Calcofluor white fungal cell wall staining

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CFW (CalcoFluor White; Ex: 347 nm – Em: 450 nm)

Principle

Calcoflour white stain may be used for direct examination of most specimens using fluorescent microscopy. The cell walls of the fungi bind the stain and fluoresce blue-white or apple-green depending on the filter combination used. The use of calcofluor white (CFW), a fluorescent brightener used in the textile industry, with the addition of potassium hydroxide (KOH) will enhance the visualization of fungal elements in specimens for microscopic examination. The CFW nonspecifically binds to the chitin and cellulose in the fungal cell wall and fluoresces a bright green to blue.

Used with Evans Blue as a counterstain the fungal elements light up green with a red background (420-490 nm excitation, suppression filter 520 nm emission) or
blue against a red background (340-380 nm excitation, suppression filter 430 nm)

Preparation of reagents

Mix in a 1:1 ratio 10% KOH (Potassium hydroxide) solution with the CFW solution. Calcofluor white is light sensitive and breaks down in KOH, so prepare solutions not too long in advance and keep the solution in the dark.

10% Potassium Hydroxide Solution

  • potassium hydroxide 10 g
  • glycerol 20 ml
  • distilled water 80 ml
  • Store at room temperature in the dark and discard if a precipitate forms.

0.1% Calcofluor White M2R (W/V) Solution

  • Use commercially available solution from Fluka (18909-100mL-F) Calcofluor White-M2R 1g/L
  • Gently heat if precipitate develops. Filter if precipitate persists.
  • Store at room temperature in the dark.

Microscope filter system

For use with the confocal microscope use excitation around 370 nm and excitation at 440 nm

An epifluorescent microscope equipped with a mercury vapor lamp and either an ultraviolet (UV) or blue-violet (BV) excitation filters to achieve radiation on the slide below 412 nm should be used since the maximum absorbance of CFW is 347 nm. Several broad band or barrier filters are available to achieve the radiation required (300-412 nm) from the microscope manufacturers.

Depending on which barrier filter is used, a blue-white or a bright green color will be observed staining the fungal cell walls. A microscope with selective filters that will prevent radiation below 490 nm should not be used for CFW; neither should a microscope with a quartz halogen bulb since the energy output is too low.

Procedure

  • Add an equal amount of KOH-CFW to the specimen
  • Mix and place a cover glass over the material
  • If necessary, allow the KOH preparation to sit at room temperature for a few minutes until the material has been cleared. The slide may be warmed to speed the clearing process.

Based on the method by doctorfungus.org accessed on 7 April 2014 a website that no longer exist...