34 METHODS IN PLANT HISTOLOGY 



the surface. Even then, it must be remembered that a waxy or cu- 

 tinized or suberized surface presents an almost impassable barrier to 

 the chromic series. 



Some objects, although small, cause trouble in various ways. Many 

 buds are hairy and will not sink; if such things are dipped quickly in 

 strong alcohol, they will usually sink. If rather large air bubbles pre- 

 vent the material from sinking, as in case of perichaetical leaves of 

 some mosses and involucral leaves of liverworts, a little dissection or 

 a careful snip with the scissors will often obviate the difficulty. If an 

 air-pump is available, some bubbles are easily removed, but air bub- 

 bles in cells may resist even the air-pump. An aspirator fastened to a 

 water tap is very efficient in removing bubbles. Heating followed by 

 rapid cooling is recommended by Pfeiffer and Wellheim for removing 

 air, but, for cytological work, the remedy is worse than the bubbles. 



It is often asked whether fixing agents really preserve the actual 

 structure of cell contents. It must be admitted that some things — 

 notably the liquid albuminoids — are much modified in appearance, but 

 the most competent observers are now inclined to believe that such 

 delicate objects as chromosomes, centrosomes, the achromatic figure, 

 and even the structure of protoplasm, can be studied with confidence 

 from material which has been fixed, imbedded, and stained. Extensive 

 investigations upon various objects in the hving condition have 

 strengthened this confidence. 



It is certain that we have not yet found the ideal fixing agent for 

 cell contents. Such an agent must not be a solvent of any of the cell 

 contents, must penetrate rapidly, must preserve structures perfectly, 

 and must harden so thoroughly that every detail shall remain un- 

 changed during the subsequent processes of dehydrating, clearing, 

 imbedding, sectioning, and staining. 



DEHYDRATING AGENTS 



Objects which are to be imbedded in paraffin or celloidin, and also 

 all other objects which are to be mounted in balsam or Venetian tur- 

 pentine, must be dehydrated, i.e., they must be freed from water. The 

 slightest trace of water is ruinous. Alcohol is used almost exclusively 

 for dehydrating. The process must be gradual. If material has been 

 fixed in an aqueous solution, it must pass through a series of alcohols 

 of increasing strength, beginning with about 3 per cent alcohol. Twen- 



