78 METHODS IN PLANT HISTOLOGY 



will slightly affect the other cell contents, but the erythrosin will give 

 the cytoplasm such a dense stain that the cell walls will be seriously 

 obscured. It would be better to use haematoxylin alone. For counting 

 chromosomes, it is better to stain in iron-alum haematoxylin alone, or 

 in safranin alone. The same suggestion may well be observed in trac- 

 ing the development of antheridia, archegonia, embryos, and similar 

 structures. 



In using combinations, it must be remembered that the second 

 stain often affects the first, e.g., if safranin is to be followed by Dela- 

 field's haematoxylin, in staining a vascular bundle, it will not do to 

 make the safranin just right and then apply the haematoxylin, for the 

 acid which must be used to differentiate the haematoxylin and to 

 avoid precipitates will also reduce the safranin, and the red will be too 

 weak. You must overstain in safranin so that the reduction will finally 

 leave it just right. The same hint will apply if safranin is to be followed 

 by anilin blue, since, here, also, acid must be used; if light green is to 

 follow the safranin, the stain itself is so acid that the safranin must be 

 rather strong before the light green is applied. Orange, whether in 

 water or in clove oil, reduces many stains and, consequently, such 

 stains must be strong enough to allow the weakening. These hints are 

 only samples: the student must observe the behavior of the various 

 stains when used singly and when used in various combinations. 



With perfect confidence, we can give advice to the beginner and 

 to the seasoned investigator: master a few stains. With Haidenhain's 

 iron-alum haematoxylin, stain sections of root-tips until you can see 

 the finest details of the chromatin. When you can make a perfect 

 preparation of a root-tip with this stain, you will have made a good 

 start toward a satisfactory histologial technique. Gentian violet is 

 often used to stain the ciha of sperms; but Haidenhain's iron-alum 

 haematoxylin will stain the ciha of cycad sperms so critically that the 

 base of the cilium, as it passes through the Hautschicht (plasma mem- 

 brane), will be differentiated from the part projecting beyond the 

 Hautschicht. Master this haematoxylin stain and then, with sections 

 of root-tips, practice the safranin, gentian violet, orange combination 

 until you can stain the chromosomes red, the spindle violet, and the 

 cytoplasm orange. For vascular anatomy, learn to stain xylem with 

 safranin; and, for a contrast, stain cellulose walls with Delafield's 

 haematoxylin, gentian violet, light green, or anilin blue. For filamen- 

 tous algae and fungi, master the iron-alum haematoxylin method; and 



