Microscopy of Fungi Using Lactophenol Cotton Blue

 

Microscopy of Fungi Using Lactophenol Cotton Blue

Theory

Fungi are eukaryotic, heterotrophic organisms that play vital roles in decomposition, fermentation, disease, and antibiotic production. They exist as unicellular (yeasts) or multicellular (molds) forms or dimorphic forms and are distinct from plants due to the lack of chlorophyll and presence of chitin in their cell walls. The general cell Structure of Fungi includes a cell wall that is composed mainly of chitin (a polymer of N-acetylglucosamine). The Cytoplasm Contains membrane-bound organelles like the nucleus, mitochondria, ER, etc, and a true nucleus with a nuclear membrane is present (eukaryotic feature). Fungi, especially molds, are composed of long, thread-like filaments called Hyphae that make up the mycelium (vegetative body of molds). The hyphae may be

o   Septate hyphae: Divided by cross walls (septa) (e.g., Aspergillus)

o   Aseptate or coenocytic hyphae: No septa; continuous cytoplasm (e.g., Rhizopus)

 

Classification of Fungi (Based on Sexual Spores)

Class	Key Features	Sexual Spores	Examples
Ascomycota (sac fungi)	Produce spores in a sac-like structure called ascus	Ascospores	Aspergillus, Penicillium, Saccharomyces
Basidiomycota (club fungi)	Spores are formed on a club-like structure called basidium	Basidiospores	Mushrooms, Cryptococcus
Zygomycota	Coenocytic hyphae, spores in zygosporangia	Zygospores	Rhizopus, Mucor
Deuteromycota (Fungi Imperfecti)	No sexual reproduction observed	Asexual spores (conidia)	Candida, Alternaria

Fungi produce both asexual and sexual spores

Asexual Spores

Produced by mitosis; help in rapid spread.

·        Conidia: Formed externally on conidiophores (Aspergillus, Penicillium)

·        Sporangiospores: Formed inside a sporangium (Rhizopus)

·        Chlamydospores: Thick-walled resting spores (Candida)

·        Blastospores: Budding spores (yeast)

Sexual Spores

Produced through fusion of nuclei during sexual reproduction.

·        Zygospores: Thick-walled spores from fusion of hyphae (Rhizopus)

·        Ascospores: Formed in asci (Aspergillus, Saccharomyces)

·        Basidiospores: Formed on basidia (mushrooms)

To observe fungal structures like hyphae and spores, staining techniques are essential. Lactophenol Cotton Blue (LPCB) is a commonly used stain which contains:

• Lactic acid (preserves fungal structures),
• Phenol (kills the fungi),
• Cotton blue (stains chitin in fungal cell walls),
• Glycerol (acts as a mounting fluid).

The Scotch tape method allows easy collection and transfer of fungal mycelium with minimal disruption of structure, making it ideal for microscopic observation.

 Requirements

• Fungal culture (e.g., Aspergillus, Penicillium) on agar plate
• Clean glass slide
• Coverslip
• Lactophenol Cotton Blue (LPCB) stain
• Transparent Scotch tape (small strip)
• Forceps
• Needle or applicator stick
• Microscope
• Tissue paper

Procedure

·       Place 1–2 drops of LPCB stain in the center of a clean glass slide.

o   Take a small piece of transparent Scotch tape (about 2–3 cm).

o   Gently press the sticky side of the tape onto the surface of the fungal colony (usually from the aerial mycelium).

• Avoid pressing too hard to prevent damaging the structure.
• Place the sticky side of the tape directly onto the LPCB drop on the slide.
• Smooth out gently to avoid air bubbles.
• Observe under low power (10x) and then high power (40x) objectives.
• Focus on hyphae, spores, and reproductive structures.

 Observation

S.N	Sample	Hyphae	Special reproductive structures	Inference
		Septate or non-septate hyphae	like conidiophores or sporangiospores

 

Result

• The fungal hyphae and reproductive structures were visible and stained blue.
• Structures such as conidia and sporangia were distinguishable, aiding in the identification of the fungal genus.

Discussion

The Scotch tape method is quick, simple, and effective for fungal microscopy. It maintains the natural arrangement of fungal elements better than traditional smears. LPCB stain is ideal for staining chitin, highlighting the cell wall and allowing easy observation. However, it is not suitable for wet fungi or heavily sporulated colonies due to the risk of overloading the tape.

Conclusion

Fungal Morphology and its identification were done by using the Scotch tape method

Precautions

• Use only a thin layer of fungal growth to avoid overloading.
• Always handle phenol-containing stains like LPCB with care; it is toxic.
• Ensure slides and coverslips are clean and dry.
• Dispose of fungal culture and used materials properly.
• Avoid direct inhalation of spores; work in a biosafety cabinet if available.

References

1. Alexopoulos, C.J., Mims, C.W., & Blackwell, M. (1996). Introductory Mycology. John Wiley & Sons.
2. Tortora, G.J., Funke, B.R., & Case, C.L. (2016). Microbiology: An Introduction. Pearson.
3. De Hoog, G. S., Guarro, J., Gené, J., & Figueras, M. J. (2000). Atlas of Clinical Fungi.