48 METHODS IN PLANT HISTOLOGY 



8. Haematoxylin. 



Many objects, like the archegonia of gymnosperms and the embryo 

 sacs of angiosperins, will stain sufficiently in 5 or 6 hours; most algae re- 

 quire at least 20 hours. 



9. Wash in water, 5 minutes, changing as often as the water shows any color. 



10. Iron-alum, 2 per cent solution. 



No time can be indicated here. The preparation must be watched 

 under the microscope. After some experience, one can form some judg- 

 ment from the color tone, as the slide stands in the Stender dish of iron- 

 alum, but the finishing must always be done under the microscope. In 

 general, if it requires more than 2 hours to secure good differentiation, use 

 a stronger iron-alum; if the differentiation is reached in less than | hour, 

 use a weaker iron-alum. A 3 /x section of a root-tip from material fixed 

 in chromo-acetic-osmic acid, with 8 c.c. of 1 per cent osmic acid to 100 c.c. 

 of the stock solution, should be perfectly differentiated in 2 hours. If the 

 stain comes out too rapidly, use 2 per cent iron-alum for an hour and 

 finish in a 1 per cent solution. The less the proportion of osmic acid, the 

 faster will the stain be extracted. If the stain is coming out rather slowly, 

 as it should, one can handle from 6 to 10 slides at one time. Put the 

 slides on a 5X7 glass plate and put the plate on the stage of the micro- 

 scope. The iron-alum can be added or removed with a pipette. As slide 

 after slide reaches the proper differentiation, it is placed in water. 



11. Water, 30 minutes. 



The water should be changed several times. If this washing is not 

 thorough, the preparation wdll fade, on account of the continued action 

 of the iron-alum. If an aqueous counter-stain is used, apply it at this 

 point. 



12. Thirty-five, 50, 70, 85, 95, and 100 per cent alcohol, 5 minutes in each. 



If an alcoholic counter-stain is used, apply it near the alcohol of the 

 same strength as the stain. 



13. Absolute alcohol and xylol, equal parts, 5 minutes. 



14. Xylol, 2-5 minutes. 



15. Balsam. 



While this schedule should enable the student to apply the method 

 not only to sections but to objects to be mounted whole, like filamen- 

 tous algae and fern prothallia, an additional schedule for such things 

 is given in chapter viii on "The Venetian Turpentine Method." 



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

 to wash 3 or 4 times as long after the first immersion in iron-alum. 

 Some think that 4 hours is enough for the entire process. In staining: 



