Hydrolysis and staining
The cell wall was softened using an acid to make the cells easier to squash.
The acid used in this study was 5N HCl and the hydrolysis performed for 30 minutes.
Additional softening with enzymes can be employed in the later step depending on the
schedule used (14). In this study, 4% cellulase and pectinase are used after the staining
stage at 60°C for 30 minutes. Feulgen which is a dye made mainly from pararosaniline,
is used to stain the chromosomes. The dye is light sensitive and thus the staining is
carried out in dark room, for 3 hours. DNA is stained a deep magenta colour while the
other cell components remain unstained.