Epidermophyton floccosum in LPCB mount and Its LPCB Preparation with Related Videos

Epidermophyton floccosum in LPCB mount

Epidermophyton floccosum in LPCB Mount

Epidermophyton floccosum in LPCB mount is showing smooth thin-walled macroconidia which are often produced in clusters growing directly from the hyphae. Numerous vegetative spores ( chlamydospores) are formed in older cultures and microconidia are not formed as shown above picture.

Keynotes

  1. Asexual spores are of two types- vegetative and aerial spores. Vegetative spores are further 3 types- arthrospores, blastospores, and chlamydopores. Arthrospores are formed by the production of cross-septa into hyphae resulting in rectangular thick-walled spores. Blastospores are formed by budding from the parent cells, as in yeasts where as chlamydospores are thick-walled resting spores developed by rounding up and thickening hyphal segments.
  2. Aerial spores are conidiospores, microconidia, macroconidia, and sporangiospores. Conidiophores – Spores borne externally on sides or tips of hyphae are called conidiospores or conidia. Small  and single conidia are microconidia. Large and septate conidia are macroconidia and are often multicellular. Sporangiospores are spores formed within the sporangium. They develop on the ends of hyphae called sporangiophores. e.g. Mucor, Rhizopus.

Asexual spores related videos

Arthrospores of Trichopsporon as shown below-

Arthrospores of Geotricum as shown below-

Blastospores of Cryptococcus neoformans as shown below-

Chlamydopores of Candida albicans as shown below-

Conidiospores or conidia of Penicillium cheresanum showing long chains of single-celled conidia as shown below-

Micrconidia and macroconidia of dermatophytes (Trichophyton rubrum) as shown below-

Sporangiosphores of Rhizopus as shown below-

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 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 magnetic stirrer until the phenol is dissolve 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
  • Cover slip
  • Dropper or bamboo sticks
  • Fungal growth  in 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 cover slip 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.
  • Epidermophyton floccosum growth on SDA and its structures in LPCB preparation

  • 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 microscope

Fungus, Acremonium on SDA and lactophenol cotton blue preparation

Aspergilus flavus on Czapek Dox agar, Corn meal 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 microscope in lactophenol cotton blue preparation
showing following structures-conidia, conidiophores and septate hyphae
Conidia in clusters

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

Arthroconidia of Trichosporon inkin – Long Cylindrical in Shape

 Keynotes

  1. LPCB wet mount is always examined at least 30 minutes after preparation.
  2. A wet mount preparation should neither be too thick or too thin.
  3.  In this preparation, both bile stained and non bile stained helminthic eggs are stained blue.
  4. LPCB kills the trophozoites of Entamoeba and Trichomonas, hence , can not be demonstrated by this.
  5. In LPCB wet mount of stool phenol and lactic clear faecal debris.
  6. In LPCB wet mount of stool, glycerol  provides a semi permanent preparation. Cyst of intestinal protozoa and ova takes blue color while ova of helminths are stained deep blue. Additional advantage of the this stain is that it can also detect blue colored Cyclospora and Isospora oocyst.

Limitations of LPCB Stain

Even though LPCB stain is being very useful has some shortcomings  like-

  1. It is only applicable for presumptive identification method of fungi.
  2. The ingredient of LPCB solution may disrupt the original morphology of the fungi.
  3. The stain can only be usable to identify mature fungi and their structures and not the young vegetative forms of fungi.
  4. A wet mount preparation should neither be too thick or too thin.
  5. Application of LPCB stain in Parasitology is not preferred  because it kills the trophozoites of Entamoeba and Trichomonas.
  6. This LPCB stain has expiry date and thus can only use before expiry.

Bibliography

  1. Medical Mycology. Editors:  Emmons and Binford, 2nd ed 1970, Publisher Lea and Febiger, Philadelphia.
  2. Clinical Microbiology Procedure Hand book, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  3. A Text Book of Medical Mycology. Editor: Jagdish Chandar.  Publication Mehata, India.
  4.  Practical Laboratory Mycology. Editors: Koneman E.W. and G.D. Roberts, 3rd ed 1985, Publisher Williams and Wilkins, Baltimore.
  5. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
  6. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1706009/
  8. https://mycology.adelaide.edu.au/laboratory/lacto/
  9. http://himedialabs.com/TD/S016.pd
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