APPENDIX A 133 



the seeds in flask B by means of a funnel, to prevent contact be- 

 tween the seeds and the neck of the flask. With the vacuum pump 

 draw the mercuric chloride from C into B ; then close with a screw 

 clip and exhaust B untn the solution begins to boil. By this means 

 the disinfectant comes into direct contact with and is able to act on 

 all portions of the seeds. Allow sterilization to go on for four to 

 six minutes, then invert B and withdraw, by means of the pump, 

 the mercuric chloride ; after this allow sterilized water to flow from 

 D into B, and wash the seeds weU two or three times with this water. 

 The seeds may then be transferred to Petri dishes and a sterilized 

 layer of 1.25 per cent solution of beef-peptone agar poured over them, 

 or they may be carried to dextrose-beef bouillon, one seed in each 



Fig. 43. Arrangement of bottles for seed sterilization 

 For explanation see text 



tube. If the bouillon remains clear until the eighth day when kept 

 in an incubator at 25° C, it indicates that aU bacteria were killed. 

 When agar is used, allow the plates to solidify, invert, and place 

 in incubator at 20°-25° C. At the end of three or four days the 

 majority of the seeds will germinate and form roots 1~1J in. long. If 

 these are free from molds and bacteria, they may be carried to 

 sterile wide glass test tubes containing about 10 cc. of Tollen's me- 

 dium or distUled water, over which cotton plugs have been placed. 

 On the cotton plugs the seedlings will grow several inches long, and 

 if no subsequent infection is noted, they may be carried over to cul- 

 ture bottles. There is less danger of infection, however, when the 

 seedlings are carried direct to the culture mediujn or soil in which 

 they are intended to grow. 



Schroeder ^ recommends a very simple method for sterilizing seed. 

 He found that the seed coats of the Graminese — for example, 



1 Centralbl. f. Batt., 2te Abt., 28 : 492-505. 1910. 



