Use of dimethylsulfoxide to prevent clumping during feulgen staining of ceratocystis ulmi

The dimorphic fungus Ceratocystis ulmi is the causative agent of Dutch Elm Disease. As part of a study on the regulation of this developmental phenomenon, we attempted to stain the nuclei of cells growing vegetatively in the yeast phase by a modification of the Feulgen technique described by Gauger (1975). The cells were harvested by centrifugation, washed twice, and resuspended in 0.05 M phosphate buffer (pH 6.5). A small portion of this cell suspension was placed on a clean No. 2 glass coverslip (22 ± 22 mm) and allowed to air dry. The coverslip was flamed briefly to heat fix the cells whereupon they were fixed in glacial acetic acid: 95% ethanol (1:3 v/v) for one hour, hydrolyzed in 1 N hydrochloric acid at 60 C for 5 minutes, and stained for 30 minutes. The Feulgen stain was prepared according to Stevens (1974). Subsequently, the coverslip was rinsed briefly with distilled water and dehydrated for 30 seconds in 70% ethanol. After air drying, the coverslip was mounted on a glass microscope slide with Permount (Fisher Scientific Co.) and examined.