DS 21.14-DS 21,15 DYE STAINS OF SPECIAL APPLICATION 391 



21.14 Semichon 1920 see DS 12.222 Semichon 1920 



21.14 Smith 1926 9940, 14:171 



formula: phenol 100, sat. ale. {circ. 1.5%) ethyl eosin 12.5 



method: [potash-cleared skeletons of arthropods] — > stain, 15 mins. at 60°C. — > balsam, 

 via clove oil 



21.14 Vaulx 1920 see DS 21.14 Racovitza (1942) (note) 



21.15 PLANT SKELETAL TISSUES 



There is a general confusion among beginners unaccustomed to botanical micro- 

 techniques between those destined to show only the skeletal structures and those which 

 are designed to show the nuclei and cytoplasmic constituents of plant material. For the 

 latter purpose any nuclear stain and any counterstain employed for zoological purposes 

 can equally well be used, and many of the triple staining methods common in zoological 

 techniques give admirable results. The staining methods discussed in this section are 

 those which are used to differentiate (usually for teaching purposes) between cellulose, 

 callose, lignified, and suberized tissues. These tissues should be freed from protoplasmic 

 material before being stained, and the customary method of doing this is to immerse 

 them, after sectioning, in a solution of sodium or potassium hypochlorite. They should 

 remain in this solution until the cellular contents have been leeched away, and should 

 then be thoroughly washed in distilled water before staining techniques are applied to 

 them. 



In general these techniques may be divided into two sections. First, there are those 

 which are destined to distinguish between lignified and cellulose tissues, which is the 

 purpose of almost all the formulas here given ; second, there are the few genuine botanical 

 triple stains in which some fat-staining constituent is employed for the purpose of 

 bringing the suberized tissues into contrast. For the former purpose the only choice 

 between the different methods rests in the color in which it is desired to differentiate the 

 elements. It is interesting to note that the stain universally referred to as "Benda's 

 Stain" in zoology is equally employed in botanical microtechnique as "Land's Stain." 

 The best technique, if it is desired to include the suberized tissues, is that of Bugnon 

 1919. A good method for class-demonstration purposes in Langeron 1902, in which 

 advantage is taken of the relative densities of the schlerenchyma and parenchyma in 

 order to distinguish between the two. For those who prefer a single solution, there are 

 the methods of Darrow 1940 and Chamberlain 1915a. The stain of Petit 1903, though 

 obsolete, is included for its interest and for the reason that it gives more permanent 

 results than other staining methods for plant skeletal tissues. 



21.15 Bugnon 1919 light green-Sudan II I-hemalum 4999,66:919 



REAGENTS REQUIRED: A. 70% alc. 100, light green to sat, Sudan III to sat.; B. 11.122 



Delafield 1885 

 method: [sections] -^ 70% alc. -^ A, 10 mins. -♦ water, rinse -> B, 10 mins. -♦ water, 



wash -^ balsam, via usual reagents 

 result: lignified tissues, green; suberized tissues, red; other tissues, blue. 

 note: Langeron 1942, 1269 suggests the substitution of Petit 1903 (sols. C and D) for 



B above. 



21.15 Chamberlain 1915a acid fuchsin-iodine green Chamberlain 1915, 62 

 STOCK solutions: I. 0.2%, acid fuchsin, II. 0.2% iodine green 



WORKING solutions: a. stock I 50, stock II 50; B. abs. alc. 100, acetic acid 1, iodine 0.1 

 method: [sections] -> water -^ A, 24 hrs. ->■ B, till differentiated -^ balsam, via xylene 

 result: lignified tissues, green; cellulose tissues, red. 

 note: a detailed description of the use of this stain is given under DS 21.10 above. 



21.15 Chamberlain 1915b cyanin-erythrosin Chamberlain 1915, 01 



^■^ reagents required: A. 1% cyanin in 50% alc; B. 1% erythrosin 



method: [sections] ->■ water ->■ A, 5-10 mins. -♦ 50% alc, rinse -^ B, \i to 1 min. -» 

 abs. alc, least possible time -* balsam, via xylene 



