Bipolaris on SDA: Introduction, Classification, Clinical Features, Lab Diagnosis and Treatment

Bipolaris on SDA

Bipolaris on SDA

Bipolaris on SDA  showing colonies are moderately fast-growing, effuse, grey to blackish brown, suede-like to floccose with a black reverse. It is a large genus of dematiaceous hyphomycetes with more than 100 species, most of them being saprobes in soil and pathogens of plants, while some of the saprobic species are potentially able to infect humans and animals.

Microscopic morphology shows sympodial development of hyaline to deep olivaceous pigmented, pseudoseptate conidia on a geniculate or zig-zag rachis. Conidia mostly curved, canoe-shaped, fusoid or obclavate, rarely straight, 2–14 pseudoseptate (usually more than 6), germinating only from the ends (bipolar).

Classification of Bipolaris

Shoemaker (1959)

  • Kingdom: Fungi
  • Division: Ascomycota
  • Class: Dothideomycetes
  • Order: Pleosporales
  • Family: Pleosporaceae
  • Genus: Bipolaris

Medically important Bipolaris species are B. australiensis, B. hawaiiensis, B. spicifera, and, to a lesser extent, B. papendorfii.

Clinical Features of Bipolaris Infection

They are allergic sinusitis, keratitis, endophthalmitis, onychomycosis, peritoneal dialysis-associated peritonitis, lung and skin infections, and, less frequently, central nervous system (CNS) infections.

Laboratory Diagnosis 

Specimen: It depends on the site of infection. e.g. nasal swab in case of allergic sinusitis whereas eye swab in case of keratitis and skin scrapping in case of skin infections.

KOH mount: For observation of fungal elements

Fungal culture: SDA or PDA can be used for fungal culture.

LPCB Preparation:  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.

Molecular Identification: Internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rDNA) sequencing may be used to identify clinical species. The glycerol-3-phosphate dehydrogenase (GPDH) gene family has been determined to be the best single phylogenetic marker of Bipolaris species.

Keynotes on Bipolaris

  1. Key Features of Bipolaris: Dematiaceous hyphomycete producing sympodial, pseudoseptate, pale brown, straight, fusiform to ellipsoidal conidia, which are rounded at both ends.
  2. The typical morphological features of Bipolaris species include rapidly growing dark colonies, geniculate conidiophores with sympodial conidiogenesis, and large conidia with transverse distosepta, usually without a protuberant hilum and with bipolar germination. Morphologically similar anamorphic genera are Drechslera, Curvularia, and Exserohilum.
  3. Morphological differentiation of the genera relied upon a combination of characters including conidial shape, the presence or absence of a protruding hilum, the contour of the basal portion of the conidium and its hilum, the point at which the germ tube originates from the basal cell, and, to a lesser degree, the sequence and location of the first three conidial septa.
  4. There is no clear morphological boundary between genera Bipolaris and Curvularia and some species show intermediate morphology and thus using a combined internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rDNA)  and GPDH gene analysis for definitive identification of species.

#Bipolaris  in LPCB mount#

Bipolaris in LPCB mount is showing sympodial development of pale brown pigmented, pseudseptate conidia on a geniculate or zig-zag rachis. Conidia are produced through pores in the conidiophore wall called proconidia and are straight, fusiform to ellipsoidal, rounded at both ends, smooth to finely roughed, germinating only from the ends, and thus called bipolar as shown below the video.

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.
  • Bipolaris growth on SDA and its structures on lactophenol cotton blue 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

#Trichosporon on SDA and lactophenol cotton blue preparation under the microscope

#Geotrichum growth on SDA and its fungal 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

#Aspergilus 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-
Helical pattern 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 fecal debris.
  6. In the 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. An 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 the 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. https://mycology.adelaide.edu.au/descriptions/hyphomycetes/bipolaris/
  2. https://jcm.asm.org/content/50/12/4061
  3. https://en.wikipedia.org/wiki/Bipolaris
  4. https://scialert.net/fulltext/
  5. https://www.woundsresearch.com/article/5191
  6. https://www.mycosphere.org/pdf/MYCOSPHERE
  7. Medical Mycology. Editors:  Emmons and Binford, 2nd ed 1970, Publisher Lea and Febiger, Philadelphia.
  8. 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.
  9. A Text Book of Medical Mycology. Editor: Jagdish Chander.  Publication Mehata, India.
  10.  Practical Laboratory Mycology. Editors: Koneman E.W. and G.D. Roberts, 3rd ed 1985, Publisher Williams and Wilkins, Baltimore.
  11. 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.
  12. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  13. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1706009/
  14. https://mycology.adelaide.edu.au/laboratory/lacto/
  15. http://himedialabs.com/TD/S016.pdf
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