SPECIAL METHODS 
141 
Permanent preparations may be secured by the following method: 
1. Treat the fresh sections with 1 per cent osmic acid, 5 to 7 minutes. 
2. Wash in water 5 to 10 minutes. 
3. Treat with a solution of iodine in potassium iodide (0.2 per cent 
iodine and 1.64 per cent potassium iodide), 20 to 30 minutes. 
4. Transfer to 25 per cent sulphuric acid, which should act for at least 
half an hour; 24 hours may be necessary. 
5. Bring the sections into 25 per cent sulphuric acid which has been satu¬ 
rated with iodine. Add a drop of Meyer’s pyoktanin solution (1 g. pyok- 
tanin coeruleum as sold by E. Merck in Darmstadt in 30 c.c. of water). 
In about 5 minutes the sections will be stained sufficiently and 
can be examined in glycerin. If there are annoying precipitates, 
remove them with a soft brush. 
According to Meyer, the swelling is an advantage only when 
the walls are very thin. When the walls are thick, the connections 
show better without any previous swelling. 
Try the following method with the seeds of Diospiros, Latania 
Chamerops, Phoenix, or Phytelephas: Soak in water and cut thin 
sections. Extract the oily and fatty substances with xylol; wash 
in 95 per cent, or in absolute alcohol; stain in anilin blue (Hoffman’s 
blue 1 g. dissolved in 150 c.c. of 50 per cent alcohol) for a few minutes. 
Examine in glycerin. This method succeeds very well with seeds of 
the date, which is sold at all groceries. 
Permanent preparations may be secured by the following method: 
1. Fix in 1 per cent osmic acid, or in absolute alcohol, 5 to 10 minutes. 
2. Stain for 24 hours in Delafield’s haematoxylin. 
3. Wash for a few minutes in acid alcohol (5 drops of hydrochloric acid 
in 50 c.c. of 70 per cent alcohol). 
4. Wash for a few minutes in ammonia alcohol (5 drops of ammonia to 
50 c.c. of 70 per cent alcohol). 
5. Dehydrate in absolute alcohol, clear in xylol, and mount in balsam. 
STAINING CILIA 
The cilia of the large spermatogoid of Ginkgo and the cycads 
stain beautifully in iron-alum haematoxylin, which not only stains 
the cilia but even differentiates the free portion from the part between 
the blepharoplast and the surface. 
The cilia of sperms of Bryophytes and Pteridophytes stain better 
with gentian-violet or crystal-violet. The periods are long; not 
less than 30 minutes, and often several hours will be required. 
