Isolation of Azospirillum Associated with the Roots of Grasses

Exercise Contributed by

Ralph Tanner

The University of Oklahoma

Adapted by Jodi Naugle

 

Azospirillum species may be isolated from rhizosphere biofilms associated with the roots of various grasses, cereals, and tuber plants. Azospirilla are generally gram-negative rods which are motile by means of a single flagellum. Azospirilla are also capable of fixing nitrogen. Their isolation is based on the premise that these organisms can grow in concentrations of nitrogen to low to support growth of most microorganisms.

Isolation Procedure
  1. Excise the major root of a grass plant and wash the root in four changes of sterile distilled water. Cut a 2 cm section from the top of the root, not from the tip, and place this in a tube of ASP soft agar. Incubate the tube at 37°C. Growth will be apparent in two to three days.
  2. Streak the growth from the top of the ASP soft agar onto two plates of ASP solid agar. Incubate at 37°C for an additional two to three days. Azospirilla colonies may be either white and flat or pink and raised.
  3. Streak one of the Azospirillum colonies onto a plate of ASP solid agar for isolation.

ASP Medium/100 mL distilled H2O

1 g Sodium succinate

    1. mL Trace Metals

Solution

0.01 g yeast extract Trace Metals Solution/1.0 L distilled H2O

0.07 g KH2PO4 2.0 g Nitrilotriacetic acid (adjust PH

0.02 g MgSO4·7H2O to 6.0 with KOH)

0.005 g CaC12·2H2O 1.0 g MnSO4·H2O

pH 7.0 0.8 g Fe(NH4)2(SO4)2·6H2O

0.2 g CoC12·6H2O

For soft agar: add 0.3 g 0.2 g ZnSO4·7H2O

purified agar 0.02 g CuC12·2H2O

For solid agar: add 1.5 g 0.02 g NiC12·6H2O

purified agar 0.02 g Na2MoO4·2H2O

0.02 g Na2SeO4

0.02 g Na2WO4

Store at 5 C