) gms of Congo Red in 99 (98) cc water; filter the excess dye.
Formula 2
Saturate concentrated Ammonium hydroxide with Congo Red.
Note: The QMS currently has supplies of Formula 2.
Procedure
Place the material to be studied in a mixture of one drop of Congo Red. Place a cover slip over the material and you may now examine the section. For a better preparation, which improves the contrast of the staining reaction, place a few drops of KOH or NH4OH on one side of the cover slip and draw the liquid under the cover slip by placing a piece of absorbent material on the opposite side of the cover slip. The excess dye under the cover slip will dissolve in the KOH or NH40H and will be removed by the absorbent material (kleenex, paper towels, etc.). Continue adding KOH or NH40H until all the excess dye is removed; the sections will then appear reddish on a colorless background. Take great care not to allow any alkali on top of the coverslip or on to your lens!
Use
Congo Red will stain the walls of hyphae and is thus the best stain for seeing the shapes of cells. Sometimes used in combination with Phloxine and water (for fresh material) or ammonium hydroxide or potassium hydroxide (for dried material).
Agaricologists have used the saturated Congo Red – Ammonium hydroxide solution to study the spore wall of some agarics, e.g. Chlorophyllum, by placing the spore in a drop of this solution and a drop of 5% KOH; the complex spore wall will appear reddish in color.
Clemencon’s Reagent
Formula
80 parts IMS
20 parts 0.88 ammonia
1 part Glycerol
Procedure
Place a piece of dry tissue on a slide and then allow one or more drops of the solution to soak in to the tissue so that it is entirely covered. Wait until the solution has been absorbed and become soft but waxy. Normally this takes about the time which is required for you to go and make a cup of tea and return to your workbench. Now cut your section as if you were working with fresh material, place it on a clean slide and proceed to stain it as necessary.
Use
Many European mycologists believe that Clemencon’s reagent allows revived material to be sectioned more easily than immersion in KOH.
Cresyl Blue
A dilute aqueous solution; try 0.5 – 1.0%
Formula
Dissolve 0.5 – 1.0 gm cresyl blue in 99.5 – 99.0 ml H2 O. Allow to stand
for 5-10 minutes then filter out the excess dye.
Procedure
Place the material to be studied in one drop of cresyl blue on a glass slide; study after covering with a cover slip.
Use
Certain spore walls and various hyphae turn reddish to violet when placed in Cresyl Blue. Such a reaction is called Metachromatism and the structures reacting positively are called Metachromatic. Some examples are: the spores of Macrolepiota, the hyphae of the stipe of some species of Mycena, gloeocystidia of the Corticiaceae, walls of the cystidia of Melanoleuca, the trama of many species of Agrocybe, the basidia of Tricholoma, and the spore ornamentation in certain species of Russula. Cresyl Blue is invaluable in locating gelatinized areas of the basidiocarp, particularly the pileipellis.
Ferrous Sulphate
Formula
Dissolve 10 gms FeSO4 crystals in 100 ml distilled water, shake vigorously.
Procedure
Place one drop of the solution directly on the tissue to be studied, generally applied to the stipe or cut flesh of a basidiocarp, and observe the colour change.
Use
Commonly used in the identification of Boletus, Clavaria, Lactarius, Russula, Tricholoma and some other genera. The most common reactions are a change to salmon, green or no change in the Russulaceae, and to various shades of olivaceous green, black and purple in Boletus.
Notes
Not generally used in microscopic preparations. Information for Australian macrofungi is very poorly developed at this stage.
Ferric Alum Crystal
Ferric Alum crystals, grown by suspending small crystals in a saturated ferric alum solution are a more practical solution for determining reactions to FeSO4 in the field. They must be kept dry, usually in a sealed glass tube. Rubbing tissues with the crystal gives to same reactions as the application of Ferrous sulphate solution.
Immersion Oil
Formula
Pure Cedarwood oil with a refractive index of approximately 1.515.
Procedure
Mount your tissue section on a slide in water, KOH or your chosen stain, place a cover slip on top and ensure that there is no excess liquid around the cover slip, if there is, remove it with a tissue or blotting paper. Align the section using a low magnification lens (X40 or X60), then lower the stage, swing the lens out and place one drop of immersion oil on the cover slip over the portion of the section you wish to examine. Swing the immersion oil lens (X100) in to position and then raise the stage very slowly until your section comes in to focus. On completion of work wipe any surplus oil off your oil immersion lens with a lens cleaning tissue following your microscope manufacturer’s instructions.
