362 



METHODS AND FORMULAS 



DS 13.5-DS 13.50 



13.5 Techniques Employing Safranin as the Nuclear Stain 



Safraiiiu is scarcely employed in the 

 English-speaking countries as a histolog- 

 ical nuclear stain, and no current staining 

 technique for animal tissues which re- 

 quires such a procedure is known to the 

 author. Most of the stains placed in this 

 class were developed for plant purposes 

 and there is no doubt that few finer botan- 

 ical stains can be produced. 



13.50 TYPICAL example 



Preparation of a transverse section of 



a Ranunculus stem using the 



safranin-crystal violet-fast 



green-orange II stain of 



Conant (1940) 



The inexperienced microtomist must 

 clearly distinguish between those methods 

 of staining plant tissues, such as the pres- 

 ent, which are designed to stain both the 

 cytoplasm and the cell walls, and those 

 such as the example given in the next 

 chapter, which are designed to stain only 

 the cell walls. The latter are much simpler 

 and may be advantageously employed for 

 class-demonstration purposes. The present 

 technique, which is one of the simplest of 

 the many quadruple stains which have 

 been suggested for plant tissues, may be 

 used as a general-purpose stain for all 

 plant material, and the selection, in the 

 present instance, of a Ranunculus stem as 

 a demonstration object is designed solely 

 because of its relative commoness and be- 

 cause of the ease with which it may be 

 prepared. This staining method has the 

 disadvantage that it does not always yield 

 reproducible results, hence it is better for 

 demonstration tlian for research purposes. 

 If it is of primary importance that sections 

 show identical staining when prepared at 

 different times from sliglitly different ma- 

 terial, it is recommended that the tech- 

 nique of Johansen 1940 (DS 13.5 Johansen 

 1940) be substituted. 



The species of Ranunculus selected for 

 demonstration purposes is not of great 

 importance, but it must be remembered 

 that the fame which the stem of this plant 

 enjoys for teaching purposes is based upon 

 the structure of Ranunculus acris, the 

 commonest European buttercup, which 



is, however, so widely dispersed in the 

 northeastern United States that it may 

 be gathered without trouble. So many 

 textbook diagrams are based on the sec- 

 tion of this species that it is well worth 

 the trouble to collect it. 



The choice of fixative is far less critical 

 in botanical than in zoological micro- 

 techniques, and it will be quite sufficient 

 for the present purpose to use any of those 

 fixatives, known to botanists as AAF fixa- 

 tives, which will be found in Chapter 18 

 under the heading of F 0000.1010. These 

 alcohol-acetic-formaldehyde mixtures keep 

 indefinitely, and no two botanists have 

 ever agreed as to what are the best con- 

 centrations to be employed. 



Stems should be collected, so far as pos- 

 sible, toward the middle of the summer, 

 when the structural detail will be found 

 at its best, and the fixative should be 

 taken into the field. The fresh stem should 

 be severed from the plant, and about the 

 lower ^4 of an inch cut off a few moments 

 later to get rid of the air which will have 

 been drawn into the vessels by the tur- 

 gidity of the specimen. The remainder of 

 the stem is then cut into about i-^-inch 

 lengths and placed in a 250 cc. bottle of 

 the fixative. It should remain in this fixa- 

 tive, which may be changed as rapidly as 

 it becomes discolored, until the chloro- 

 phyll has been thoroughly removed, and 

 should then be washed in 70% alcohol 

 until the washings no longer smell of 

 acetic acid. The specimens may then be 

 stored in 70% alcohol, though it has been 

 suggested that slightly better preservation 

 of detail may be secured by the addition 

 of 1 % glycerol to the preserving fluid. 

 This is not, however, a matter of any 

 importance provided less than a year 

 elapses between the time of the collection 

 of the material and the cutting of sections. 



The stem presents no difficulties in em- 

 bedding either by the dioxane technique 

 or by the more conventional routine of 

 alcohol-xylene. It is suggested that sec- 

 tions be cut at about 15 microns in thick- 

 ness, since these rather thicker than nor- 

 mal sections give a much better idea of 

 general structures when used for teaching 

 purposes. The sections are attached to the 

 slide, deparaffinized in the usual manner, 



