98 Methods in Plant Histology 



While this is the general outline, it is not sufficiently definite for 

 a working introduction. The following concrete examples, describ- 

 ing the use of Venetian turpentine with an aqueous stain, with an 

 alcoholic stain, and with a combination of aqueous and alcoholic 

 stains, will be more practical than general directions. The steps 

 from fixing to mounting, as used with an aqueous stain, will be 

 described first, since this will introduce the method in its least com- 

 plicated form. 



Haidenhain's Iron-Haematoxylin. Using Spirogyra as a type, 

 proceed as follows: 



1. Fix 24 hours in chromo-acetic acid. 



1 per cent chromic acid 70 c.c. 



Glacial acetic acid 3 c.c. 



Water 90 c.c. 



The volume of the fixing agent should be at least 100 times that of 

 the material to be fixed. 



2. Wash in water, 24 hours. 



3. 2 per cent aqueous solution of ammonia sulphate of iron, 2 hours. 



4. Wash in running water, 20 minutes. If running water is not avail- 

 able, wash in a large quantity of water and change frequently. 



5. Stain over night, or 24 hours, in per cent aqueous solution 

 haematoxylin. 



6. Wash in water, 20 minutes. 



7. 2 per cent aqueous solution of ammonia sulphate of iron, until the 

 stain is satisfactory. This can be determined only by examining 

 frequently under the microscope. 



8. Wash in water, 2 hours. If this washing is not thorough, the con- 

 tinued action of the iron-alum will cause the preparations to fade. 



9. Transfer to 10 per cent glycerin, and allow the glycerin to concentrate 

 until it has the consistency of pure glycerin. It is not necessary to 

 use an exsiccator. Merely put the glycerin into shallow dishes, and 

 leave it exposed to the air, but protected from dust. If the material 

 is in Petri dishes or other dishes with a large surface, 3 or 4 days 

 will be sufficient. This process should not be hastened by warming. 



10. Wash out the glycerin with 95 per cent alcohol. It will be necessary 

 to change the alcohol several times. From 10 to 20 minutes will be 

 sufficient if the alcohol is changed frequently. 



11. Complete the dehydration in 100 per cent alcohol: 10 minutes 

 should be sufficient. 



12. M ost failures are now ready to occur. 



