THE GLYCERIN METHOD 101 



material must be bleached in hydrogen peroxide or in chlorine before 

 proceeding with the stain (see p. 27). After bleaching and washing in 

 water, treat with 2 per cent iron-alum, 4 hours; wash in water, 30 

 minutes; stain in § per cent haematoxylin overnight or 24 hours; wash 

 in water, 30 minutes; and transfer again to iron-alum. This time, the 

 iron-alum will extract the stain. The rapidity of the action of the n-on- 

 alum will now depend upon the fixing agent. If it contained 6 or 7 c.c. 

 of 1 per cent osmic acid to 100 c.c. of the solution, it may require an 

 hour, or even more, to make a satisfactory differentiation of the stain. 

 If there was no osmic acid in a chromic fixing agent, the differentiation 

 may be complete in 10 minutes. If the stain becomes too weak in 4 or 

 5 minutes, use 4 per cent iron-alum as a mordant, stain longer, and 

 use 1 per cent iron-alum for the second treatment. Haidenhain's iron- 

 alum haematoxylin gives its most brilliant results when chromic mix- 

 tures with 5-7 c.c. of 1 per cent osmic acid to 100 c.c. of the chromo- 

 acetic acid have been used. 



Different species of Spirogyra and even different collections, fixed in 

 the same reagent, will differ in their reaction to stains; and different 

 unicellular and filamentous forms in different fixing fluids, will present 

 so much difference in times that only general suggestions can be given. 

 When the stain is satisfactory, wash in running water for an hour. If 

 this second iron-alum is not washed out thoroughly, its continued 

 action will cause the preparation to fade. 



Put the material into 10 per cent glycerin (1 part glycerin and 9 

 parts water), and then allow the water to evaporate gradually in a 

 place as free from dust as possible. Nothing is better than a Petri dish 

 for this purpose, because it presents a large surface for the evaporation 

 of the water in the mixture. If there is much dust, cut a piece of filter 

 paper just the size of the dish and let it float on the 10 per cent glycer- 

 in. The liquid will soak through the paper and evaporate without ex- 

 posing the material itself to the dust. The process may be hastened, 

 safely, by warming up to 35° C. The temperature of a paraffin bath — 

 45° to 52° C. — causes such rapid evaporation that the material is likely 

 to shrink. The concentration from 10 per cent glycerin to pure glycerin 

 should not require less than three days. This time is easily regulated 

 by the amount of 10 per cent glycerin and the size of the exposed sur- 

 face. 



When the glycerin has become about as thick as pure glycerin, the 

 material is ready for mounting. Place a small drop of glycerin, with 



