A Versatile Giemsa Protocol for Permanent Nuclear Staining of Fungi

Abstract

A variety ofcytological stains and staining procedures including Giemsa-HCl (23), acetic-orcein (11), propionic-carmine (17), iron haematoxylin (20), safranin 0 (1), aniline blue or trypan blue (5), toluidine blue (9), and basic fuchsin or Feulgen stain (4, 16) have been used to investigate the nuclear condition of reproductive and somatic structures of fungi. Fluorescent stains such as acriflavin (19), acridine orange (22), 2,5bis(4-aminophenyl)1 ,3,4-oxadiazole (BAO) (14), and especially 4,6-diamidino-2-phenylindole (DAPI) (3, 13, 18) have been employed more recently to differentiate and improve the resolution of nuclear DNA from other cellular constituents and to quantitate genomic DNA through microspectrophotometric analyses. Most of these staining procedures were developed to produce temporary slides. Destaining solutions often are used as mounting media for temporary slides and usually cause stained nuclei to fade or become less differentiated within several hours to several days after preparation. Consequently, slide preparations from such procedures must be viewed and photographed during the short period when they are optimally differentiated. Since many nuclear-staining procedures are useful when only temporary slides are required, there is a need for a protocol that provides stable nuclear staining on permanent slides. Giemsa stain has been used successfully for many years on a large diversity of fungi with various procedures and modifications. This paper describes a protocol that allows preparation of slides for permanent nuclear staining of fungal structures with minimal fading. The procedure is useful for revealing numbers and positions of nuclei and providing tentative indications of ploidy as inferred from the numbers and sizes of nuclei within cells. It provides the capability of