Calcofluor White Staining: Principle, Procedure, and Application

Calcofluor white staining uses fluorescent dyes to stain the chitin and cellulose in the fungi, plants, and algae cell walls. Calcofluor binds non specifically to the chitin and cellulose, and exposure to the long-wavelength ultraviolet and short-wavelength visible light helps in colored observation.

Although KOH wet mount is the recommended method for direct examination of fungal stains, it has less sensitivity and can not differentiate the hyphae from the collagen fibers and other artifacts. So, calcofluor white staining is preferred nowadays.

This technique helps visualize a mixed fungal infection as well. When coupled with the KOH, the calcofluor white stain provides better sensitivity to detect fungi in the clinical specimen. The KOH clears the cell debris and calcofluor and then colors fungus, which has a bright green color when observed under a fluorescence microscope.

Principle of Calcofluor White Staining

Calcofluor white (CFW) is a water-soluble, colorless dye and fluorescent whitener used in the textile and paper industry. When the CFW comes in contact with clinical specimens, it binds with the 1-3 beta and 1-4 beta polysaccharides on the chitin and cellulose if fungal elements are present. Then it fluoresces into green color when exposed to ultraviolet light. It can be observed under a fluorescent microscope, and the differentiation of infectious compounds is based on color and morphology.

The CFW excites at 380 nm wavelength. The absorption in calcofluor white stain occurs over the 300-412 nm range. Its absorbance peak is 347nm, and its emission peak is 475 nm. Using violet or blue light also gives good results, but one prefers UV light because maximum excitation and fluorescence occur with it. During the fluorescent microscopic observation, fungi and other organisms like a cyst of Pneumocystis fluoresce brilliant apple-green. The green coloration is due to barrier filters in the fluorescence microscope. Other elements present in the sample will fluoresce as reddish-orange. Yellowish-green background fluorescence can also be observed when a tissue sample is used. Observing the slide under the blue light and using various combinations of exciter and barrier filter can diminish background fluorescence.

Evans blue is used as a counterstain and diminishes the background fluorescence by using the blue light excitation (not UV). The addition of 0.1 % of Evans blue minimizes the non-specific background fluorescence. Evans blue counter stain also produces the contrasting orange to ruby-red background and aids in clearly demonstrating the fungi in the surrounding tissue.

Preparation of the Calcofluor White Stain

The preparation of calcofluor white stain requires calcofluor white powder, which is dissolved in distilled water. 1% (w/v) calcofluor white stain is prepared by properly dissolving the 1 g powder of calcofluor white and 100 ml distilled water. The best way to use the solution is to dilute the 1% CFW solution to 0.1%. Storage should be done at room temperature and in the dark helps preserve the solution for years.

Adding 10% KOH solution (10 g KOH powder in 90 ml distilled water and 10 ml glycerol) to the CFW just before use is helpful while handling skin and nail samples.

Likewise, 0.05% to 0.01% Evans blue solution (w/v) can be added to the CFW as counter stain. Evans blue solution can also be used after using a calcofluor white stain.

Calcofluor White Staining Overview

Procedure

1. Firstly take a clean, grease-free glass slide and place the sample in the center.
2. Then add one drop of calcofluor white stain or CFW with Evans blue solution on top of the sample.
3. For nail and skin scrapings, add 10% KOH in the slide.
4. Then, put the coverslip on top and let it stand for a minute.
5. After that, observe the slide under UV light at 100X to 400X magnifications.

Result and Interpretation of Calcofluor White Staining

• Fungi and the parasites fluoresce apple-green while its other elements fluoresce reddish-orange.
• Cotton fibers fluoresce more intensely than the fungal hyphae, so one must carefully observe them.
• In the case of amoeba, its trophozoite will fluoresce, but its cysts will fluoresce.
• Cysts of the Pneumocystis are observed as a round cell having a uniform shape with a 5-8 µm diameter. It is differentiated from yeast cells based on budding characteristics and deep internal staining.
• The reaction may be non-specific in the tissue samples.

Application of the Calcofluor White Staining

Calcofluor white staining is one of the methods being used frequently for the rapid detection of fungal infections. Some of the more detailed application of calcofluor white staining are as follows:

• Calcofluor white is a sensitive stain that helps to visualize the hyphae, pseudohyphae, and yeast. Since the chitin concentration is higher in the budding yeasts, calcofluor white stains the bud scars more intensely.
• Calcofluor white stain helps detect non-culturable fungus like Pneumocystis jirovecii. 
• Calcofluor stain can be applicable for observing non-fungal agents such as free-living amoebae (Acanthamoeba, Naegleria, and Balamuthia) and larva of Dirofilaria. Calcofluor white stain helps in the rapid diagnosis of Acanthamoeba keratitis from the corneal scrapings and keratectomy specimens.
• Calcofluor white stain can be incorporated into the growth media because it can withstand the autoclave conditions (121°C for 15 minutes). So, a calcofluor white stain can be used as the vital stain growing fungus in the slide culture.
• Calcofluor white stain stains the vegetative cells but not the ascospores. So, it helps in differentiation when incorporated into the ascospore-inducing media.

Vital stain means the process of adding the stain on living cells without killing it.

Limitations of the Calcofluor White Staining 

Although Calcofluor white staining is a technique with a wide range of applications, it has a limitation; it is an expensive method. All routine laboratories cannot afford the fluorescence microscope with a wavelength filter of 390-420nm.

References

• Monheit, J. E., Cowan, D. F., & Moore, D. G. (1984). Rapid detection of fungi in tissues using calcofluor white and fluorescence microscopy. Archives of pathology & laboratory medicine, 108(8), 616–618.
• Wilhelmus, K. R., Osato, M. S., Font, R. L., Robinson, N. M., & Jones, D. B. (1986). Rapid diagnosis of Acanthamoeba keratitis using calcofluor white. Archives of ophthalmology (Chicago, Ill.: 1960), 104(9), 1309–1312. https://doi.org/10.1001/archopht.1986.01050210063026
• Harrington, B. J., & Hageage, G. J. (2003). Calcofluor White: A Review of its Uses and Applications in Clinical Mycology and Parasitology. Laboratory Medicine, 34(5), 361–367. https://doi.org/10.1309/eph2tdt8335gh0r3
• Chander, J. (2018). Textbook of Medical Mycology (Fourth edition). Jaypee Brothers Medical Publishers Ltd.