Improved Methods for Acetocarmine and Haematoxylin Staining to Visualize Chromosomes in the Filamentous Green Alga Zygnema (Charophyta).

Genome sizes of Zygnema spp. vary greatly, being unknown whether polyploidization occurred. The exact number of chromosomes in this genus is unknown since counting methods established for higher plants cannot be applied to green algae. The massive presence of pectins and arabinogalactan proteins in the cell wall interferes with the uptake of staining solutions; moreover, cell divisions in green algae are not restricted to meristems as in higher plants, which is another limiting factor. Cell divisions occur randomly in the thallus, due to the intercalary growth of algal filaments. Therefore, we increased the number of cell divisions via synchronization by changing the light cycle (10:14 h light/dark). The number of observed mitotic stages peaked at the beginning of the dark cycle. This protocol describes two methods for the visualization of chromosomes in the filamentous green alga Zygnema. Existing protocols were modified, leading to improved acetocarmine and haematoxylin staining methods as investigated by light microscopy. A freeze-shattering approach with liquid nitrogen was applied to increase the accessibility of the haematoxylin dye. These modified protocols allowed reliable chromosome counting in the genus Zygnema. Key features Improved method for chromosome staining in filamentous green algae. Optimized for the Zygnema strains SAG 698-1a (Z. cylindricum), SAG 698-1b (Z. circumcarinatum), and SAG 2419 (Zygnema 'Saalach'). This protocol builds upon the methods of chromosomal staining in green algae developed by Wittmann (1965), Staker (1971), and Fujii and Guerra (1998). Cultivation and synchronization: 14 days; fixation and permeabilization: 24 h; staining: 1 h; image analysis and chromosome number quantification: up to 20 h.