272 



METHODS AND FORMULAS 



DS 10-DS 11.10 



be entirely confined to the study of the 

 spermatogenesis of rats, and that the 

 majority of the carmine stains, now 

 widely emploj-ed for staining whole- 

 mounts, would l)e confined to the pre- 

 staining of tissues prior to embedding, so 

 that the nuclei might be stained in sec- 

 tions subsecjuently to be cut from them. 



The dj-e staining techniques of general 

 application are here divided into the three 

 broad headings of those ])rimarily in- 

 tended for nuclear staining (DS 11), those 

 primarily intended for providing a con- 

 trast to a jM-eviously ai)i)lied nuclear stain- 

 ing (DS 12), and those intended for ap- 

 plication to sections in which both the 

 nuclei and background are to be stained in 

 the same operation (DS 13). 



DS 11 NUCLEAR STAINING 

 TECHNIQUES 



This section includes those techniques 

 customarily em])loyed for staining nuclei 

 in general, and they are to be kept distinct 

 from those special techniques intended for 

 the studv of the nucleus as such, which are 

 to be found in Chapter 21 (DS 22.1). 



Nuclear staining techniques are here 

 classified solely on the liasis of the re- 

 agents employed and of the manner of 

 their employment. Prior to the use of 

 stains in microtomy, hematoxylin was 

 widely employed in the dye industry for 

 obtaining purples and blacks, the former 

 with the aid of alum mordants, and the 

 latter with the aid of iron mordants. This 

 division has been fairly closely followed in 

 microtomy and is used as the basis for the 

 division into mordant staining and direct 

 staining which follows. The selection be- 

 tween these two methods must be made 

 entirely on the purpose for which the 

 method is intended. 



The use of carmine is of greater antiq- 

 uity than is the use of hematoxylin. The 

 original use of carmine in textile dyeing 

 was with the aid of a whole range of mor- 

 dants, which enabled the dyer to select 

 colors ranging from a brilliant scarlet to a 

 dead black. In general the methods of 

 microtomy utilize only the red aluminum 

 mordants and to a lesser extent, the black 

 iron mordants. The main use of carmine 

 in microtomy, however, is in direct stain- 



ing with the aid of solutions containing 

 aluminum mordants. There is consider- 

 able misapprehension about the degree of 

 alkalinity of the various formulas. Early 

 workers tended to say that a solution was 

 too alkaline on the sole basis of the fact 

 tliat it was prepared with a sodium salt 

 rather tliaii \\ith the more expensive 

 lithium compounds or with borax. The 

 rediscovery in German}' in the 1930's of 

 some of the older sodium-carmines has led 

 to their inclusion in the present work. 



Brazilin and saffron are also to a minor 

 extent employed in the staining of nuclei; 

 and they terminate the list of tlie so-called 

 natural dyes. 



The synthetic dyes form a large group 

 which are here subdivided according to 

 which dye is used. Too much attention 

 cannot be drawn to the oxazine dyes de- 

 scribed by Becher 1921, which have been 

 rediscovered at intervals. The}' have never 

 replaced hematoxylin, but it must appear 

 to most microtomists they are ultimately 

 destined to do so. They have the ad- 

 A-antage over hematoxylin stains in that 

 the}' are relatively acid-fast once they 

 have adhered to the nucleus, and therefore 

 give a whole range of dark blue and black 

 nuclear stains which can be employed 

 before counterstaining methods involving 

 acid materials. There is some confusion 

 between the use of nuclear stains in 

 zoology and botany and the use of the 

 same stains for the preparation of bacteria. 

 Nuclei and bacteria have essentially the 

 same staining reactions, and a method 

 which may be utilized for one can without 

 difficulty be adapted to the other. 



DS 11.1 Hematoxylin Stains 



DS 11.10 TYPICAL EXAMPLES 



Demonstration of spermatogenesis in 

 the rat testis using the iron hematoxy- 

 lin stain of Heidenhain 1892 



The laboratory white rat is one of the 

 best forms in which to show spermato- 

 genesis, for the reason that it has a con- 

 tinuous breeding period and all stages are 

 therefore available in almost every section 

 examined. Baker 1945, 180 recommends 

 the salamander Triturus for this purpose, 



