Modified staining protocol with Safranin O and Astra Blue for the plant histology

Many staining protocols are widely applied in botanical microtechniques and serve specific histological purposes. In particular, some dyes are used simultaneously to receive contrasting colorations of different chemical structures, e.g., lignin and cellulose. One of the most popular differential staining protocols is based on the Safranin O / Astra Blue dyes combination. Safranin O is a water-soluble basic dye that stains lignin in red. Astra Blue is also a water-soluble dye but having an acidic reaction, which stains cellulose in blue. Usually, a 1–2 % solution of Safranin O in distilled water or 50–70 % ethanol is applied in combination with the 0.5–1 % water solution of Astra Blue to detect lignified structures and obtain contrasting pictures convenient for the light microscopy. For a long time, Astra Blue was used exclusively with water solutions, and such recommendation without additional options is indicated on producers’ web sites. However, in 2002 it was proposed to use 1 % Astra Blue solution in 95 % ethanol to identify the lignified tissues. Later, such an ethanol solution of Astra Blue was also successfully applied by other researchers for different experimental purposes.We tested the modified staining protocol with the application of both 1 % Safranin O and Astra Blue solutions in a slightly lower concentration of ethanol (70 %) on the flower buds of Gagea lutea (Liliaceae) and found it working well. We believe that such a modified protocol with the solutions of these two dyes in 70 % ethanol allows simplifying the procedure of the plant material staining due to application of the same concentrations of dissolvent and reducing the difference in solvent concentration between two following contrasting staining solutions. Such differential staining can be effectively applied for plant histology purposes, especially where there is a need to distinguish lignified structures and secretory tissues.