Normally in measuring the nitrogen–fixing abilities of rhizobia, it is desirable to grow the host plant in a nitrogen–free medium. Several nitrogen–free nutrient solutions are in common use and perhaps each has its advantages and disadvantages.

The important point is that the host plant be well nourished but not overly nourished with all nutrients needed for growth except nitrogen and the various elements should be in balance for the specific host.

Since nutrient requirements of plants vary with the species and with environmental factors, it is unlikely that the perfectly balanced medium can often be obtained. Bryan's modification of Crone's salt formulation (Soil Sci. 13:279) appears to be the most commonly used and has generally worked out satisfactorily. It is usually supplemented with micronutrients.

The Nitragin modification is given below:

pH of nutrient solution (100 ml. 4 Normal H₂SO₄) = 2.00

pH of sand moistened with above solution and autoclaved ranges from 7.6 to 8.2.

• Mix all salts and grind to a fine powder with a mortar and pestle.
• Add 1.5 g. of the salt mixture to one liter of distilled water.
• Supplement with Micronutrient solution and iron as indicated below.
• Presently 100 ml. of 4 Normal of H₂SO₄ added to 9 liters of nutrient solution brings about the desired pH with sand alone.
• When "Terra–Lite" is used in the glass jars, only 50 ml of 4 Normal H₂SO₄ is needed.

Micronutrient stock solution should be added at the rate of 5 ml. per liter or 45 ml. per 9 liters of nutrient solution.