Lactophenol Cotton Blue staining preparation for fungi Principle: The Lactophenol Cotton Blue preparation is used to stain

fungal elements, which aids in the microscopic identification of mycotic agents. The cotton blue dye stains the chitin, a nitrogenous substance present in the cell walls of most fungi. The phenol kills any organisms and the lactic acid preserves fungal structures. Materials: Glass slides Wooden applicator sticks 22 x 22 coverglass

Reagents: Lactophenol Cotton Blue Stain

Precaution Store at room temperature. Protect from light. Avoid contact with skin.

VII. Procedure: Teasing Preparation: o Place a clean glass slide on a sheet of white paper. o Place a small drop of Lactophenol Cotton Blue in the center of the slide. o Break a stick in half so that a rough tapered edge is formed. o Remove a fragment of a fungal colony (approximately 1-2 mm from the periphery) with the broken stick and place in the Lactophenol Cotton Blue. o Gently tease the fragment until it has been separated. o Gently lower a coverslip over it. Do not tap or push down as this may dislodge conidia from conidiophores. o Examine microscopically for presence of fungal elements. o For a permanent preparation, rim the coverslip with clear nail polish or Permount. Adhesive Tape Preparation: o Place a clean glass slide on a sheet of white paper. o Place a small drop of Lactophenol Cotton Blue in the center of the slide. o Touch the adhesive side of a small length of transparent tape to the surface of the colony. o Adhere the length of the tape to the surface of the slide with the Lactophenol Cotton Blue.

Observe microscopically for the characteristic shape and arrangement of the spores.