Crystal Violet Staining Procedure for Root-Tip Chromosomes.
L. F. Randolph
- Place slides in xylol to remove the paraffin. Flush with fresh xylol, then with absolute alcohol. Pass the slides successively through 95%, 60% and 30% alcohol to water; 3-5 minutes for each step.
- 1% potassium permangenate, 2-3 minutes. Rinse in tap water.
- 5% oxalic acid, until the sections are bleached - usually 1-3 minutes. Prolonged treatment with oxalic acid sometimes causes the sections to come off the slide. Wash in tap water 15 minutes. The bleaching process in permangenate and oxalic is not always necessary, but it usually adds contrast.
- Mordant in 1% chromic, 20 minutes. Rinse in tap water and then in 2 or 3 changes of distilled water.
- 1% aqueous solution of crystal violet, 4 hours. It is often desirable to vary the staining period. If the stain comes out too rapidly in the alcohols and clove oil, leave the slides in tne stain longer. If destaining is prolonged, shorten the period. Rinse in tap water.
- Treat with iodine-potassium iodide (iodine 1 gm., potassium iodide 1 gm., 80% alcohol 100 cc.) until the color of the sections changes from blue to brown, usually 1-2 minutes.
- Rinse in 95% alcohol and pass through 3 changes of absolute alcohol to clove oil. Differentiate in the alcohols and clove oil, ordinarily 1-3 minutes. Watch the process in the final stages under the microscope. The metaphase chromosome groups under a 16 mm. objective should stand out sharply against a practically colorless background of cytoplasm.
- Pass through several changes of xylol to remove all of the clove oil. Mount in thin xylol-balsam. After the cover glass is in place invert the slide on paper toweling and apply mild pressure to force the excess balsam from under the cover glass. Add a few drops of xylol to the edges of the slide, cover with another paper towel and a piece of heavy glass, or other suitable weight. As soon as the slides are dry they may be examined. This method of mounting removes all excess balsam and brings the cover in close contact with the material, so that high-power objectives may be used with greater safety.