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Trichophyton mentagrophytes 

Trichophyton mentagrophytes in LPCB mount is showing  microconidia, macroconidia, chlamydospores and spiral hypahe of this organism as shown above figure. Trichophyton mentagrophytes complex consists  five species T. mentagrophytes, T. interdigitale, T. erinacei, T quinckeanum, and T. benhamie—as well as nine different genotypes of T. mentagrophytes / T. interdigitale associated with the geographical origin and the source of infection. Phenotypic features ( growth on fungal medium LPCB preparation and other biochemical tests) alone can not differentiate to species level and thus genotyping is mandatory i.e. at the molecular level, the species could only be identified as T. mentagrophytes complex.

Pathogenecity of Trichophyton mentagrophytes 

The Trichophyton mentagrophytes complex is the second most common causal agent of dermatophytosis after T. rubrum. It has  the enzyme keratinase; thus, it can cause infections in the skin, hair, and nails in both humans and animals.

Key Notes

1. Trichophyton mentagrophytes  is urea hydrolization test positive.
2. It also shows hair perforation test positive.

Introduction of LPCB stain

LPCB  stands Lactophenol Cotton Blue and  it is a combination of  fixative , staining and clearing agent. 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) and

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 colour 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 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

• Compound light microscope
• LPCB stain
• Test slides
• Cover slip
• Dropper or bamboo sticks
• Fungal growth of Trichophyton

Procedure

Scotch tape preparation

1.  Take a clean and grease free slide and place a drop of LPCB on it.
2. Touch the adhesive side of the tape of transparent scotch tapes on the surface of the colony  at a point intermediate between its centre and periphery.
3. Fix the adhesive side of the tape over an area on the glass side containing the LPCB.
4. Focus the preparation at 10X and finally observe at 40X objective of light microscope.

Tease mount preparation

1. Put a drop of LPCB on a clean grease free glass slide.
2. Take a small portion of the colony and the supporting agar at a point between the centre and the periphery and place it in the drop.
3. With the help of a needle,tease the fungal culture first and spread in the LPCB.
4. Wait for normally 30 minutes i.e. sufficient time for the structures to take up the stain.
5. Focus at 10X objective and finally examine at 40X objective under microscope.

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 as shown above picture.

References

1. Collier L, Balows A, Sussman M. Topley and wilson’s Microbiology and MIcrobial infections. 9th Edition.Mycology Volume 4
2. Jagdish Chander. A textbook of Medical Mycology.
3. Garcia LS. Diagnostic Medical Parasitology. ASM press,Washington D.C. 4th Edition
4. Medical Mycology. Editors:  Emmons and Binford, 2nd ed 1970, Publisher Lea and Febiger, Philadelphia.
5. Rippon’s JW: Medical Microbiology. The pathogenic fungi and the pathogenic Actinomycetes. 3^rd ed 1988 Publisher WB saunder co, Philadelphia.
6. Clinical Microbiology Procedure Hand book Vol. I & II, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6945559/