BY R. GREIG SMITH. 665 



Although a culture of the nodule oreianism was obtained from 

 the lupin nodules by smearing the surface of set agar, the method 

 did not recommend itself as one at all well adapted for easily- 

 getting the organism. 



■ Beijerinck's method of sowing drops of nodule emulsion was 

 just as useless, because the places were in a few days swarming 

 with other bacteria. 



Better results were obtained by washing the nodules and 

 passing them successively througli mercuric chloride, alcohol and 

 ether, holding them with sterile forceps until the ether evaporated 

 and placing each into a Freudenreich tlask containing 10 c.c. 

 sterile, 0*6 per cent, potassic chloride. In the flasks the nodules 

 were crushed with stout sterile glass rods. The emulsion thus 

 obtained was blown by means of a sterile glass spray upon the 

 surface of set gelatine medium in a Petri dish. From six to 

 twelve plates should be prepared from the same number of 

 nodules, as some of the nodules may contain foreign organisms 

 Avhich grow quickly and generally liquefy the gelatine. One 

 objection to spraying the plates is that the air is washed at the 

 same time, and moulds and aerial bacteria carried to the gelatine 

 surface. The usual method of obtaining pure cultures by inocu- 

 lating the gelatine, previous to pouring into plates, is not to be 

 recommended, as the nodule-formers are then chiefly in the l)ody 

 of the gelatine film, and grow ^■ery slowly indeed, especially when 

 taken directly from the nodule where they are presumably in a 

 somewhat enfeebled condition. The passage through the potas- 

 sium chloride seems to act as a stimulant, for the colonies grow 

 ^faster than when distilled water is employed. 



A better method than spraying consists in sterilising a small 

 <;amers-hair brush or pencil b}' passing it successively through 

 mercuric chloride, alcohol and ether, allowing the ether to evapo- 

 rate and washing in sterile potassium chloride. The moist sterile 

 brush is then pushed about in the nodule emulsion and painted 

 over the set gelatine surface. Confluent or isolated colonies 



appear in from six to ten days^ and from these a pure culture 

 43 



