Epidermophyton floccosum in LPCB mount and Its LPCB Preparation with Related Videos
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.
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.
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.
Conidiospores or conidia ofPenicillium 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
Clean and grease-free microscopic slides
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.
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
LPCB wet mount is always examined at least 30 minutes after preparation.
A wet mount preparation should neither be too thick or too thin.
In this preparation, both bile stained and non bile stained helminthic eggs are stained blue.
LPCB kills the trophozoites of Entamoeba and Trichomonas, hence , can not be demonstrated by this.
In LPCB wet mount of stool phenol and lactic clear faecal debris.
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-
It is only applicable for presumptive identification method of fungi.
The ingredient of LPCB solution may disrupt the original morphology of the fungi.
The stain can only be usable to identify mature fungi and their structures and not the young vegetative forms of fungi.
A wet mount preparation should neither be too thick or too thin.
Application of LPCB stain in Parasitology is not preferred because it kills the trophozoites of Entamoeba and Trichomonas.
This LPCB stain has expiry date and thus can only use before expiry.
Medical Mycology. Editors: Emmons and Binford, 2nd ed 1970, Publisher Lea and Febiger, Philadelphia.
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.
A Text Book of Medical Mycology. Editor: Jagdish Chandar. Publication Mehata, India.
Practical Laboratory Mycology. Editors: Koneman E.W. and G.D. Roberts, 3rd ed 1985, Publisher Williams and Wilkins, Baltimore.
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.
Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.