284 STATE BOARD OF AGRICULTURE. 



Method No. 1. 



I. "Wash nodule free from soil. 



II. Place in a 1:500 solution of acid mercuric chloride for two minutes. 



III. Rinse in sterile water and dry between folds of sterile filter paper. 



IV. Hold nodule with flamed forceps and cut upon with flamed knife edged 



platinum needle. 

 V. Make transfer with flamed straight needle to melted media. 

 VI. Make two single loop dilutions from original transfer tube and plate. 

 Set plates in temperature room at 25° C, if possible. 

 VII. After colonies appear (24-72 hours) isolate organism from characteristic 

 branched colony to slant tubes and into liquid media. Note — The 

 characteristic branched colony will predominate over any possible con- 

 taminations in plates. 



Method No. 2. 



To be recommended when nodules are very small. Wash, sterilize, rinse, and 

 dry as in I, II, and III, Method No. 1. 



IV. Place nodule between flamed one-half cover slips and press slips firmly 



together. 



V. Transfer these slips into tube of melted media and break them apart with 



sterile platinum needle. 

 Make proper dilutions, grow, and isolate as in VI and VII, Method No. 1. 



Both methods have been found, in our work, to be very satisfactory in isolating 

 a pure culture. 



The final step is to establish the supposed identity of a culture thus isolated. 

 To do this, we employed the following method: 



Seedlings of the species of Leguminosae from which the organism in question 

 was isolated, were grown in sterile quartz and watered with a dilute sterile soil 

 solution. As soon as the first pair of leaves were formed, a 24-hour liquid culture 

 of the isolated organism was poured about the stems of seedlings in one pot; a 

 second pot, without pure culture, being kept for control. 



If culture is true to name, nodules will appear within 14 days after inocula- 

 tion. 



In our work, better results were obtained when nodules were collected from 

 nearly mature plants. The reason for this is not determinable at present with 

 us. After the circulation in the plant ceases, the centres of the nodules rapidly 

 break down into a doughy mass, hence the necessity for collecting nodules for 

 future use, before this stage is reached, becomes apparent. 



Special culture media best serve the purpose for cultivating this organism 

 satisfactorily, although it will grow well on nutrient agar and in bouillon. 

 Various combinations have been tested by Mr. H. F. Tuttle and myself, the fol- 

 lowing proving most satisfactory: 



I. Extract 450 gr. beef with 1000 cc. lime water. 



II. Strain out meat and boil extract down to 500. 

 III. Add 1% peptone and dissolve. 



IV. Add 1Vj% agar predigested in 500 cc. distilled H-Q. 

 V. Establish acidity at 8°+, cook one hour, and filter. Tube and sterilize. 



Two per cent glucose added to this medium permits a more rapid growth, but 

 a fermentation takes place in slant cultures and interferes with a characertistic 

 growth. One other, however, gave good results, and might well be mentioned: 



• 



Ordinary beef extract 1,000.0 cc. 



NaCl 5.0 gr. 



MgSO^ 0.1 gr. 



KH„PO, 0.5 gr. 



Maltose 15.0 gr. 



Agar 15.0 gr. 



Acidity established at 8° + 



