LACTOPHENOL COTTON BLUE (LPCB) STAINING

 LACTOPHENOL COTTON BLUE (LPCB) STAINING

AIM

           To identify the Microscopic morphology of Fungi by Lactophenol Cotton Blue (LPCB) Staining.

PRINCIPLE  

            Fungi are the Eukaryotic microorganisms which are classified into two main group viz., (i) Molds (Multicellular) and (ii) Yeasts (Unicellular). The Lactophenol Cotton Blue (LPCB) is a stain used for making semi-permanent microscopic preparation of Fungi. The LPCB stain has three components: (i) Phenol – Act as a Disinfectant or Fungicide and kills any living microorganisms; (ii) Lactic acid – Preserves the Fungal structure and (iii) Cotton blue stain - Stains the chitin and cellulose of the fungal cell wall intensely blue. LPCB solution is a mounting medium and staining agent used in the preparation of slides for microscopic examination of fungi. In LPCB staining, fungal elements are stained intensely in Blue colour.

MATERIALS REQUIRE

• Fungal culture plate
• Lactophenol Cotton Blue (LPCB) Stain
• Bunsen burner
• Teasing needle
• Glass slide
• Coverslip
• Microscope.

PROCEDURE

• Place a drop of Lactophenol Cotton Blue reagent on a clean, grease free and dry slide. The stain imparts a blue colouration on hyphae.
• By using a Teasing needle, carefully tease the small portion of fungal culture into a thin preparation and form the Fungal mycelial mat.
• Place a coverslip gently on the Fungal mycelial mat without air bubbles and wait for about 3 - 5 minutes.
• Observe under microscope at 40 X objective lens for detailed examination of Fungal morphology and Spores.

OBSERVATION AND RESULTS

        The morphology of the fungi appears (mycelium and spores) as dark blue stained mycelium under microscope in 45 X objective.

Figure – 1: Microscopic morphology of Aspergillus niger

Figure –  2: Microscopic morphology of Aspergillus flavus

Figure – 3: Microscopic morphology of Aspergillus fumigatus

 

Figure – 4: Microscopic morphology of Penicillium chrysogenum

Figure – 5: Microscopic morphology of Rhizopus sp. (Rhizoids present) 

Figure – 6: Microscopic morphology of Mucor sp. (Rhizoids absent)