Mycelium, hypahe and conidia on LPCB tease mount: Introduction and

Mycelium, Hypahe and Conidia on LPCB Tease Mount: Introduction and LPCB Preparation in Details

Mycelium, Hyphae, and Conidia under Microscope

Molds grow as branching filaments called hyphae usually 2-10 µm in width.
Hyphae may be septate or non sepatate/ aseptate.
The hyphae continue to grow and branch to form a tangled mass of growth known as mycelium.
Conidia (singular conidium), sometimes termed an asexual chlamydospore or chlamydoconidia is an asexual, non-motile spore of a fungus. The name comes from the Greek word for dust and they are also called mitospores due to the way they are generated through the cellular process of mitosis.
Asexual reproduction in ascomycetes is by the formation of conidia, which are borne on specialized stalks called conidiophores.

Note: The hyphae of Aspergillus species range in diameter from 2.5 to 4.5 µm and having septate hyphae, but these are not always apparent, and in such cases, they may be mistaken for Zygomycota. Aspergillus hyphae tend to have dichotomous branching that is progressive and primarily at acute angles of around 45° which is key identification property of Aspergillus as shown above image.

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

Take a clean and grease-free slide and place a drop of LPCB on it.
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.
Fix the adhesive side of the tape over an area on the glass side containing the LPCB.
Focus the preparation at 10x and finally observe at 40x objective of a light microscope.

Tease mount preparation

Put a drop of LPCB on a clean grease-free glass slide.
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.
With the help of a needle, tease the fungal culture first and spread in the LPCB.
Wait for normally 30 minutes .i.e. sufficient time for the structures to take up the stain.
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.

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