Sectioning Unembedded Tissues 95 



and rinse the sections Avith two changes of water. Proceed with the 

 staining process. 



The most easily controlled stain combination consists of a self- 

 mordanting hematoxylin (Chap. 7) followed by an aniline dye. 

 Assume that sections of a firm woody subject are to be stained. Drain 

 off the water in which the sections are floating in a watch glass, and 

 flood the sections with hemalum or similar stain. After 5 min. remove 

 a section with a brush, rinse in distilled water and then in tap water 

 and examine with a microscope. Lignified cells, such as tracheids and 

 phloem sclerenchyma, should be practically colorless. Soft tissues like 

 pith, cortex, and cambium should have blue-stained walls. Nuclei 

 should be blue-black. If the sections are overstained the entire 

 protoplasts become blackened, obscuring cellular detail, and the walls 

 of bonified cells become stained. Sections can be destained with 1/2% 

 HCl and thoroughly washed in tap water. When the correct intensity 

 of blue is attained, cover the sections with safranin. Try an interval 

 of 15 min. in safranin. Locate this point in the staining schedule 

 given in Staining Chart III. Rinse the sections in water, and cover 

 with 50''/ alcohol. The safranin will dissolve out of the nonlignified 

 tissues faster than out of lignified cell walls. Slower destaining can be 

 obtained with 50% acetone. When the nonlignified cells are still deep 

 red, rinse the sections quickly in anhydrous alcohol or acetone. 

 Acetone stops destaining action better than does alcohol. Complete 

 the process as shown in Staining Chart III. Mount the sections on 

 slides as described in Chap. 7. 



Having gained some familiarity with the above stain and with the 

 use of staining schedules, study the discussion of staining in the 

 paraffin method (Chap. 7) and the celloidin method described 

 earlier in this chapter, and try some of other stain combinations 

 described in those chapters. 



Sectioning by Encasing in Water-Soluble Waxes 



These little-known methods are intermediate between embedding 

 methods and sectioning without embedding. As a matrix, use one of 

 the water-soluble wax-like synthetics, such as glycerol monostearate, 

 which melts at 55°C. The living or fixed material is transferred 

 directly from water to the melted matrix, which is then hardened, 

 fastened to a wood mounting block, and sectioned. Very little 

 infiltration occurs, but the material is encased and held with sufficient 

 rigidity to make fairly good sections. This method has been used 

 successfully with nearly mature clover seeds and shows much promise 



