Killing, Fixing, and Storing Plant Tissues 17 



Materials killed in the fluids given in Table 3.1 should be washed 

 in ruiuiing Avater. Various devices may be used tor accomplishing 

 this prolonged washing. The simplest method is to tie a strip of 

 cheesecloth over the wide mouth ot the bottle containing the tissues 

 and to allow a slow stream of water to How into the bottle. More 

 vigorous washing action can be obtained by inserting the water inlet 

 tube to the bottom of the specimen bottle. These fluids do not have 

 good hardening action, so it is best to avoid violent motion of the 

 pieces. Firm materials can be washed in a vertical length of 1-in. 

 glass tube with a stopper at the lower end, admitting a stream of 

 water through a small tube, the waste water leaving through cheese- 

 cloth tied over the upper end of the large tube. 



Osmic acid is used in a class of formulas known as the Flemming 

 fluids. These fluids are indispensable for cytological studies but are 

 seldom justifiable for histological work. Osmic acid is expensive, its 

 vapors are highly irritating, and it blackens tissues, making it neces- 

 sary to bleach sections before staining. Osmic acid preserves chromo- 

 some details with great fidelity, but has no special virtues for the 

 preparation of slides of such subjects as corn stem or apple leaf for 

 anatomical or histological study. Osmic acid has poor penetrating 

 ability and is therefore not satisfactory for bulky objects. The formu- 

 las given in Table 3.1 will serve for preliminary tests, subject to 

 experimental variation of proportions. Because of the blackening 

 action and poor hardening properties of the Flemming fluids, mate- 

 rial should be washed in water and processed immediately after kill- 

 ing. Ihe intervals for killing are approximately those given for 

 chrome-acetic. 



Table 3.2 gives several formulas based on the Nawaschin formula, 

 containing chromic acid, acetic acid, and formaldehyde. Numerous 

 modifications may be found in the literature. The name Craf has 

 been coined for this widely used type of fluid. For critical work on 

 specific subjects, experiment with variations of the formulas in the 

 table. The acetic acid should be varied from 0.7 to 5% glacial acetic 

 acid equivalent by volume. The optimum chromic acid and formalde- 

 hyde concentrations for many subjects are the proportions given in 

 formula V. The other formulas in the table, including Nawaschin's 

 original formula, also give good results with specific subjects. The 

 formaldehyde should be added immediately before using. If one of 

 these formulas is to be used for making extensive collections in the 

 field, it will be found convenient to make up the desired mixture of 

 the chromic and acetic acids, adding the measured volume of for- 



