Lactophenol cotton blue mount

Principle

• Phenol kills the fungus, lactic acid preserves fungal structures and clears, glycerol prevents drying, and cotton blue (aniline blue) stains fungal chitin blue.
• Used to see the microscopic features of molds grown in culture.

Specimen

• Fungal growth from culture plates or slants (mold colonies), or slide cultures.

Reagents and equipment

• Lactophenol cotton blue (LPCB) reagent; slides, coverslips, teasing needles, clear adhesive tape.
• Class II biological safety cabinet for handling molds.

Procedure

• Tease mount: place a drop of LPCB on a slide, take a small piece of colony (from between center and edge), tease apart gently with needles and add a coverslip.
• Tape mount: touch the sticky side of clear tape to the colony surface and lay it onto a drop of LPCB on a slide.
• Slide culture: grow the fungus on a small agar block under a coverslip; mount the coverslip on LPCB. This best preserves the natural arrangement of conidia.
• Examine with 10× and 40× objectives; seal edges with nail polish for a semi-permanent mount.

Results

• Hyphae and reproductive structures stain blue on a pale background.
• Aspergillus: conidiophore ending in a swollen vesicle with phialides and chains of conidia.
• Penicillium: brush-like branching (penicillus).
• Rhizopus: sporangia with rhizoids at the base of sporangiophores; Mucor: no rhizoids.
• Microsporum: rough, thick-walled spindle-shaped macroconidia; Epidermophyton: smooth club-shaped macroconidia, no microconidia; Trichophyton: mainly microconidia with smooth, pencil-shaped macroconidia when present.

Quality control and pitfalls

• Vigorous teasing breaks conidial chains and distorts arrangements.
• Young cultures may not have sporulated; re-examine older growth.
• Phenol is toxic and corrosive; wear gloves.
• Handle possible dimorphic pathogens (e.g. Histoplasma, Coccidioides) only in a BSC; spore release is a serious hazard.

Clinical use

• Standard mount for morphological identification of molds and dermatophytes.