DS 21.10 



DYE STAINS OF SPECIAL APPLICATION 



381 



the sections are produced, they are ac- 

 cumulated in a small dish of distilled 

 water. These sections will, of course, re- 

 tain the cell contents, which must be re- 

 moved in order that the section may be 

 turned into a true skeleton. 



The best reagent to use for skeleton- 

 izing a section of plant tissue is either 

 potassium or sodium hypochlorite; the 

 ordinary bleaching solutions sold for 

 household purposes under various trade 

 names are not suitable, since they contain 

 considerable quantities of calcium hypo- 

 chlorite. If, however, the pure salts are 

 not available, the household solution may 

 be employed by adding to it enough of a 

 solution of potassium or sodium carbonate 

 to precipitate the calcareous contents, and 

 then filtering the solution before use. If 

 the pure salts are available, a 1 % solution 

 may conveniently be employed. 



The sections are removed from the dis- 

 tilled water on a section lifter and trans- 

 ferred to a watch glass of the sodium or 

 potassium hypochlorite solution. If the 

 sections are made from material wliich has 

 been preserved in alcohol, this solution 

 should be used cold, but it must usually 

 be warmed if it is to have the desired 

 effect on materials which have been resur- 

 rected from a dried condition. In either 

 case the operation should be watched very 

 carefully under a low power of the micro- 

 scope, and the action of the hypochlorite 

 should be discontinued as soon as the cells 

 are free of their contents. If the mounter 

 is completely inexperienced in this field, 

 and is unable to determine the point at 

 which the operation should be stopped, it 

 is recommended that a single section 

 should be taken and the skeletonizing 

 followed under a microscope wliile it is 

 timed. When the operation has gone too 

 far, the finer of the cell walls present will 

 be dissolved by the solution. If the period 

 at which the first of the cell walls dissolves 

 is carefully recorded, and one-half of this 

 time taken for the subsequent sections, 

 they will be perfectly cleaned without 

 the shghtest risk of damage to their walls. 

 After they are removed from the hypo- 

 clilorite solution, the sections should be 

 thoroughly washed in several changes of 

 distilled water and then passed into 1% 



acetic acid in which they are rinsed 

 several times. They are then rewashed in 

 ordinary water until the wash water no 

 longer smells of acetic acid. The skeleton- 

 ized sections from as many roots as it is 

 desired to cut at one time should be ac- 

 cumulated in water until one is ready to 

 stain them, or they may be preserved in- 

 definitely in alcohol. 



The stain which is recommended in the 

 present case (DS 21.15 Chamberlain 

 1915a) is freshly prepared when required 

 by mixing equal parts of a 0.2% acid 

 fuchsin solution and a 0.2 '^o iodine green 

 solution. The mixed stains do not remain 

 usable for much longer than one day, but 

 the separate stock solutions may be kept 

 indefinitely. The differentiating solution, 

 which is 1 % acetic acid in absolute alco- 

 hol containing 0.1% iodine, is also stable. 

 The staining solution should be placed in 

 a small capped vial or stoppered bottle, 

 and the sections transferred to it from the 

 water. They should remain in stain for 

 about 2-4 hours, and it is recommended 

 that they should not be left longer than 

 36 hours or they may suffer from a precipi- 

 tate over the surface. For this reason it is 

 desirable to accumulate as many sections 

 as possible before starting the process. 

 When the staining period is concluded the 

 contents of the vial should be tipped out 

 into a large watch glass. Usually some of 

 the sections will remain stuck to the side 

 of the vial from which the^^ are removed. 

 Under no circumstances should the vial 

 be rinsed with anything except the stain- 

 ing solution, which should be poured back 

 from the watch glass (the sections will 

 have settled to the bottom), swirled 

 around, and returned to the watch glass. 

 A second watch glass, or even a small 

 crystallizing dish, is now filled with the 

 differentiating solution. Each section is re- 

 moved individually with a section Hfter 

 from the stain and placed into the differ- 

 entiating solution, where it may be 

 watched under the low power of a micro- 

 scope as the dish is rocked gently from 

 side to side. Differentiation will usually 

 take place within two or three minutes 

 and is terminated when the lignitied tis- 

 sues are a bright, clear green, leaving a 

 bright red in the nonlignified tissues. This 



