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Aspergillus Structure

Aspergillus structures in LPCB preparation has the following structures-

• Septate hyphae
• Conidiophore or stipe
• Vesicle
• Phialides
• conidia
• metulae
• dichotomous branching ( at  an angle of  approximately 45°)
• Note: Phialides and metulae are not showing on the picture because of not clear images.

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 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) 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 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 this stain is that it can also detect blue-colored Cyclospora and Isospora oocyst.

Requirements

• Compound light microscope
• LPCB stain
• Test slides
• Coverslip
• 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 tape on the surface of the colony at a point intermediate between its center 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 a 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 center 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 on 10X objective and finally examine at 40X objective under a 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.

#Aspergillus fumigatus Colony on SDA and LPCB tease mount under the microscope as shown below-

#Aspergilus flavus on Czapek Dox agar, Cornmeal agar, and LPCB tease mount as shown below-

#Aspergillus niger– black pigmentation of Conidiaspores on SDA as shown below-

#LPCB preparation demo and observation of fungal elements under the microscope as shown below-

Further Readings

1. Collier L, Balows A, Sussman M. Topley, and Wilsons’ 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 Handbook Vol. I & II, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.