338 METHODS IN PLANT HISTOLOGY 



into melted paraffin; and then dip into cold water to harden the paraf- 

 fin. While there is no infiltration, the paraffin holds the needles in 

 place for cutting. Fasten in a sliding microtome and cut with the 

 knife placed obliquely. Place the sections in water as they are cut and 

 the paraffin can be easily skimmed away. Then fix the sections in 95 

 per cent alcohol for half an hour; transfer to 70 per cent alcohol, 

 where they should remain for about 5 minutes; then to 50 per cent al- 

 cohol, where they should be kept until the green color, due to chloro- 

 phyll, disappears. Stain in safranin and light green. 



The young leaves cut easily in paraffin. But the mature leaves 

 which, we used to think, must be cut freehand, can be got into paraffin. 

 Then thinner and better sections can be made than are possible by 

 the freehand method. 



Take leaves at the end of their first growing season. They will have 

 all the structures of leaves two or three years old and will cut better. 

 With a safety razor, cut the needles into pieces about 1 cm. long. Fix 

 in formalin acetic alcohol for several days : 70 per cent, overnight or 24 

 hours; 85 per cent, 2 days; 95 per cent, overnight or 24 hours; 100 per 

 cent, overnight or 24 hours. Then use the close series of xylols. The 

 time in the bath is likely to be 24 or 48 hours. 



Spermatogenesis. — In October the clusters of staminate cones 

 which are to shed their pollen in the coming spring are already quite 

 conspicuous. The cones should be picked off separately, and the scales 

 should be carefully removed so as to expose the delicate greenish cone 

 within. At this time the sporogenous cells are easily distinguished. 

 Material collected in January, or at any time before growth is re- 

 sumed in the spring, shows about the same stage of development. If 

 it is desired to secure a series of stages with the least possible delay, a 

 branch bearing numerous clusters of cones may be brought into the 

 laboratory and placed in a jar of water. Growth is more satisfactory 

 in case of branches broken off in the winter than in those brought in 

 before there has been any period of rest. The material can be ex- 

 amined from time to time, and a complete series is easily secured. The 

 mitotic figures in the pollen mother-cells furnish exceptionally instruc- 

 tive preparations. The two mitoses take place during the last week in 

 April and the first week in May. Staminate cones which will yield mi- 

 totic figures can be selected with certainty by examining the fresh 

 material. Crush a microsporangium from the top of the cone and one 

 from the bottom, add a small drop of water and a cover to each, and 



