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Aspergillus in LPCB mount

Aspergillus species  in LPCB mount showing conidia, phialides, vesicle. stipe, conidiophore as shown above picture.

Introduction

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

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. 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.

Requirements

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

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.