[HARRISON &BAKLOW] NODULE ORGANISM OF THE LEGUMINOS A E 173 



10. Culture's of Leguminosce in flasls containing Ash-maJtose-agar. 

 A^arious means were tried in order to kill the bacteria and moulds on le- 

 guminous seed without killing the seeds. The agents employed alone and 

 in combination with dry heat, moist heat, sulphuric acid, and calcium 

 hydrate, formalin, and mercuric chloride, — but only in a few instances 

 were living seeds obtained free from living bacteria, never when the seeds 

 were first inoculated with spores of bacteria. The seeds either refused 

 to germinate after treatment or else after germination they proved to be 

 still infected with bacteria. We then gave up trying to kill bacteria 

 when these were already present and found a way to get seeds free from 

 bacteria. 



The pods were picked from the plants and such were selected as were 

 sound, well-filled and mature, but not yet dry. The pods were washed 

 and immersed in mercuric chloride solution, 1 : 1000, for an hour or more, 

 then placed between folds of sterile cotton. A pod was held in for- 

 ceps and passed through the flame on all sides and the ends were well 

 burnt. AVith flamed forceps the pod was opened and the seeds placed 

 bet^'een folds of sterile cotton. After a few days the seeds were dry 

 and they were then taken in flamed forceps and put in test-tubes which 

 had been plugged Avith cotton and sterilized. The tubes of seeds were 

 then kept at room temperature until used. 



Seeds were thus obtained in the summer of 1905 and 1906 from 

 pea, vetch, bean and soy bean. These seeds were examined for bacteria 

 by plating some of them in ordinar}^ gelatin or agar media and in ash- 

 maltose-agar and by dropping some into tubes of sterile bouillon. The 

 seeds so treated imbibed moisture and in some instances germinated. 

 Some of these cultures were kept for weeks, yet remained sterile and free 

 from bacteria and fungi. Plate cultures showed occasional colonies, 

 mostly moulds from the air, but occasionally the seeds contained living 

 bacteria. This occurred more often with seeds which were discoloured or 

 infested with weevils, or which came from broken or diseased pods. 



Germination of the Seeds. • From one to three seeds were dropped 

 into about 3 c.c. of boiling sterile water in a test tube. The tubes were 

 immediately cooled and incubated at 37 °C and then at 25° C until the 

 seeds germinated. After the first 24 hours, the tubes were so inclined 

 that the seeds were only partially in the water. Most of the pea and 

 vetch seeds germinated in three days, but the beans did not germinate 

 so well. The water in the tubes remained clear and bright and free 

 from bacteria, except in an occasional tube in which the liquid became 

 very turbid and contained bacteria, sometimes small and slender rods 

 and at other times large, motile bacilli in chains. The seed germinated 

 well in a tube of ash-maltose-agar prepared as for the growth of Legu- 



