Staining Paraffin Sections 71 



serve as excellent cytological stains for many subjects, primarily for 

 the preparation of classroom materials. 



THE TRIPLE STAIN (FLEAAMING) 



The triple stain is of considerable historical interest and is still 

 in high favor in some laboratories. The three components are safranin, 

 crystal (gentian) violet, and orange G (or gold orange) . Safranin is 

 intended to stain chromatin, lignin, cutin, and in some cases chloro- 

 plasts. Gentian violet should stain spindle fibers, nucleoli during some 

 phases, and cellulose walls. The orange dye acts as a differentiating 

 agent, serves as a general background stain, and stains cytoplasm and 

 in some subjects cellulose walls. All three components are highly 

 soluble in the reagents used in the staining process and are subject 

 to changes of intensity and mutual interaction during most of the 

 process. The correct balance of relative intensities is, therefore, very 

 difficult to control. The process yields spectacularly beautiful slides 

 from the hands of an expert. However, an attractive or gaudy poly- 

 chrome effect is not adccjuate justification for the use of an elaborate 

 and time-consuming process. The real test of the desirability of a 

 multiple stain is the specific selectivity of its color components for 

 definite morphological or chemical entities in the cell. 



The sphere of usefulness of the triple stain may be jndged by a 

 consideration of the stains used in modern cytological research. It is 

 noteworthy that the most critical modern work on chromosome 

 structure and behavior has been done with the iron-hematoxylin 

 stain, with the gentian violet-iodine stain, and with acetocarmine 

 smears. The most reliable work on the spindle-fiber mechanism and 

 spindle-fiber attachment also has been done with the first two stains. 

 As an illustration in the field of anatomy, it will be obvious that in 

 studies of vascular tissues a stain is required primarily to show a 

 xylem-phloem contrast, distinguishing between lignified and un- 

 lignified cell walls. This usually is done adequately with a two-stain 

 combination. There is no special virtue in having a delicate orange 

 background for a study of the organization of a vascular bundle or in 

 a section of pine lumber. However, in many cytological problems 

 involving the entire cell rather than merely the actively dividing; 

 chromosomes, the triple stain is an indispensable tool. Another legiti- 

 mate sphere is in pathological studies in which it is desirable to 

 produce polychrome contrasts between a parasite and its host. The 

 object of the above discussion is to emphasize again the view that any 

 elaboration that does not serve a definite, useful function is a waste 



