Isolation of Nitrogen-fixating bacteria

Goal

N₂ is a gas that ubiquitously available in the atmosphere. Some special bacteria are capable of fixating it and thereby make it available for other life forms. In this experiment we will isolate them from soil.

Materials

• 100 mL of Azotobacter medium
• 1 Azotobacter Agar plate
• 3 100 mL erlenmeyer flasks
• Soil
• Nitrogen test strips

Method

1. Prepare 2 Erlenmeyer flasks by transfering 25 mL into each.
2. Add some soil to one of the Erlenmeyer flasks.
3. Save the other Erlenmeyer as negative sterile control.
4. Clearly label the flasks: name, date & content.
5. Incubate the flasks at 30° C.
6. Inspect the flasks every day or two.
7. As soon as (most likely after one week) a small film can be observed on the surface of the medium, transfer a small amount into a fresh Erlenmeyer flask.
8. Incubate this flask again at 30° C.
9. Look at the biofilm under the microscope.
10. Take a sample from the flasks and check the NH⁺ concentration with the test strip.
11. As soon as growth can be observed in the 3^rd flask, streak a Azotobacter agar plate and incubate at 30° C. Lable the plate: name, date & content.
12. Look at a sample under the microscope.
13. Check after 24h.

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