Pteridophytes Filicales 239 



good supply of antheridia in three weeks after sowing;, and the 

 archegonia appear soon after, but it is well to make sowings 6 weeks 

 before material is needed for use. In P. aquilina and in many others 

 if the spores are sown too thickly only antheridial plants will be 

 obtained (Fig. 79). If prothallia are to produce archegonia, they 

 must have sufficient room and nutrition. If there are no green- 

 house facilities and the prothallia must be grown in the laboratory, 

 it is a good plan to take a glass dish, 10 or 12 inches in diameter and 

 about 2 inches deep, put a layer of broken pieces of flower pots on the 

 bottom, cover this with a layer of rich loam, and over this sprinkle 

 a layer of fine, clean sand, since sand is much more easily washed 

 away from the rhizoids than is the loam. The whole should now be 

 thoroughly wet, but not so as to have water standing on the bottom. 

 Sow the spores and cover with a tightly fitting pane of ground glass. 

 There should be no need for moistening the culture again, for pro- 

 thallia can be kept fresh and vigorous for several months, or even for 

 a year, without any wetting. When it is desired to secure fertilized 

 material, sprinkle the prothallia with water, and the young sporo- 

 phytes will soon appear. 



While there should always be a study from living material, it is 

 worth while to make permanent mounts, even for habit study. 

 For such study, the prothallia should be mounted whole. Fix in 

 about 5 per cent formalin and stain some in iron-haematoxylin and 

 some in Magdala red and anilin blue. Use the Venetian turpentine 

 method. Each mount should show the filamentous stage, the 

 apical cell stage, and the group of initials stage, and also antheridia 

 and archegonia. 



For paraffin sections, it is a good plan to grow the prothallia upon 

 Sphagnum, since they can be cut without removing them from the 

 substratum. Rotten wood is also a good substratum, since it cuts so 

 easily that it is not necessary to remove it from the rhizoids. 

 Damp pieces as large as one's fist may be placed under a bell jar, 

 where a damp atmosphere can be maintained. 



For the development of antheridia and archegonia, fix in chromo- 

 acetic acid with the addition of some osmic acid. If the osmic acid 

 is used as strong as in Flemming's weaker solution, allow it to act 



