186 Direct Assimilation of Ammonium Salts hy Plants 



pump, sterilised water was allowed to flow in from D and the seeds 

 well washed in 2 — 3 changes of water. They were then transferred 

 to Petri dishes and a sterilised 1'25 per cent, solution of agar was 

 poured in ; solidification of the medium occurred in a few minutes, 

 the plates were inverted and placed in the incubator at 20° C. 



At the end of 3 — 4 days, the majority of the seeds had germinated 

 and formed roots 1 — 1^ inches in length, and if sterile, remained quite 

 free from mould or bacterial growth, and were then transferred to 

 sterile wide glass test-tubes containing 10 c.c. distilled water over 

 which a small plug of cotton wool had been placed. On this cotton 

 wool the seedlings were allowed to grow until the shoot was approxi- 

 mately 3 inches long, and if they ftxiled to show any subsequent 

 infection, were then carried over to the culture bottles at the end of 

 7 — 8 days. 



Culture Bottles. Many forms of apparatus have been suggested for 

 the cultivation of plants under sterile conditions ; but the majority 

 are either too complicated or do not allow sufficient facilities for the 

 exclusion of micro-organisms at all stages of the plant's growth. The 

 apparatus used in these experiments has the advantage of being com- 

 paratively simple, is compact enough to allow of sterilisation in any 

 ordinary autoclave, and may be used either for soil-, sand-, or water- 

 cultures. 



For the reception of the plant a three-necked Woulff's bottle A of 

 750 — 1500 c.c. capacity was taken, and rubber corks were placed in 

 each of the side necks. One of the corks held a straight glass tube 

 which had at its upper end a small adapter B filled with cotton wool, 

 while the lower end almost touched the bottom of the bottle ; this tube 

 served to filter the air used for aerating the bottle from time to time. 

 The other cork held a short glass tube bent at right angles which was 

 connected to a Pasteur-Hansen flask C, filled with distilled water, in 

 order that the culture solution in the Woulff's bottle could be kept to 

 the same level throughout the course of the experiment. A few drops 

 of concentrated sulphuric acid were placed in the side tube D. In many 

 cases the flask G was attached to two or three Woulff's bottles by 

 means of three- or four-way glass tubes. The middle neck of the 

 culture bottle was firmly plugged with cotton wool and the whole 

 apparatus heated in the steam steriliser at 99° for three hours. As 

 soon as the sterile seedlings had formed shoots about 2^ — 3 inches in 

 length they were taken from the test-tubes with sterilised forceps and 

 the roots introduced through the middle neck of the Woulff's bottle 



