Nitrogen-fixing bacteria free-living in the soil
Class practical
In this activity, students will culture a free-living nitrogen-fixing bacterium (Azotobacter) from the soil. This will reinforce understanding of the role of bacteria in the nitrogen cycle.
This practical is based on an investigation called published in Practical Microbiology for Secondary Schools © Society for General Microbiology.
Lesson organisation
Make students aware of procedures for safe handling of microbial material when dealing with their incubated plates.
You may be able to set up this investigation alongside the investigation of nitrogen-fixing microbes from legume root nodules in the same lesson. If so, make sure the students do not mix up the plates of different media from the two activities.
Apparatus and Chemicals
For each group of students:
Nutrient agar plate, 1 (see Standard technique Pouring an agar plate)
Nitrogen-free mineral salts agar plate, 1 (see Standard technique Making up nutrient agars)
Spatula or forceps to dispense soil, 1
Marker pens
Adhesive tape
For the class – set up by technician/ teacher:
Sterile specimen bottles containing samples of a soil that readily forms small crumbs, no more than 1 bottle per group
Nitrogen-free mineral salts agar medium, sterilised in McCartney bottles or poured as plates
Nutrient agar medium, sterilised in McCartney bottles or poured as plates
Nitrogen-free mineral salts agar plate, to be left unopened as a control, 1
Nutrient agar plate, to be left unopened as a control, 1
Disinfectant to clean the work area before and after the investigation (Note 2)
Health & Safety and Technical notes
Carry out a full risk assessment before planning any work in microbiology (see note 1 for more details).
Check the standard techniques for more details of Making up nutrient agars, Pouring an agar plate, Aseptic techniques and Incubating and viewing plates.
1 Before embarking on any practical microbiological investigation carry out a full risk assessment. For detailed safety information on the use of microorganisms in schools and colleges, refer to Basic Practical Microbiology – A Manual (BPM) available free from the Society for General Microbiology (email This email address is being protected from spambots. You need JavaScript enabled to view it.) or go to the safety area of the SGM website www.microbiologyonline.org.uk/safety.html, or refer to the CLEAPSS Laboratory Handbook, section 15.2 and 15.12.
2 Suitable disinfectants include sodium chlorate(I) (hypochlorite) at concentrations greater than 1% (refer to CLEAPSS Hazcard 89), or Virkon used according to manufacturer’s instructions. Wear eye protection when handling disinfectants.
Procedure
SAFETY:
Tape plates to keep them closed but not sealed.
Incubate up to but not above 25°C.
Wear eye protection when handling disinfectants.
Preparation
a Calculate the quantity required. Prepare just enough nitrogen-free mineral salts, agar medium, and nutrient agar medium for the investigation (12-15 ml for normal depth in a 90 mm Petri dish). Refer to Standard technique: Making up nutrient agars for the details of the recipes. Any surplus will keep for 6-12 months in tightly-sealed screw-top bottles if sterile and stored out of direct sunlight. Or you can buy prepared plates (see Suppliers).
b Distribute the agar into individual McCartney bottles. Sterilise them if you want the students to pour their own plates - see Standard techniques: Pouring an agar plate. Mix to disperse the CaCO3 in the nitrogen-free mineral salts agar medium before pouring.
c Prepare a suitable solution to disinfect the work area for the investigation and afterwards.
d Collect sterile sample bottles or Petri dishes (or sterilise some) for the soil samples.
Investigation
a Leave one plate of each type of culture medium unopened as a control. (See also answers to student notes Q1 for other controls that could be run.)
b Collect one of each type of agar plate. Label one with initials, date and ‘nutrient agar’. Label the other with initials, date, and ‘N-free’.
c Using the spatula or forceps, place 10-20 small crumbs of soil evenly over the surface of each plate.
d Tape the lids closed with adhesive tape and incubate with the lid uppermost at 20-25°C for 2-3 days. Incubating this way up is unusual, but otherwise the soil crumbs will fall off the agar media. See Standard technique Incubating and viewing plates.
e Next lesson, describe the appearance of the colonies on the two types of agar media. Do not open the plates. Mucoid (sticky) colourless growth oozing from soil particles on the nitrogen-free mineral salts agar medium indicates the presence of Azotobacter. Look for any other types of growth you think are likely to be of nitrogen-fixing microorganisms.
Teaching notes
Free-living nitrogen-fixing bacteria fix nitrogen by reducing gaseous nitrogen in the air to ammonia. This is incorporated into organic compounds which can be used by plants. An enzyme complex called nitrogenase catalyses this reaction. Nitrogenase activity is sensitive to the presence of oxygen. Azotobacter (an aerobic nitrogen fixer) has the highest respiratory rate of any organism. This enables it to remove oxygen rapidly from its surroundings through its own respiration. The mucoid slime material produced by the bacteria also aids protection from oxygen.
Nitrogen-fixing bacteria will be unable to compete with other non-nitrogen-fixing soil microbes for nutrients on the nutrient agar medium, but will have an advantage on the selective nitrogen-free mineral salts agar medium. Therefore, colonies of nitrogen-fixing bacteria will grow well on the nitrogen-deficient medium.
Health & Safety checked, June 2008
Downloads
Download the student sheet 
Nitrogen-fixing bacteria free-living in the soil (345 KB) with questions and answers.
Web links
www.microbiologyonline.org.uk/teachers/resources
Society for General Microbiology – source of Basic Practical Microbiology, an excellent manual of laboratory techniques and publishers of Practical Microbiology for Secondary Schools, a selection of tried and tested practicals using microorganisms. These include the original procedure for Nitrogen-fixing bacteria which is also available here.
(Website accessed October 2011)