Nuclei of vegetative hyphae of the puffball species Lycoperdon pyriforme are small and difficult to stain. A simple mitotic staining technique utilizing a modified version of Ziehl-Neelsen's carbol-fuchsin stain, originally developed for examining bacteria and small chromosomes of plants, has proven useful for observing fungal nuclei in this gasteromycete species. Cells of vegetative hyphae were pre-treated for 2 hours with an aqueous solution saturated with naphthalene, fixed in a 3:1 mixture of 100% ethanol and glacial acetic acid, and hydrolyzed for 10 minutes in a 1:1 mixture of 100% ethanol and 12 M hydrochloric acid. Cells were rinsed with distilled water before exposure to the carbol-fuchsin stain. Microscopic examination of hyphae revealed that the nuclei were darkly stained, whereas the rest ofthe tissue remained relatively clear. Examination of 800 apical and adjacent subapical cells from six isolates revealed that the hyphae are predominantly binucleate. This nuclear staining technique may prove useful in studies of the life cycle and genetics of this and other fungal species.
Huss, Martin J.
"Application of a Modified Ziehl-Neelsen's Carbol-Fuchsin Stain for Observing Nuclei in Vegetative Hyphae of the Puffball Species Lycoperdon pyriforme,"
Journal of the Arkansas Academy of Science: Vol. 50
, Article 14.
Available at: http://scholarworks.uark.edu/jaas/vol50/iss1/14