universe84a

Conidia of Curvularia in LPCB Mount

Conidia of Curvularia in LPCB mount as shown above picture. Conidia are pale brown, with three or more transverse septa ( phragmoconidia) and are formed apically through a pore ( poroconidia) in a sympodially elongating geniculate conidiophore. Conidia are cylindrical or slightly curved, with one of the central cells being larger and darker, germination is bipolar and some species may have a prominent hilum.

Introduction of LPCB Stain

LPCB stain stands for lactophenol cotton blue and it is a combination of fixative, staining, and clearing agent. LPCB uses both as a mounting fluid and a stain. This is used for staining and microscopic identification of fungi. Its contents functions are as follows-

Lactic acid: It helps in preserving the morphology of the fungal elements.

Phenol: It acts as a disinfectant.

Cotton blue: It stains the fungal elements as well as intestinal parasitic (cyst, ova, and oocyst) and non-parasitic structures (vegetable cells, mucus, muscle fibers, and other artifacts).

Glycerol: It is a hygroscopic agent that prevents drying.

Principle of LPCB Stain

Ingredients of LPCB stain like lactic acid acts as a clearing agent and aids in preserving the fungal structures. Similarly, phenol kills the organism and fixes it while glycerol prevents drying. Cotton blue stains the chitin in the cell wall of fungi and identification of filamentous fungi is made by their characteristic microscopic morphology such as shape, size, arrangement of spores, and hyphae providing color to the structure. It can be used alone or in conjunction with KOH.

Composition of LPCB Stain 

For 50 ml
Lactic acid : 10 ml
Phenol : 10 ml
Glycerol :20 ml
Cotton blue (Poirier blue or Aniline blue): 0.025 g
Distilled water : 10 ml

• Dissolve phenol in lactic acid, glycerol, and distilled water.
• Finally, add cotton blue and mix well.
• But this LPCB stain is prepared over two days.
• On the first day, dissolve the cotton blue in the distilled water and leave it overnight to eliminate insoluble dye.
• On the second day, wearing gloves add the phenol crystals to the lactic acid in a glass beaker. Place on a magnetic stirrer until the phenol is dissolved or do manually.
• Add the glycerol.
• Filter the cotton blue and distilled water solution into the phenol/glycerol/ lactic acid solution.
• Mix and store at room temperature.

Requirements for Test

• Compound light microscope
• LPCB stain
• Clean and grease-free microscopic slides
• Coverslip
• Dropper or bamboo sticks
• Fungal growth  in the medium

Procedure of LPCB Preparation

• Take a clean and grease-free glass slide.
• Put a large drop of LPCB with a Pasteur pipette or dropper.
•  Transfer a small quantity of the culture to the drop.
• Tease the culture (in case of a mold) well with teasing needles
  so as to get a uniform spread.
• Put on a coverslip gently to avoid entrapment of air bubbles.
•  Examine under low  (10 X) and high-power (40 X) objectives.
• Observe the morphological features carefully as shown below.
  

  Observation

  Fungi appear as dark blue stained mycelium.

  Results and Interpretations

  Different fungi under LPCB wet mount will show different types of morphological structures including hyphae and spores. We concern with Aspergillus as shown below.

• Fungal spores, hyphae, and fruiting structures: Takes stain blue
• Background: stains pale blue.
  

  Application of LPCB Stain

•  For staining and microscopic identification of fungi observing fungal spores, hyphae, and fruiting structures.
• It is also applicable in parasitology for the observation of Cyst of intestinal protozoa and ova takes blue color while ova of helminths are stained deep blue.
• Various fungi and their structures ( yeast cells, budding yeast, hyphae, pseudohyphae, mycelium, spores) in LPCB preparation are as follows-

Aspergillus fumigatus Colony on SDA, LPCB tease mount under microscopy

LPCB Mount of Curvularia species

Trichosporon on SDA and lactophenol cotton blue preparation under the microscope

Geotrichum growth on SDA and its fungal structures on lactophenol cotton blue preparation

Bipolaris growth on SDA and its structures on lactophenol cotton blue preparation

Syncephalastrum in lactophenol cotton blue preparation under the Microscope

Penicillium colonial morphology and its microscopic features in lactophenol cotton blue tease mount under the microscope

Fungus, Acremonium on SDA and lactophenol cotton blue preparation

Aspergillus flavus on Czapek Dox agar, Cornmeal agar, and lactophenol cotton blue tease mount

Fusarium growth on SDA and its structures in lactophenol cotton blue preparation

Cryptococcus neoformans in lactophenol cotton blue tease mount

Candida albicans in LPCB tease mount

Cladosporium on SDA and its fungal structures on lactophenol cotton blue preparation

Mucor in LPCB mount

Lactophenol cotton blue tease mount procedure and observation under the Microscope

Sporothrix schenckii under the microscope in lactophenol cotton blue preparation
showing the following structures-conidia, conidiophores, and septate hyphae
Conidia in clusters

Trichophyton mentagrophyte Isolated: features-
Helical pattern on lactophenol cotton blue Mount seen
Urease test_Positive
Hair perforation test-Positive

Arthroconidia of Trichosporon inkin – Long Cylindrical in Shape