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Penicillium species in LPCB Mount

Penicillium species in LPCB mount shows chains of single-celled conidia which are produced in basipetal succession from a specialized conidiogenous cell called a phialide. The term basocatenate often uses to describe such chains of conidia where the youngest conidium is at the basal or proximal end of the chain. In Penicillium, phialides may be produced singly, in groups, or from branched metulae, giving a brush-like appearance called penicillus which is the identification feature of Penicillium. The penicillus may contain both branches and metulae (penultimate branches which bear a whorl of phialides). All cells between the metulae and the stipes of the conidiophores are referred to as branches. The branching pattern may be either simple, one stage branched,  two-stage branched or even three- to more-staged branched. Simple branched is (non-branched or monoverticillate), one-stage branched is (biverticillate-symmetrical) while two-stage branched is  (biverticillate-asymmetrical). Conidiophores are hyaline, smooth, or rough-walled. Phialides are usually flask-shaped, consisting of a cylindrical basal part and a distinct neck, or lanceolate. Conidia are in long dry chains, divergent or in columns, are globose, ellipsoidal, cylindrical or fusiform, hyaline or greenish, smooth or rough-walled as shown above picture. Sclerotia are produced by some species which are a compact masses of hardened fungal mycelium containing food reserves. They help to survive environmental extremes.

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.
• Penicillium growth and its structures in LPCB preparation having phialides, penicillus, conidiophores as shown below-
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  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

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 a 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

LPCB Mount of Curvularia species

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-
The helical pattern on lactophenol cotton blue Mount seen
Urease test_Positive
Hair perforation test-Positive

Arthroconidia of Trichosporon inkin – Long Cylindrical in Shape