B. Phloxine (particularly good for Homobasidiomycetes & filamentous fungi):
*Stain: 1% aqueous phloxine.
*Accompaniments: 70% ethyl alcohol; 5% aqueous KOH.
C. Melzer's iodine reagent (specifically for amyloid reaction):
100 g chloral hydrate;
5 g potassium iodide;
1.5 g iodine;
100 ml distilled water.
A. General method:
*Tease a small amount of tissue or hyphae into a few drops of alcohol (removes air); blot near edge of material or allow to evaporate
*Add a drop of KOH (expands dry material)
*Gently rinse preparation with distilled water; blot near edge of material
*Follow with a drop of stain
*Add a drop of mounting medium (water for temporary; Karo or Hoyer's for permanent - see below)
*Carefully place a coverslip on the material for microscopic examination
B. Clear polyester adhesive tape method (particularly good for showing the natural orientation of dry spores)
*Note. Transparent polyester film tape 850 is available from 3M. It is very thin, unaffected by water, and optically clear; cellophane tapes are generally unsatisfactory on all of these counts.
1. Put a small drop of either distilled water or clear permanent mounting medium on a clean slide
2. Using scissors and forceps, cut off a small piece of tape (a couple of mm's square), from the roll of tape: BE CAREFUL NOT TO TOUCH THE TAPE WITH YOUR FINGERS
3. Working under the dissecting microscope, hold the small piece of tape with the forceps and touch the sticky side gently to the fungal colony
4. Next, place the piece of tape sticky side up on the drop on the slide
5. Add a small drop of stain to the fungal material;
6. Add a larger drop of distilled water or permanent mounting medium
7. Carefully lay a cover slip on the preparation
AQUEOUS STAINED PREPARATIONS (do not need to be ringed)
C. Hoyer's fluid (see Cunningham, 1972)
1. Ingredients: 15g gum arabic or guiac; 100 g chloral hydrate; 10g glycerin; 25ml distilled water.
2. Formulation: [a] soak the gum in the water for ~24 hrs (to prevent bacterial growth add a crystal of chloral hydrate about the size of a small pea); [b] add the rest of the chloral hydrate and let the solution stand several days until all material is dissolved; [c] add the glycerin.
~Use like water to mount materials,
~Tends to remove stains used for hyaline materials,
~Excellent for phase contrast microscopy,
~Lasts indefinitely, i.e., years.
D. Karo syrup (Stevens, 1974; Johansen, 1940; Patrick, 1936).
Note. This is an American product made from maize that is a mixture of dextrose, dextrin, and maltose and whose sugars, unlike those of cane syrup (treacle), do not crystallize on drying; when hard it is as firm as balsam and ringing is not necessary except in very humid climates.
1. Formulation: 100ml Karo syrup (white); 300ml distilled water; 4ml (40%) formalin (to prevent molding).
2. Use in combination with aqueous stains (e.g., phloxine)
E. Archive quality slides (See Volkmann-Kohlmeyer & Kohlmeyer, 1996).
Cunningham, J. L. 1972. A miracle mounting fluid [Hoyer's] for permanent wholemounts of microfungi. Mycologia 64: 906-911.
Dring, D. M. 1971. Techniques for microscopic preparation. in C. Booth, ed. Methods in Microbiology, vol. 4, pp. 95-11. Academic Press.
Hadzic, I. 1992. How to preserve a spore-print. The Mycologist 6: 188-9.
Johansen, D. A. 1940. Plant Microtechnique. McGraw-Hill.
Omar, M. B., Bolland, L. & Heather, W. A. . A permanent mounting medium for fungi [PolyVinylAlcohol+lactic acid/cotton blue].
Stain Technology 53(1978): 293-294;
Bulletin of the British Mycological Society 13(1979)31-32.
Patrick, R. 1936. "Karo" as a mounting medium. Science 83: 85-86.
Stevens, R. B., ed. 1974. Mycology Guidebook. University of Washington Press.
Twomey, D. G. 1977. The rapid preparation of micro-fungi for microscopic observations. Bulletin of the British Mycological Society 11: 148-149.
Volkmann-Kohlmeyer, B. & Kohlmeyer, J. 1996. How to prepare truly permanent microscope slides. Mycologist 10: 107-108.