[HARRISON A barlow] NODULE ORGANISM OF THE LEGUMINOSAE 161 



Heat each lot in steam and boil over the flame or heat in the 

 autoclave at 10 pounds steam pressure, filter, tube and sterilize in steam, 

 or in autoclave at 10 pounds steam pressure for 30 minutes to 5 hours. 

 The media improve with age. Agar media filter slowly through filter 

 paper, and we prefer to filter through absorbent cotton by means of a 

 vacuum pump. 



Aga7' media for Leguminosae. — Water-culture fluids were not tried^ 

 but other workers have stated that nodules are not formed beneath the 

 surface of liquids. Crushed quartz with water-culture fluids did not 

 fully meet all requirements. Gelatin media were found favourable for 

 growth, but as gelatin itself is highly nitrogenous, it was unsuitable for 

 experiments in nitrogen fixation. We then tried agar and found it 

 suitable in almost every respect. 



Ash-maltose-agar sufficient for 12 flask cultures of Leguminosae 

 may be conveniently prepared as follows : — To 4,500 c.c. of distilled or 

 tap water and 18 grams of wood ashes, heat in steam, boil one minute 

 and filter through absorbent cotton by means of a vacuum pump or 

 through filter paper. To 4,000 c.c. of the clear filtrate add 40 grams 

 of agar and 16 grams of maltose, heat in steam until dissolved, boil 

 a minute and filter as above. To each of 12 Erlenmeyer flasks of 

 1,500 c.c. capacity add 250 grams of the filtered medium, plug with 

 cotton and sterilize in flowing steam one-half to one hour on each of 

 three successive days, or better, in the autoclave at 10 pounds pressure 

 for one-half to one hour. After sterilization, tie a piece of parchment 

 paper, wet with mercuric chloride solution 1-1000, over the mouth of 

 the flask, attach a card to receive data and weigh each flask. It will 

 be seen that each flask contains approximately 250 grams of water, 

 2.0 grams of agar, 1 gram of maltose and the soluble part of 1 gram 

 of ashes. As soon as the flasks are sterilized some sterile litmus solu- 

 tion may be added to some of them by means of a slender pipette 

 thrust between the glass and the cotton. The medium remaining after 

 preparing the 12 flasks may be tubed and used as a medium for Ps. 

 radicicola to furnish cultures for inoculating the flasks and for isolating 

 the cultures from the nodules which develop. Such tubes are useful 

 also in germinating the leguminous seeds. 



The ash-maltose-agar in Erlenmeyer flasks has many advantages 

 over quartz and water-culture fluids. It is true tha.t the quartz medium 

 may be made nitrogen free, but the seed itself is highly nitrogenous 

 and the ash-maltose-agar is a nitrogen-poor medium, the 1 per cent 

 agar containing very little combined nitrogen. The agar medium con- 

 tains only 1 per cent of inert material, i.e., lagar; but the crushed 

 quartz, if saturated, contains about 60 per cent by volume of inert 



