a _second hardening_ stage has been the cause of the divergence in meaning of the two terms. The best fixing agents are those that kill the cells at once, but cause a slow coagulation with little or no shrinking. They must penetrate and diffuse through the tissues rapidly so that the deepest cells are quickly reached. Acid media, especially those containing small percentages of acetic acid, are therefore better than alkaline solutions. The tissue-elements, particularly the nuclei, must be preserved as perfectly as possible so that they will not be affected by further procedures of microscopic technique. The chemic properties of physiologic and pathologic substances must likewise be preserved. The preservation of karyokinetic figures is a criterion of good fixation. In pathologic work it is also desirable that the fixing agent should preserve the red blood cells, and permit of the staining of bacteria in sections. Since fixing media are more or less selective in their action, it follows that there is no one fixative that gives equally good results in all cases. Especial fixing reagents must be used for the demonstration of certain substances (fat, etc.), or for the use of certain staining methods. Some stains cannot be used at all after certain fixing agents have been employed. For general use that fixing agent having the widest range of usefulness should be employed; and for this reason fixing media composed of several fixing agents are often employed in preference to the use of a single one. GENERAL RULES FOR FIXING AND HARDENING.