168 CARL DOWNEY LA RUE 



isolated. Finally a special method was devised which has been described 

 in the Botanical Gazette (LARuE 1920). 



In this method a selecting device was used which consisted of a brass 

 cone threaded at one end, and turned into a small tube at the other, the 

 end of the wall of the tube being made thin so as to form a sharp cutting 

 edge. The device was screwed into the nosepiece of a microscope in place 

 of one of the objectives. The spores to be examined were mixed in a 

 sufficient dilution in a tube of 1 -per cent LIEBIG'S beef -extract agar and 

 sterile slides were spread with a thin layer of this agar. When the agar 

 cooled the spores were firmly fixed in a thin transparent agar matrix and 

 could easily be located and measured under the microscope. When a 

 spore of the desired size was located it was carefully centered in the field 

 of vision and the nose-piece of the microscope was turned so as to place 

 the selecting device, of which the tip had just been sterilized by flaming 

 with a gas or alcohol flame, immediately above the spore. In a good 

 microscope, made so that the objectives center properly, this is an entirely 

 mechanical process requiring no skill on the part of the operator. The 

 microscope tube was now lowered until the sharp tube entered the agar 

 and cut a ring completely around the spore. The agar disk containing the 

 spore was now examined to see that only the chosen spore was contained 

 in it, and then lifted with a flattened platinum wire and transferred to a 

 tube of nutrient agar. 



The agar used for imbedding the spores on the slides was filtered very 

 carefully to render it as transparent as possible so that it might not inter- 

 fere with a clear view of the spores. After selection the spores were grown 

 in Hevea agar of the same composition as that used in the former experi- 

 ments. However, after five selections had been made the cultures were 

 transferred from Sumatra to Michigan, and the supply of Hevea decoction 

 taken with them having been lost in transit, prune-juice agar was used 

 for the remaining cultures of the experiment. The prune agar, which 

 gave satisfactory results, was made by adding 2 percent of agar-agar to a 

 decoction of prunes. Since only one lot was made, and used for all the 

 cultures the exact proportions are of no special significance in the experi- 

 ment, and are therefore omitted. Care was taken, however, to see that the 

 medium was as uniform as possible for all the cultures. 



Experiment 3 



The line used for this experiment was No. 17, an isolation made from a 

 leaf-spot of a seedling of Hevea brasiliensis, which had been grown in 

 culture and measured for nine generations prior to the initiation of the 



