200 BOTANICAL MICROTECHNIQUE 



B. Methyl green. A saturated solution in 1% acetic acid keeps well, or it 

 may be made up simply in distilled water. Dilute if desired. This is 

 a good stain for living cells, but it is especially valuable in combination 

 with acid fuchsin, forming an effective double stain for the tissues of 

 higher plants. Sections from preserved material may be stained at once, 

 those from fresh material must be fixed in absolute alcohol or chrom- 

 acetic acid (Sec. 194). Stain first with methyl green for two hours or 

 more and wash in distilled water until the green remains in the ligni- 

 fied cell walls alone. Then stain with acid fuchsin for a few minutes, 

 — not long enough to affect the lignified tissues, — and pass through 

 95% alcohol to absolute alcohol and into clove oil and balsam (Sec. 187). 



C. Erythrosin. This stain is similar to eosin and may be used, in saturated 

 solutions in water or 70% alcohol. It is a good counterstain following 

 haematoxylin or green and blue anilin dyes. 



MOUNTING IN BALSAM AND GLYCERIN 



187. Mounting in balsam, Canada balsam is the most satisfactory medium 

 for permanent preparations. It should be used whenever possible, but there 

 are some subjects, such as delicate filamentous algse and fungi, which cannot 

 easily be carried into balsam without shrinkage, or which cannot be teased 

 apart when brought into that medium because the clearing agents such as 

 xylol or clove oil render the filaments much less flexible. For such subjects 

 glycerin, glycerin jelly, or Venetian turpentine are better media. 



Material is carried into balsam from absolute alcohol through a clear- 

 ing agent. It must first be brought up through the grades of alcohol to 95% 

 (Sec. 178), where it is best left several hours (it may remain in 95% alco- 

 hol indefinitely). Material is then placed in absolute alcohol to remove all 

 trace of water (dehydration). Dehydration takes from thirty minutes to 

 an hour or more, according to the size of the object, and it is well to change 

 the alcohol once or twice if there is much material. From absolute alcohol 

 the material is carried into a clearing agent, clove oil or xylol being the 

 simplest. Clove oil removes the absolute alcohol rapidly and is the better 

 clearing agent following anilin dyes, but should never be used after hcBmatoxy- 

 lln, for its acid quality fades that stain. Xylol acts more slowly and does not 

 affect hsematoxylin stains. Microtome sections on the slide are handled 

 much more rapidly through the alcohols and clearing agents, as is described 

 in Sec. 199. 



With delicate material xylol is always the safest clearing agent, because 

 it mixes more slowly and less violently with absolute alcohol. The danger of 

 shrinkage is lessened greatly by preparing three mixtures of absolute alcohol 



