xxx INTRODUCTION. 



small portions of large ones. Use a relatively large bulk of 

 the fixing fluid, and wash for several hours according to the 

 fixative employed. 



2. Dissociating or macerating consists in dissolving the 

 intercellular substance that binds the tissue together. By 

 the aid of this method, followed by teasing, the separate cells 

 of a tissue may be isolated. Any fixative used in a diluted 

 form (about one-tenth) acts as a dissociator. Weak alcohol 

 (30 per cent.), osmic and acetic acids, and Muller's fluid 

 are the most useful. For Hydra, osmic-acetic acid is specially 

 useful. 



3. Hardening is necessary in the case of soft animals or 

 of tissues to be cut into slices. Grades of alcohol 50 per 

 cent., 70 per cent., and 90 per cent, are successively em- 

 ployed after fixation and washing. In other cases (e.g. the 

 fresh-water mussel) a weak solution of chromic acid (J per 

 cent.) in water serves well. For the brain of the rabbit and 

 pigeon ordinary methylated spirit is convenient. 



4. Staining not only makes the tissues more distinct, but 

 also affords insight into their minute structure. 



Most stains will not penetrate a living organism or tissue. 

 Methylene blue, however, is an exception, and therefore of 

 great value. It is employed in very dilute solutions (see 

 Appendix) for the study of small transparent forms (e.g. 

 Paramecium, Hydra) and for the tracing out of nerves and 

 nerve-cells in higher animals. For rapidly staining dead 

 but fresh tissues and such as have been fixed and washed in 

 water, safranin and dahlia are useful, especially for Pro- 

 tozoa, developing spermatozoa of the earthworm, leech, and 

 crayfish. The stain must only be allowed to act for two or 

 three minutes, and then the surplus carefully removed by 

 distilled water. 



Carmine is a generally useful stain, especially Grenacher's 

 borax-carmine. As this is an alcoholic stain, specimens 

 should be ^placed in it from 50 per cent, alcohol. After the 

 staining is completed, the object should be transferred to 

 acid-alcohol for a few minutes or hours according to its size. 



Picrocarmine is an aqueous solution and directly applicable 



