Staining Paraffin Sections 69 



clearly, it may be necessary to compromise by leaving too much red 

 in the cellulose walls. It a finished preparation is found to be un- 

 satisfactory, the cover glass can be removed, and the material destained 

 or restained. However, alterations in the intensity of the safranin can 

 be made best after the slide has been examined from carbol-xylene. 

 Carbol-xylene has a very slow destaining action on safranin. 

 Preparations left in carbol-xylene for 4 to 12 hr. show highly critical 

 differentiation of structures having varying degrees of lignification, 

 such as the stratifications in the walls of xylem cells and sclerenchyma. 



SAFRANIN-FAST GREEN 



The next type of stain combination to be considered has two 

 components, both of which are subject to differential destaining and 

 which react upon each other during dehydration. This staining 

 process is obviously more difficult to control than the preceding 

 processes. As shown in Staining Chart IV, the first stain to be applied 

 is aqueous safranin, in which the preparation is strongly overstained. 

 One hour in safranin is occasionally enough; some woody materials 

 stain well in 5 min. Your previous experience with the hemalum- 

 safranin combination will indicate the safranin-holding capacity of 

 tested materials. The safranin begins to dissolve out during passage 

 through 30, 50, and 95% alcohol. The counterstain, fast green FCF 

 in 95% alcohol, is now applied. Both the green stain and its solvent 

 have a differential solvent action on the safranin, and remove it from 

 the unlignified tissues more rapidly than from the lignin, cutin, and 

 chromatin. 1 he interval in green is usually a matter of seconds, rarely 

 as much as 2 min. Correct contrast has been attained when lignin, 

 chromatin, and in some cases cutin are brilliant red, chloroplasts pink 

 to red, and cellulose walls and cytoplasm are green. 



> The two stains of this combination can be manipulated until the 

 desired contrast and intensities are obtained. If alcohol is used in the 

 dehydrating series, the slide may be placed on a microscope, kept wet 

 with 50% alcohol, and observed until only the lignified elements 

 remain red. The slide is then rapidly carried through the subsequent 

 processes. Acetone is too volatile to permit such examination. With 

 some experience it is possible to judge when the safranin has been 

 destained sufficiently to add the green counterstain. If the stock 

 solution of fast green acts too rapidly for a given subject, the green 

 color will mask or remove the red, and all cells may become stained 

 deep green. In such cases dilute the green stain with 1 to 5 volumes of 

 95% ethyl alcohol. The slide may be examined best out of carbol- 



