Germination of Helianthus annuus. If. 891 
seedlings had reached the desired stage of development, they were taken up 
and washed free from the sand. The hypocotyls were then separated from 
the cotyledons, and the two parts then ground up separately in an ordinary 
meat chopper. The ground material was then covered with 95 per cent, 
alcohol and left until it could be treated further. 
Previous experience had shown that the ether extract of the sunflower 
seed and seedling is very easily oxidized. In order to prevent oxidation, 
or to reduce it to a minimum in the preparation of the dry material of the 
seed and seedling, the following method was used : 
The alcoholic material was placed in two litre glass jars, the lids of 
which were provided with ‘Bunsen valves’. These jars were placed on the 
steam bath, and the alcohol and water driven off as nearly as possible. 
The material was then transferred from the jars to large desiccators and 
dried in vacuo over sulphuric acid. When the material was dry it was 
thoroughly pulverized and placed again in vacuo over sulphuric acid until 
ready for use. 
II. Preparation of the Ether Extract. The dry powdered material 
was placed in a large Soxhlet extraction apparatus and extracted for sixteen 
hours with absolute anhydrous ether. The ethereal solution was then placed 
on the water bath, and a portion of the ethereal solution distilled off. The 
concentrated ethereal solution was then placed in a desiccator, and the 
ether evaporated in vacuo over sulphuric acid until no trace of it remained 
in the oil. The ether extract was then transferred to small tightly corked 
bottles until ready for use. 
III. Preparation of the Neutral Oil. It was thought advisable to 
examine the neutral oil at the various stages of development of the seedling, 
in order to ascertain what changes, if any, it might undergo as compared to 
those taking place in the entire ether extract. The method used for 
obtaining the neutral oil was as follows : 
95 per cent, alcohol was added to the ether extract in the proportion 
of 10 grammes of ether extract to 50 c.c. of alcohol. The mixture was placed 
N 
on the steam bath and heated to boiling, and then titrated with — sodium 
hydroxide, using phenolphthalein as an indicator. To the cooled mixture 
was then added an equal volume of petroleum ether, and the mixture shaken 
gently in a separatory funnel. In this manner is obtained a separation of 
a solution of soap in 50 per cent, alcohol, and a solution of neutral oil in 
petroleum ether. The solution of soap was separated from the petroleum 
ether solution, and the latter again washed with 50 per cent, alcohol to 
remove all the soap. The petroleum ether solution was then placed on the 
water bath and most of the petroleum ether driven off. The concentrated 
solution was then placed in a closed vessel which was connected with the 
filter pump, and the vessel heated on the water bath until all trace of the 
