194 BOTANICAL MICROTECHNIQUE 



A. Weak chrom-osmo-acetic acid (Weak Flemming): 



1% chromic acid 25 cc. making .25% chromic acid. 



1% acetic acid 10 cc. making .1% acetic acid. 



1% osmic acid 10 cc. making .1% osmic acid. 



distilled water ■ 55 cc. 

 100 cc. 

 This well-known formula is used for the algse and fungi and delicate 

 tissues of the higher plants. It has the same strength as weak chrom- 

 acetic acid but with osmic acid added. Half the amount of osmic acid 

 in the above formula gives, according to our experience, better results 

 with many algse and fungi. 



B. Strong chrom-osmo-acetic acid {Strong Flemming) : 



1% chromic acid 75 cc. making .75% chromic acid, 



glacial acetic acid 5 cc. making 5% acetic acid. 



2% osmic acid 20 cc. making .4% osmic acid. 



100 cc. 

 This formula has a medium strength of chromic acid but an exceptional 

 strength of acetic and osmic acids. It may easily be modified by vary- 

 ing the amounts of its components. Thus Mottier recommends for 

 anthers the following proportions : 1% chromic acid, 80 cc. ; glacial 

 acetic acid, 5 cc. ; 2% osmic acid, 15 cc. Strong Flemming naturally 

 finds its use on the same sort of subjects as require the medium or 

 strong formulae of chrom-acetic acid, as for example the firmer tissues 

 of the higher plants. 

 Material fixed by Flemming's fluids must be washed to remove the chrom- 

 acetic acid, as described in the previous section. The osmic acid always 

 blackens the material, but this discoloration is not treated until just be- 

 fore staining (Sec. 198), when the preparations are bleached with hydrogen 

 peroxide. The chromic acid must be dissolved in sea water, when these 

 fluids are used upon marine algse, and the material also washed in sea 

 water. 



174. Absolute alcohol. The fixing fluids based on chromic acid penetrate 

 rather slowly, and consequently very dense tissues or structures with heavy 

 hard cell walls are sometimes not at all well fixed by them, the protoplasts 

 appearing shrunken. There is also occasional difficulty in immersing or 

 wetting material in these water solutions. For such material some of the 

 fixing fluids based on alcohol are preferable. The best of these are absolute 

 alcohol and Carnoy's fluid. 



Absolute alcohol alone is not an especially good fixing agent except for 

 very small objects, which it can penetrate almost instantly. Material is, of 

 course, ready very quickly for preservation in 85% alcohol, or for the process 

 of imbedding in parafBn. 



