For mounting in glycerin, glycerin jelly, or in Venetian turpentine, fix in 10 per cent formalin and stain in iron-haematoxylin.
For sections, fix in Flemming's weaker solution, but omit the osmic acid for spermatogenesis and germination of carpospores. The time should be very short) 5 to 40 minutes being sufficient. If material is left too long, it goes to pieces. Wash in a gentle stream of sea-water for 24 hours. Stain in iron-haematoxylin and then for 2 to 3 minutes in safranin. This short stain in safranin gives a faint rosy tinge to mucilaginous structures, but does not obscure the fine nuclear detail. In the nucleus of the sperm, the chromosomes remain distinct, so that the number, 20, can be counted from the time the sperm is formed up to fertilization.
With very delicate forms, like Callithamnion and Griffithsia, the washing may be in part or even wholly omitted, and the chromic acid extracted by the lower alcohols, the material being kept in the dark.
Corallina.-Corallina and other forms whose surface is incrusted with lime need special treatment. The following solution is good:
Chromic acid..............................1 g. Glacial acetic acid.......................1 c.c. Sea-water...............................100 c.c.
Fix 24 hours, changing the fixing agent 2 or 3 times. Wash 24 hours in sea-water.
If carefully applied, the following is a good method: Put the material into 5 per cent glacial acetic acid (in sea-water) and watch it. As soon as the vigorous effervescence begins to subside, rinse in sea-water and transfer to Flemming's weaker solution, and fix 24 hours. Iron-haematoxylin is best for figures, but for general structure the safranin, gentian-violet, orange combination gives beautiful results.
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