Instructions for Calcofluor use in fluorescence (Harrington and Hageage Jr 2003.)

Aqueous solutions of CFW have an absorption peak at 347 nm. Maximum excitation and fluorescence occurs with UV light. Excitation with violet or blue violet (BV) light, (BG3 and BG12 exciter filters) also give good results. A 50 or 100 watt mercury vapor lamp used in an epi-fluorescence microscope is recommended.

(Narrow bandwidth blue light exciter filters used for acridine orange or auramine O excitation cannot be used for CFW staining because they block wavelengths below 490 nm. Quartz halogen bulbs have too low energy output in the range required to fluoresce CFW and should not be used.)

The fluorescence of CFW is bluish/white, too intense for unaided eyes; but with barrier filters commonly used in fluorescence microscopy (i.e.: 510, 520, 530), the fluorescence is bright green.

Evans blue can be used as a counterstain—incorporated in the CFW solution at 0.01% to 0.05% (w/v). BV excitation imparts a reddish coloration to background material, (and may suppress non-specific background fluorescence). Evans blue excites maximally at ~500 nm. With UV excitation it does not fluoresce and a reddish color is not seen.