Warning
Do not attempt to re-examine your section with a dry lens once you have oil on the slide, you may damage your lenses.
Use
Oil immersion procedures are an essential part of microscopic work in mycology. Many structures, particularly spores, cystidia and cells in the pileipellis require a magnification of 1000 using your X 100 oil immersion lens. All spore measurements are also carried out at this magnification.
Melzer’s Reagent
(Affectionately known as “Melzer’s”.)
Formula
Add Iodine (1.5 gm), Potassium-Iodide (5.0 gm), and Chloral Hydrate (100 gm(ml)) to H2 0 (100 ml). Warm but do not boil.
Procedure
Place the material to be studied in a drop of Melzer’s then cover.
Warning
Do not mix Melzer’s with any type of alkali, as a cloudy precipitate will develop immediately.
Notes
A positive reaction usually occurs immediately, but in doubtful cases it is best to leave the material in the solution for 20 minutes. Frequently it’s best to turn the light source of the microscope to high in order to enhance the contrast of the reaction. Three color reactions of material mounted in Melzer’s can occur. These are:
- Amyloid, the material turns blue to black, this is sometimes called a positive reaction.
- Dextrinoid, in this case the material turns brownish to red-brown.
- Inamyloid, in this case there is no reaction or occasionally a slight yellowing.
The following Genera have Dextrinoid spores: (SS = Some Species only) Crinipellis SS Cystolepiota SS Hebelornina Hygrophoropsis Paxillus SS Pseudobaeospora (most) Rozites
The following Genera have Amyloid spores: Amanita Baeospora Cantharerulla Catathelasma Clitocybula Crinipellis SS Cybellostereum Cystoderma SS Dermoloma SS Fayodia SS Hydropus SS Lactarius Lentinellus Lepiota SS Leucopaxillus Melanoleuca Mycena SS Myxomphalia Panellus Porpoloma Pseudoclitocybe Pseudoomphalina Russula Xeromphalina
Methylene Blue
1% aqueous solution
Formula
Dissolve 1 gm of Methylene Blue (Methyl Blue) in 99 cc water.
Procedure and Use
Same as for Cresyl Blue. Some agaricologists think that the resolution of the hyphal characters is better in this reagent than in Cresyl Blue.
Note
Very useful for examining cystidia, including dermatocystidia in the Russulaceae and thus avoiding the use of aggressive chemicals like sulphovanillin.
Potassium Hydroxide (KOH)
3-5% aqueous solution
Formula
Dissolve 3(-5) gms of potassium hydroxide in 97(-95) ml water.
Procedure
Place the material to be studied in a drop of potassium hydroxide on a glass slide; add Congo Red and/or phloxine if desired.
Use
3% KOH is the reagent used by most American mycologists to revive the hyphae of dried basidiocarps. It softens tissues. It is also often used to mount spores from prints and dried specimens, for examination and measurement.
Some examples of specific microchemical reactions with potassium hydroxide are as follows: incrustations of certain hyphae turn faintly yellow, internal contents of chrysocystidia turn yellow (as in Hypholoma), the pileus hairs of Crinipellis turn grey and the hyphae of the pileipellis of certain species of Cystoderma turn reddish brown.
Water
Formula
Pure distilled water
Procedure
Many mycologists like to mount a section in water when they first examine a fungus. The great advantages of a water mount are that there is generally no distortion of colours, or of the size of spores or hyphae in this mountant. It is also relatively safe for the microscope. However many microscopic structures can only be seen accurately and described using stains (see above).
References
- Charbonnel, J. Les Reactifs Mycologiques Tome 1. 1995. A good guide to all the chemicals used in mycology covering formulas, procedures, and some of the commonly used tests for separating various species. In French.
- Largent, D., Johnson, D. and Watling, R. How to Identify Mushrooms to genus III: Microscopic Features. Mad River Press 1977. ISBN 0-916-422-09-7. Contains an outline of the chemicals used in Mycology and a great deal of useful information on microscopic characters of the fungi.
- Watling, R. 1973. Identification of the Larger Fungi. Hulton Group Keys. ISBN 0 7175 0595 2. This book contains a very useful guide to preparing sections for microscopic examination. It is in the QMS Library.
Pat Leonard September 2008