Calcofluor-white

Calcofluor-white or CFW is a fluorescent blue dye that is used to bind to the polysaccharide polymers of amebic cysts. It functions by being able to bind to 1-3 beta and 1-4 beta polysaccharides on chitin and cellulose that is present in cell walls on fungi, plants, and algae.

The fluorescent staining of yeast with calcofluor-white. The yeast shows a vivid blue color for the cell walls.

In plant cell biology research, it is used for the staining of cell walls of both algae and higher plants.[1][2]
It is also useful in medicine and animal biology for the identification of fungi in the tissue,[1][3][4] (it binds to the chitin in the cell walls of the fungi).

Usage

Medicine

CFW can be used in both clinical mycology and parasitology. Free-living amoebae in ocular species can have cysts that can be vividly seen with the stain. These have been seen in eye-wash stations in laboratories[5]. CFW can be used with the Papanicolaou stain to help strengthen the response of yeasts in Pap smears[6].

Research

In research, calcofluor-white is also used to stain bud scars of yeast cells because the bud scars have a higher content of chitin, which stains them more than the rest of the cell membrane. Due to this stain it is possible to count the bud scars which is an indication for the age of the cell. It shows similar staining patterns as Wheat Germ Agglutinin (WGA). It is especially useful for the identification of Mucor and the causative agents of zygomycosis. Calcofluor-white can be used to stain thecal plates in armoured dinoflagellates.[7] Calcofluor is also used as the dye in white-colored clothing, and results in the characteristic appearance under "black-lighting".

Textiles

CFW is a fabric brightening dye. It is water-soluble and colorless at first. It has an optical brightening effect that has been used since the 1940s to give a whiter appearance to textiles. It is also added to laundry detergents commercially as a brightener[8].

Fluorescence
The cross section of an Eagle Fern shows the bright green and bright blue fluorescence seen with CFW.

Solutions that are aqueous for calcofluor-white have an absorption spectrum from 300 to 412 nm with a peak at 347 nm. The peak of fluorescence occurs best with ultraviolet light, but violet/blue violet also gives great results of fluorescence. Calcofluor-White is an fluorescent dye, meaning it has an excitation and emission wavelength that differs from one another. The excitation wavelength is 380 nm while the emission is 475 nm. The fluorescence can best be seen with ultraviolet light, but it can also be seen with violet or blue violet.

Fungi tend to display a bright green color under UV/violet/violet-blue light due to the barrier filters used in microscopy for fluorescence. Calcofluor white has to careful interpretation of staining as some non-specific reactions can make the background look a fluorescent yellow-green color. This can be reduced by looking at the cells under blue light or with other filters to see the best picture possible. Evans blue is a counterstain for calcofluor white as it can help diminish the background tissues and cells with blue light[9].

Comparative Studies

In a comparative study of KOH, Calcofluor-White and Fungal cultures for diagnosing Fungal Onychomycosis, CFW was seen as the most sensitive method in detecting the infection in humans. The study saw that out of 150 patients CFW showed positive in 95 of them. The study also reviewed key advantages for using the Calcofluor-White stain over KOH. It discussed that CFW requires a quick screening time as it can be seen on low power objectives and can be done within a minute while KOH requires about 3-4 minutes for each slide. It is a quicker and more sensitive method of screening than KOH, and the background material can be diverted using CFC[10].

In a study of methods to diagnose mycoses, a fungal infection in humans, CFW was compared with KOH and Chicago Sky Blue 6B. CFW showed positive testing in 53.4% of cases that had suspected mycoses infections while Chicago Sky Blue 6B had 55% accuracy. The study showed that the CFW is less effective in quick detection compared to Chicago Sky Blue 6B, but it is an effective way to detect the fungal infection[6].

A study that investigated the detection of Candida in cancerous or precancerous lesions using CFW with fluorescent microscopy. The study wanted to see the effectiveness of Calcofluor-White against a species of mycotic infections that can cause death or serious injury against patients who are immunocompromised. It also investigated the sensitivity of the dye compared to Gram staining and PAS staining. The study concluded that it was a quick and easy way to diagnosis in cytopathology and histopathology, but it was relatively expensive. The study also showed that no special techniques were needed to make the staining effective. The dye also does not interfere with PAS or Gram staining. For cytology, Pap-CFW gave a positive response 50 out of 135 times, and for histopathology, it gave a positive response 43 out of 135 for just the CFW stain. The study showed that the staining improved with a pairing of Pap and CFW. The results gave that the dye was incredibly effective in terms of quick diagnosis and effectiveness, making it superior to Gram positive and PAS in this particular study[11]. Overall, the stain has key advantages to research and diagnostic purposes, but it has an issue of being slightly expensive and can not be as accurate for certain species.

