44 Methods in Plant Histology 



13. Wash several hours in water. 



14. Alcohol series: 10, 30, 50, 70, 80, 95, 100 per cent. 



If a counter-stain is desired, introduce it in one of the alcohols 

 of this series. 



15. Absolute alcohol and xylol, equal parts. 



16. Xylol. 



17. Mount in balsam. 



While these two schedules would enable the, student to apply 

 the method in case of objects to be mounted whole, like filamentous 

 algae, fern prothallia, etc., a complete schedule is given in chap, 

 viii on "The Venetian Turpentine Method." 



The times given above must not be accepted as final. Many 

 prefer to wash in water for several hours after the first immersion in 

 iron-alum. Some think that 4 hours is enough for the entire process. 

 Many put the slide into iron-alum in the morning and finish the 

 process in the afternoon. These short schedules are not likely to 

 prove satisfactory with mitotic figures. A plan which has proved 

 convenient and very successful is to put the slide into the iron-alum 

 in the morning, let it wash in water during the afternoon, leave it in 

 the \ per cent of haematoxylin over night, and finish the prepara- 

 tion the next morning. It is a long process, requiring care, patience, 

 and judgment, but it is worth the effort. 



Chromosomes, centrosomes, and pyrenoids take a ^brilliant black, 

 or, if the second treatment with iron-alum be more prolonged, 

 a blue black or purple. Achromatic structures stain purple, but 

 the stain can be extracted while it is still bright in the chromosomes. 

 Lignified, suberized, and cutinized structures stain lightly or not at 

 all. Cellulose does not stain so deeply as with Delafield's haema- 

 toxylin. Archesporial cells and early stages in sporogenous tissue 

 stain gray. Many details which are not so brilliantly colored often 

 show good definition. 



If a counter-stain is desired, anything which gives a serviceable 

 contrast may be used. In any case, the haematoxylin stain must be 

 complete and the washing thorough before the second stain is applied. 

 An aqueous stain should be applied just after the final washing in 

 water; an alcoholic stain should be applied during the process of 

 passing the slides through the alcohols, staining in a solution of saf- 

 ranin in 50 percent alcohol from the 35 or 50 per cent alcohol; and 



