Simple staining methods for botanical sections

Chemicals and dyes: Distilled water, isopropyl alcohol, xylene, mounting medium solved in xylene, DELAFIELD´s hematoxylin, chrysoidine, aniline blue, safranine, methylated spirit. You also need staining blocks and dropper pipettes.

Suitable objects: Lignified stems or twigs, about 5 mm in diameter.

Preparation: Make sections with a razor blade (brand blades!) and collect them in a staining block filled with distilled water. It should be stressed that it is impossible to get complete cross sections this way - unfortunately not only beginners try that! Try to produce small wedge-shaped sections instead. Such sections do not look very nice, but they are sufficiently thin at the "edge" of the wedge and show all details wanted.

Monochromatic staining with DELAFIELD´s hematoxylin

Put the sections into a small staining block, wash with distilled water (no tap water!) and cover them with diluted DELAFIELD´s hematoxylin for about 5 minutes. Stir gently. Other preparations of hematoxylin are inadequate here. Remove the dye stuff with a dropper pipette, then wash several times with tap (!) water. The colour of the sections will change from light brown to dark violet or dark blue with hematoxylin being fixed on the tissue at the same time. This reaction is due to the alkalinity of tap water. Therefore distilled water, rainwater and tap water with nearly no alkaline are insufficient. In that case add some sodium hydrogencarbonate. Then wash the sections three times with isopropanol (2 minutes each time), wash with xylene two times, mount with a resin solved in xylene (e.g. Canada balsam) and cover with a coverslide. If a stove is available dry for 48 hours at 60 oC.

Dichromatic staining with DELAFIELD´s hematoxilin and chrysoidine

Stain the sections with DELAFIELD´s hematoxylin as described above. Then remove the water and cover with a solution of chrysoidine (1 gram chrysoidin dissolved in a mixture of 50 ml methylated spirit and 50 ml distilled water). Wait until she sections are stained dark orange, then wash quickly three times with isopropanol. Since the sections are stained too intensively now, add some drops of water and stir gently sucking the liquid with a dropper pipette to and fro. The isopropanol, as it contains a small amount of water slowly extracts the chrysoidine. When blue and orange areas are distinctly different stop the process by removing the diluted isopropanol and adding pure isopropanol. Wash twice, then wash twice with xylene and cover with Canada balsam or a similar mountant. Non-lignified cell-walls are stained blue, lignified cell-walls are stained orange. This method is safe as the blue staining of hematoxylin is stable during the process.

Dichromatic staining with safranine und aniline bue

Dissolve 1 gram of safranine or aniline blue respectively in a mixture of 50 ml methylated spirit and 50 ml destilled water..

First stain the sections with safranine for 5 minutes until they are dark red, wash them with isopropanol three times, then add some water then to the osopropanol and extract the safranin slowly until the sections are a dark pink. Add aniline blue for about one minute, wash again with isopropanol, add some water to the isopropanol and extract aniline blue until the sections show distinct areas stained pink or blue respectively. Stop the process with pure isopropanol, wash twive, then wash twice with xylene and cover with a mountant solved in xylene. The method needs some intuition, as the extraction of aniline blue effects safranine as well, so do not remove too much of the safranine during the first step.

Since all methods given here are carried out in staining blocks and the solvents are added with a dropper pipette, the consumption of dyes and solvents is very low.

 

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