SPERMATOPHYTES— GYMNOSPERMS 339 



examine. If there are pollen tetrads at the bottom, but only undivided 

 spore mother-cells at the top, it is very probable that longitudinal sec- 

 tions of the cone will yield the figures. If a drop of methyl green be 

 allowed to run under the cover, it will enable one to see whether 

 figures are present or not. When desirable cones are found, slabs should 

 be cut from two sides, in order that the fixing agent may penetrate 

 more rapidly and that infiltration with paraffin may be more thor- 

 ough. 



The later stages, showing the germination of the microspores, fur- 

 nish better sections if the cones are cut transversely into small pieces 

 about 5 mm. thick. It is very easy to get excellent mounts of the pol- 

 len just at the time of shedding, which, in Pinus laricio in the vicinity 

 of Chicago, occurs near the middle of June. Shake a large number of 

 cones over a piece of paper, thus securing an abundance of material; 

 slide the pollen off from the paper into a bottle half-full of water and 

 shake a little to wet the pollen grains, because pollen of wind-pollinat- 

 ed plants is likely to be more or less shriveled at the time of shedding. 

 A few minutes in water will make the pollen turgid. Fix in formalin- 

 alcohol-acetic acid. If material is so abundant that you can lose much 

 of it and still have plenty left, fix in chromo-acetic-osmic acid. Stain- 

 ing, especially the staining of mitotic figures, is likely to be more 

 brilhant after fixing in the chromic series. However, most of the mi- 

 toses take place before the pollen is shed or after it reaches the nucel- 

 lus. Infiltration in the bath will not require more than 30 minutes. 

 When the infiltration is complete, pour out into a paper tray or an 

 imbedding dish, just warm enough to allow the pollen to settle to the 

 bottom. A layer of pollen 3 mm. thick, with enough paraffin to make 

 the cake about 5 mm. thick, will be satisfactory for cutting. Or, the 

 paraffin may be poured out upon a piece of cold glass. Still another 

 method is to leave the paraffin in a shell or vial during infiltration in 

 the bath, and then let it cool in the bottle. After the paraffin is hard, 

 break 'the bottle. Break the bottle carefully, cut off the lower portion 

 of the paraffin containing the pollen, mount it on a block in the usual 

 manner, and trim away some of the paraffin so that two parallel sur- 

 faces will make the sections ribbon well. Sections should not be thick- 

 er than 5 /x, and 3 ^ is better. 



Material in this stage shows a large tube nucleus, a somewhat 

 lenticular (generative) cell with a more deeply staining nucleus, and, 

 lastly, two small prothallial cells quite close to the spore wall. The 



