Unit 3: Fungi & Lichen

Lactophenol Cotton Blue Staining

This is the standard method for preparing a temporary slide of a fungus for microscopic observation.

Principle

The stain is a "4-in-1" solution:

• Lactic Acid: Acts as a clearing agent and preserves the fungal structures.
• Phenol: Acts as a fungicide/bactericide, killing the living specimen.
• Cotton Blue (Aniline Blue): This is the stain. It strongly stains the chitin present in the fungal cell walls.
• Glycerol (often included): A hygroscopic agent that prevents the mount from drying out.

Procedure

1. Place a clean glass slide on a paper towel.
2. Add one drop of Lactophenol Cotton Blue stain to the center of the slide.
3. Using a sterile needle, pick up a small portion of the fungal growth (e.g., from a bread mold or culture plate).
4. Place the fungus into the drop of stain.
5. Using two teasing needles, gently tease the fungal hyphae apart. This is crucial to avoid a thick, unobservable clump.
6. Carefully lower a coverslip, starting from one edge, to push out air bubbles.
7. Wipe away any excess stain from the edges of the coverslip.
8. Warm the slide gently (do not boil) over a flame to help stain uptake and remove air bubbles.
9. Observe under low power (10x) and then high power (40x).

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Identification of Fungi

Rhizopus (e.g., Bread Mold)

Class: Zygomycetes

[Image of Rhizopus structure with sporangium, stolon, and rhizoids]

• Vegetative Hyphae: Coenocytic (aseptate, no cross-walls), broad and ribbon-like.
• Key Structures:
  • Rhizoids: Root-like hyphae that anchor the fungus and absorb nutrients.
  • Stolon: A horizontal, aerial hypha that connects groups of rhizoids.
  • Sporangiophores: Upright, unbranched stalks that grow in clusters from the point where rhizoids form.
• Reproductive Structures:
  • Asexual: A large, black, spherical sporangium forms at the tip of the sporangiophore. It contains thousands of sporangiospores. A central sterile region called the columella is visible after the spores are released.
  • Sexual: (Observed less often) Formation of a thick-walled, black, warty zygospore where two compatible hyphae meet.

Penicillium

Class: Ascomycota (Deuteromycetes)

• Vegetative Hyphae: Septate (with cross-walls), branched, and colorless.
• Reproductive Structures (Asexual):
  • Conidiophore: The entire spore-producing structure. It is an upright, branched stalk.
  • The branching pattern looks like a "brush" or "broom" (Latin: penicillus).
  • The branches are called metulae, which bear bottle-shaped cells called phialides (or sterigmata).
  • Conidia: Long, green, spherical spores produced in unbranched chains (basipetal succession) from the tips of the phialides.

Ustilago (Smut Fungus)

Class: Basidiomycota

• Identification: This is a plant parasite. You will likely observe it as a prepared slide of infected host tissue (e.g., corn, wheat).
• Key Feature: The fungus destroys the host's ovaries, anthers, or vegetative parts and replaces them with large galls or "smut sori."
• Microscopic View: The sori are filled with a black, powdery mass of millions of thick-walled, spherical, and often spiny teliospores (also called chlamydospores or smut spores). These are the resting spores.

Puccinia (Rust Fungus)

Class: Basidiomycota

• Identification: An obligate plant parasite. You will observe prepared slides of a cross-section (T.S.) of an infected host leaf (e.g., wheat or *Berberis*).
• Key Structures (on Wheat leaf):
  • Uredosorus (Uredinium): A reddish-brown, blister-like pustule breaking through the leaf epidermis. It contains stalked, binucleate (n+n), rust-colored uredospores. This is the "red rust" stage.
  • Teleutosorus (Telinium): A black, blister-like pustule. It contains thick-walled, dark brown, two-celled, stalked teliospores. This is the "black rust" stage.

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Study of Lichen Growth Forms

A Lichen is a symbiotic association between a fungus (the mycobiont, providing structure and protection) and an alga or cyanobacterium (the phycobiont, providing food via photosynthesis). You will observe macroscopic (whole) specimens.

1. Crustose Lichen

• Appearance: Forms a thin or thick crust, like a layer of paint.
• Attachment: Tightly appressed to the substrate (rock, bark, or soil). The lower surface is fully attached and cannot be separated from the substrate without breaking it.
• Example: Graphis (Script lichen), Lecanora.

2. Foliose Lichen

• Appearance: Leaf-like, with flattened lobes and a distinct upper and lower surface.
• Attachment: Loosely attached to the substrate by root-like anchoring structures called rhizines. It can be carefully peeled off the substrate.
• Example: Parmelia, Xanthoria (orange lichen).

3. Fruticose Lichen

• Appearance: Shrub-like, hair-like, or strap-shaped. Highly branched, either upright or hanging (pendant).
• Attachment: Attached to the substrate at a single basal point or holdfast.
• Example: Usnea (Old man's beard), Cladonia (Reindeer moss).