Calcofluor White (CFW) Staining - Principle, Procedure, Result, Interpretation, Application, Advantages, Disadvantages

Introduction to Calcofluor White Stain (CFW)

Calcofluor White (CFW) is a non-specific fluorescent blue dye used in biology as well as textiles. The dye binds to 1-3 beta and 1-4 beta polysaccharides of chitin and cellulose which are present in the cell walls of fungi, algae, plants, and parasitic organisms.

Principle of Calcofluor White Stain (CFW)

Being a fluorescent dye, Calcofluor White Stain (CFW) has an excitation wavelength of 380nm and an emission wavelength of 475nm. The dye binds to 1-3 beta and 1-4 beta polysaccharides of chitin and cellulose which are present in the cell walls of fungi, algae, plants, and parasitic organisms.

Depending upon the combination of filters used, the fungal elements appear as apple green or blue-white under UV/violet/violet-blue light. Other biological elements stain reddish-orange while Yellowish-green background fluorescence may be observed with tissue samples. Background fluorescence can be reduced by the use of blue light or different combinations of emission and excitation filters.

Evans blue is also used as counterstain while using blue light excitation (instead of UV) as it reduces the background fluorescence of tissues and cells.

Calcofluor White Stain (CFW) can be mixed with potassium hydroxide for clearing up specimens and aid in the visualization of fungal elements.

Composition of Calcofluor White Stain (CFW)

Procedure of Calcofluor White Stain (CFW)

The procedure for Calcofluor White Staining (CFW) is as follows:

1. Place the specimen on a slide

2. On the slide add one drop of 0.1% calcofluor white reagent and one drop of 10% Potassium Hydroxide

3. Cover the preparation with a coverslip

4. Leave for stain in 1 minute

5. Gently remove excess fluid by gently pressing the slide with a paper towel

6. Observe and examine for flurorence under flurorescence microscope at x100 to x400 magnification

Result, Interpretation of Calcofluor White Stain (CFW)

Fungal hyphae, pseudohyphae, yeasts, amoebic cysts, and parasitic organisms (except trophozoites) appear bright green to blue against other biological materials which appear reddish orange.

A yellowish-green background fluorescence may occur in tissue samples. It can be differentiated as fungal and parasitic organisms will have a more intense fluorescence.

Application of Calcofluor White Stain (CFW)

In Medicine

In the medical field, Calcofluor White Stain (CFW) can be used in parasitology as well as clinical mycology. If used in association with Papanicolaou stain, Calcofluor White Stain (CFW) greatly helps in staining yeasts in Pap smears.

In research

In research, Calcofluor White Stain (CFW) is used to determine the age of yeast cells and AD research to study the fundamental processes of aging. Since bud scars have a higher content of chitin than the rest of the yeast cell membrane, Calcofluor White Stain (CFW) stains them more. Thus, the greater the number of bud scars on the cell, the more aged it is.

It is also used in staining of thecal plates in armored dinoflagellates as well as identification of Mucor (causative agent of Mucormycosis).

In Textiles

Calcofluor White Stain (CFW) is also used in fabric and papermaking industries as a brightening dye. The water-soluble agent is colorless at first and later develops an optical brightening effect which gives a whiter appearance to textiles. It is also one of the ingredients in laundry detergents as a brightener.

Advantages of Calcofluor White Stain (CFW)

• is a rapid test that requires a quick screening time (within 1 minute per slide) as compared to KOH preparation (3-4 minutes on each slide)

• in comparison to KOH preparation, Calcofluor White Stain (CFW) has high sensitivity and specificity

• clearing up background specimens can aid in the visualization of fungal elements

• can be used in cytopathology and by pairing with Pap

• does not interfere with PAS or Gram staining

• used in structural detection of  non-culturable fungus Pneumocystis jirovecii 

Disadvantages of Calcofluor White Stain (CFW)

• Requires fluoresce microscopy

• Is expensive to be used in routine diagnosis

• Vaginal secretions are difficult to interpret due to the presence of commensal fungi

• Less sensitivity and specificity than the Chicago Sky Blue 6B stain

• Calcofluor White Stain (CFW) poorly stains several fungi such as Coccidioides immitis, Histoplasma capsulatum, and Cryptococcus neoformans.

• Dark-pigmented fungi may hide the fluorescence emitted by the Calcofluor White Stain (CFW)

• Cotton fibers, which also fluoresce strongly, must be differentiated from fungal hyphae

• Fungal trophozoites will not stain thus fluoresce is absent