The simple method of fixing and staining, just described, while giving excellent results in many cases, is not always applicable, nor as a rule are the permanent preparations so made satisfactory. For permanent preparations, strong alcohol (for very delicate tissues, absolute alcohol, when procurable, is best) is the most convenient fixing agent, and generally very satisfactory. Specimens may be put directly into the alcohol, and allowed to stay two or three days, or indefinitely if not wanted immediately. When alcohol does not give good results, specimens fixed with chromic or picric acid may generally be used, and there are other fixing agents which will not be described here, as they will hardly be used by any except the professional botanist. Chromic acid is best used in a watery solution (five per cent chromic acid, ninety-five per cent distilled water). For most purposes a one per cent solution is best; in this the objects remain from three or four to twenty-four hours, depending on size, but are not injured by remaining longer. Picric acid is used as a saturated solution in distilled water, and the specimen may remain for about the same length of time as in the chromic acid. After the specimen is properly fixed it must be thoroughly washed in several waters, allowing it to remain in the last for twenty-four hours or more until all trace of the acid has been removed, otherwise there is usually difficulty in staining. As staining agents many colors are used. The most useful are hæmatoxylin, carmine, and various aniline colors, among which may be mentioned, besides gentian violet, safranine, Bismarck brown, methyl violet. Hæmatoxylin and carmine are prepared in various ways, but are best purchased ready for use, all dealers in microscopic supplies having them in stock. The aniline colors may be used either dissolved in alcohol or water, and with all, the best stain, especially of the nucleus, is obtained by using a very dilute, watery solution, and allowing the sections to remain for twenty-four hours or so in the staining mixture. Hæmatoxylin and carmine preparations may be mounted either in glycerine or balsam. (Canada balsam dissolved in chloroform is the ordinary mounting medium.) In using glycerine it is sometimes necessary to add the glycerine gradually, allowing the water to slowly evaporate, as otherwise the specimens will sometimes collapse owing to the too rapid extraction of the water from the cells. Aniline colors, as a rule, will not keep in glycerine, the color spreading and finally fading entirely, so that with most of them the specimens must be mounted in balsam. Glycerine mounts must be closed, which may be done with Canada balsam as already described. The balsam is best kept in a wide-mouthed bottle, specially made for the purpose, which has a glass cap covering the neck, and contains a glass rod for applying the balsam.