Problems with Calcofluor-White Staining

There have been studies showing that CFW does not fluorescent well with Coccidioides immitis despite it being a fungus. Histoplasma capsulatum and Cryptococcus neoformans also tend to have problems with showing the fluorescence of CFW. Another report saw the Pneumocystis carinii in lung biopsies had to be stained at room temperature for five minutes and then 65°C for five minutes to see the fluorescence best. Fluorescence with a darkly pigmented fungi can have misleading results as it is dependent on how pigmented the fungi is to how well the stain works[5].

References
  1. Herth, W.; E. Schnepf (1980). "The fluorochrome, calcofluor white, binds oriented to structural polysaccharide fibrils". Protoplasma. Springer. 105 (1–2): 129–133. doi:10.1007/BF01279855. ISSN 0033-183X.
  2. Bidhendi, AJ; Chebli, Y; Geitmann, A (May 2020). "Fluorescence Visualization of Cellulose and Pectin in the Primary Plant Cell Wall". Journal of Microscopy. doi:10.1111/jmi.12895. PMID 32270489.
  3. Hoch, H.C.; C.D. Galvani; D.H. Szarowski; J.N. Turner (2005). "Two new fluorescent dyes applicable for visualization of fungal cell walls". Mycologia. 97 (3): 580–588. doi:10.3852/mycologia.97.3.580. ISSN 0027-5514.
  4. Burns, Edward R.; Wittner M.; Faskowitz F. (5 April 2005). "Method for detecting chitin-containing organisms - Patent 6875421". Retrieved 10 November 2010.
  5. Harrington, Brian J.; Hageage, George (May 2003). "Calcofluor White: A Review of its Uses and Applications in Clinical Mycology and Parasitology" (PDF). your lab focus. Department of Public Health and Department of Microbiology and Immunology, Medical College of Ohio, Pathology Department, Mercy Integrated Laboratories, and Department of Microbiology and Immunology, Medical College of Ohio, Toledo, OH. Archived (PDF) from the original on May 2003. Retrieved 10 April 2019.
  6. Afshar, Parvaneh; Larijani, Laleh Vahedi; Rouhanizadeh, Hamed (2012–2018). "A comparison of conventional rapid methods in diagnosis of superficial and cutaneous mycoses based on KOH, Chicago sky blue 6B and calcofluor white stains". Iranian Journal of Microbiology. 10 (6): 433–440. ISSN 2008-3289. PMC 6414738. PMID 30873272.CS1 maint: date format (link)
  7. Fritz, L. and Triemer, R. 1985. A rapid simple technique utilizing calcofluor white M2R for the visualization of dinoflagellate thecal plates. Journal of Phycology, 21: 662-664.
  8. Hoch, H. C.; Galvani, C. D.; Szarowski, D. H.; Turner, J. N. (2005-05-01). "Two new fluorescent dyes applicable for visualization of fungal cell walls". Mycologia. 97 (3): 580–588. doi:10.1080/15572536.2006.11832788. ISSN 0027-5514.
  9. Biologicals, Daylnn (September 2005). "Calcofluor White Stain" (PDF). Dalynn Biologicals. Catalogue No. SC15.
  10. Dass, S. Manick; E. V, Vinayaraj; Pavavni, K.; Pallam, Aishwarya; Rao, M. Srinivas (2015). "Comparison of KOH, Calcofluor White and Fungal Culture for Diagnosing Fungal Onychomycosis in an Urban Teaching Hospital, Hyderabad" (PDF). Indian Journal of Microbiology Research. 2 (3): 148–153. doi:10.5958/2394-5478.2015.00004.7 – via Amazon AWS.
  11. Bhavasar, Rashmi S. K.; Goje, Santosh Kumar; Takalkar, Anant Arunrao; Ganvir, Sindhu M.; Hazarey, Vinay K.; Gosavi, Suchitra R. (2010). "Detection of Candida by Calcofluor White". Acta Cytologica. 54 (5): 679–684. doi:10.1159/000325232. ISSN 0001-5547.


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