Fungal Staining

Fungi are eukaryotic organisms with both macroscopic and microscopic characteristics.  Lactophenol Cotton Blue (LPCB) Staining is a simple histological staining method used for the microscopic examination and identification of fungi. The fungal spore cell wall is made up of chitin, which, the components of the Lactophenol Cotton Blue solution stains for identification. The lactophenol cotton blue solution acts as a mounting solution as well as a staining agent. The solution is blue in color and it is made up of a combination of three main reagents:

·        Phenol: It acts as a disinfectant by killing living organisms

·        Lactic acid: It preserves the fungal structures

·        Cotton blue: It stains the chitin on the fungal cell wall and other fungal structures

 

The stain will give a blue-colored appearance to the fungal spores and structures, such as hyphae.

 

Reagents of Lactophenol Cotton Blue (LPCB) Staining

 

A preparation of 50ml Lactophenol cotton Blue staining solution is made up of:

·        Distilled water 50ml

·        Cotton Blue (Aniline Blue) 0.125g

·        Phenol Crystals (C₆H₅O₄)  50g

·        Glycerol 100ml

·        Lactic acid (CH₃CHOH COOH) 50ml

·        70% ethanol

 

Preparation of Lactophenol Cotton Blue solution

Lactophenol Cotton Blue solution is prepared for over two days leaving the reagents undisturbed to allow dissolving and maturation.

1.     Day 1: Dissolve the cotton blue in distilled water and leave to rest overnight.

2.     Day2: Using protective gloves, add phenol crystals to lactic acid in a glass beaker and stir using a magnetic stirrer until the crystals dissolve.

3.     Add glycerol

4.     Filter the Cotton blue and the distilled water into the phenol + glycerol +lactic acid solution and mix.

5.     Store at room temperature.

 

Procedure of Lactophenol Cotton Blue (LPCB) Staining

1.     On a clean microscopic glass slide, add a drop of Lactophenol Cotton Blue Solution

2.     Add the fungal specimen to the drop of alcohol using a sterile mounter such as an inoculation needle.

3.     Tease the fungal sample using needle, to ensure the sample mixes well with the alcohol.

4.     Carefully cover the stain with a clean sterile coverslip without making air bubbles to the stain.

5.     Examine the stain microscopically at 40X, to observe for fungal spores and other fungal structures.

 

 

Results and Interpretation

Fungal spores, hyphae, and fruiting structures stain blue while the background stains pale blue.

 

          

                                     Penicillium                                 Aspergillus

Limitations

·        It can only be used as a presumptive identification method of fungi which should be followed up with other diagnostic tools such as biochemical and cultural examination.

·        The components of the solution should be used before expiry, including the use of the solution before it expires.

·        The solution may disrupt the original morphology of the fungi.

·        The stain can only be used to identify mature fungi and its structures and not the young vegetative forms of fungi.

·        The stain can not be stored for a long period of time.

 

Applications

Clinical diagnosis:

·        Identifying fungal infections in various body sites like skin, lungs, blood, and cerebrospinal fluid. 

·        Differentiating between different types of fungi based on their staining characteristics. 

·        Monitoring the effectiveness of antifungal treatments.

 

Research applications:

·        Studying fungal cell wall composition and structure 

·        Investigating fungal interactions with host tissues 

·        Analyzing fungal diversity in environmental samples 

·        Evaluating antifungal efficacy in vitro 

 

 

Common fungal stains and their characteristics:

 

·        Periodic Acid-Schiff (PAS):

Used for detection of fungi in tissue sections. Stains fungal cell walls pink, making it useful for identifying both yeast and hyphae. Oxidation of fungal polysaccharides results in the production of aldehydes that react with Schiff reagent and fungal cell walls appear pink/magenta in colour.

Procedure

1. Tissue sections are treated with periodic acid.
2. Stain with Schiff reagent.
3. Observe under bright-field microscope.

Results:

• Fungal structures appear magenta/red; tissue background lightly stained.

Applications:

• Detection of fungi in tissue sections (biopsy specimens).
• Identification of pathogenic fungi in histopathology.
• Examples: Candida albicans, Histoplasma capsulatum.

           

                                                           Candia hyphae overlying a     plaque of squamous cells- Fungal esophagitis- PAS stain

Calcofluor White:

• A fluorescent stain that binds to chitin and cellulose in fungal cell walls, allowing for rapid visualization with a fluorescence microscope. Fungi fluoresce bright blue-white under UV light.

Procedure

1. Place fungal material on a slide.
2. Add a drop of Calcofluor White.
3. Observe under fluorescence microscope.

Results:

• Fungal structures glow bright blue/white; background remains dark.

Applications:

• Rapid detection of fungi in clinical specimens (e.g., skin scrapings, sputum).
• Study of spores, hyphae, and yeast forms.
• Examples: Candida, Trichophyton.

 

Calcofluor white stained Candida albicans

showing true hyphae (*) and pseudohyphae (+).

Gomori's Methenamine Silver (GMS):

Gomori's Methenamine Silver (GMS), is a histological stain that highlights polysaccharides in tissue, primarily used to detect fungi by staining them black. It oxidises  carbohydrates in fungal cell walls to aldehydes, which then reduce silver ions to visible black metallic silver,  and is good for diagnosing fungal infections like Pneumocystis carinii pneumonia (PCP) and identifying opportunistic pathogens. GMS is considered highly sensitive for detecting fungi in tissue samples.   

 

GMS

Fungal staining methods with specific applications

Staining Method	Principle	Procedure (Brief)	Appearance / Results	Specific Applications / Examples
1. Lactophenol Cotton Blue (LPCB) Staining	Cotton Blue binds to chitin in fungal cell walls; phenol kills fungus, lactic acid preserves structure.	Mix fungal material with LCB, cover with coverslip, observe under bright-field microscope.	Hyphae, conidia, and spores appear blue; background pale.	- Identification of fungi in culture (e.g., Aspergillus, Penicillium). - Study morphology: hyphal septation, spore arrangement.
2. Calcofluor White Staining	Fluorescent dye binds to chitin and cellulose in fungal walls; fluoresces under UV light.	Mix fungal material with Calcofluor White, observe under fluorescence microscope.	Fungal structures bright blue/white; dark background.	- Rapid detection of fungi in clinical samples (e.g., skin scrapings, sputum). - Visualization of yeast and filamentous forms (e.g., Candida, Trichophyton).
3. Periodic Acid-Schiff (PAS) Staining	Periodic acid oxidizes polysaccharides to aldehydes, which react with Schiff reagent; fungal cell walls stain magenta.	Treat tissue section with periodic acid (Schiff reagent). Observe under bright-field microscope.	Fungal structures appear magenta/red; tissue lightly stained.	- Detection of fungi in tissue sections or biopsies (e.g., Candida albicans, Histoplasma capsulatum). - Diagnosis of invasive fungal infections.

In short

• LPCB → culture/fungi morphology
• Calcofluor White → rapid clinical detection, fluorescence
• PAS → tissue biopsy, invasive fungal infections