208 BOTANICAL MICROTECHNIQUE 



198. Bleaching after osmic acid. Material fixed in clirom-osmo-acetic acids 

 (Flemming's fluids) is always blackened by the osmic acid. This blackening 

 must be chiefly or wholly removed befoi-e staining. Microtome sections, 

 after the solution of the paraffin with xylol and rinsing in 95% alcohol (Sec. 

 197), are placed in wells of 5-10% hydrogen peroxide in 70% alcohol. The 

 bleaching is generally effected in an hour or less, but may require longer. 

 Stronger solutions of hydrogen peroxide can be used if necessa,ry, but it is 

 safer to employ them weak. As soon as the gray or black tint is removed 

 the slide is rinsed in 95% alcohol and is then ready for the stain. Small 

 objects which are not to be sectioned (such as filamentous algae) are treated 

 in the same manner in watch glasses. 



199. Staining on the slide. The best stains for the details of protoplasm 

 are iron-alum hsematoxylin , or saf ranin followed by gentian violet. Perhaps 

 the most successful combination is safranin, gentian violet, and orange G, — 

 a combination known as Flemming's triple stain, the use of which is, how- 

 ever, one of the most difficult of the staining methods. Delafield's hsema- 

 toxylin, and safranin followed by Delafield's hsematoxylin, are among the 

 best general stains for tissues, and methyl green followed by fuchsin is 

 also good. 



A. Iron-alum hematoxylin. This method follows the same outline as is 

 given in Sec. 182 for staining in bulk. The slide is taken from 95% 

 alcohol, dipped in 35%, and placed in iron alum (best used in 1% solu- 

 tion) for from two to four hours ; it is then rinsed in distilled water and 

 left in hssmatoxylin from four to eight hours or over night. From the 

 hsematoxylin it is returned to the iron alum to extract the stain, and 

 this process must be watched with care, the preparation being examined 

 from time to time under the microscope. At the proper point of extrac- 

 tion the slide is placed in a large dish of tap water, where it must remain 

 for fifteen minutes or more. It is then dipped in 36% alcohol (if desired) 

 and left two or three minutes in a well of 95% alcohol (it may remain 

 indefinitely in the 95%). Finally the slide is taken from the 95% alco- 

 hol, drained, and some absolute alcohol is poured over the sections 

 from a small bottle and rapidly drained off, and the slide placed as 

 soon as possible in a well of xylol (clove oil should never be used). 

 The preparation must remain in the xylol half an hour or more, since 

 the xylol and absolute alcohol do not mix rapidly, after which it is 

 removed, the superfluous xylol drained off, and the sections mounted 

 in balsam. Minute bubbles on the slide after having been in xylol 

 indicate that the dehydration has not been perfect, and they must be 

 removed by absolute alcohol and the slide again placed in xylol. 



B. Iron-alum hoematoxylin and sajranin. The h£eraatoxylin stain may be 

 extracted by iron alum until it remains practically in the nucleus alone. 



