MICROBIOLOGY 101 LABORATORY MANUAL

EXERCISE 22: SOIL MICROBIOLOGY AND THE ELECTIVE CULTURE TECHNIQUE

NAME, ID #:_______________________________________________

NAME of TA: ______________________________

REVISED: 08/04/99

INTRODUCTION TO EXERCISE

NITROGEN FIXATION

Being very clever, you will immediately realize after considering the import of that last statement, that something doesn't make sense. Namely: "If the ability to fix nitrogen is such a great thing, why don't all forms of life do it?". You all are very bright (& good looking too) to pose such an insightful question! The answer turns out to be a case of "cost/benefit" management. Nitrogen gas requires a humongous amount of energy to FIX. So a microbe that fixes N₂, can't grow or do much of anything else fast because it is expending a huge chunk of its limited supply of energy fixing N₂. It's like skiing without lifts; i.e., while it is great fun going down, if you have to climb up a hill after each run, skiing quickly lose its appeal, right? So the nitrogen fixing microbes tend to be found in places where little or no fixed N₂ is present (which actually includes much of the earth's surface), which means that there won't be too many other life forms competing with them for the other nutrients. But when they die, their nitrogen-rich remains fertilize the growth of non-nitrogen-fixers (like you and me).

ELECTIVE OR ENRICHMENT CULTURE TECHNIQUE

It turns out that quite a few prokaryotes are able to fix nitrogen, including some Cyanobacteria (Blue Green Algae), a number of non-phototrophic free-living forms and some symbiotic forms (#Rhizobium). But how can you isolate N₂-fixing microbes? To do this, you will use a technique developed by the early microbiologists for isolating microbes with particular biochemical abilities. The technique is called the ELECTIVE or ENRICHMENT culture technique. It is based on the following common sense principle. If you want to isolate a microbe that has a unique biochemical characteristic, you provide a medium made so that ONLY microbes with that characteristic can grow. For example, if you want to catch (isolate) "bank robbers" you would hang around banks, right? So if you want to isolate microbes that eat turkey feathers or an insecticide you make up a medium containing all the nutrients except those that you find in turkey feathers/insecticide etc. and then you add a few turkey feathers or a pinch of insecticide to the medium. Now where do you find "turkey-feather/insecticide-eating-microbes"? It turns out that's rather easy. You dump in a few pinches of a rich soil (e.g. from a garden). Remember, microbes have been around for a few billion years so they have had the chance to be essentially everywhere. In this exercise, you will isolate "free living, aerobic, nitrogen-fixing" bacteria. These are bacteria that require oxygen, but no fixed nitrogen. To do this a NITROGEN-FREE media is prepared as follows:

Plus tap water to 1 liter.

PURPOSE OF THE LABORATORY

1. To demonstrate elective culture technique as a means of isolating free-living nitrogen fixing bacteria.
2. To demonstrate the presence of symbiotic Rhizobium nitrogen-fixing bacteria living in association with leguminous plants.
3. To describe and explain the significance of microbial nitrogen fixation in the maintenance of soil fertility.

PROCEDURE

Materials

1. N-free medium plate, one per student.
2. Suspension of soil in water; one per table.
3. Glass spreaders.
4. Alcohol and burner; two per table.
5. Zip-locked bag; one per table.

Aerobic, free-living N-fixers

1. One N-free medium plate/student.
2. Inoculate the plate by spreading two drops of the soil suspension onto the center of the plate.
3. Dip the glass spreader into the alcohol and pass it through the flame so the alcohol catches fire. Remove the spreader from the flame and allow the alcohol to burn off. Wait about 1 minute for the glass to cool.
4. Touch the spreader on the agar away from the drops and then rub it gently through the drop so as to spread the liquid all over the plate.
5. Label it with your name and the date and place it into a ziplocked bag and incubate in your desk at room temperature.
6. Examine the plate at the beginning of each of the next two lab periods and look for the appearance of small, convex, transparent colonies become large gummy colonies that develop a dark-brown pigmentation over time.
7. Carry out negative and Gram stains of one of these isolated colonies; if there are more than one colony type, stain one of each.
8. Draw what you see and write a description of their appearance in both the wet mount and the stains.
9. Show both the colonies and the stains to you T.A.

   Figure 1. Root of a legume with nodules containing Rhizobia

Rhizobia and leguminous plants

    The instructors will supply some roots of legumes.

1. Examine the roots for the presence of root nodules described in #Chapter 21.
2. Hold the nodules up to the bright light and look for color. If a dissecting microscope is available examine the nodules with it.
3. Pick a nodule off, place it in a drop of sterile water on a slide and crush it between two clean slides until you produce a milky suspension.
4. Prepare two smears of the suspension on two slides and heat fix.
5. Stain one smear with methylene blue dye and the other with carbon-fuchsin.
6. Draw the many different shapes of the stained bacteria below. Look carefully to distinguish between plant cell debris and the smaller bacteria.
7. When you think you have the correct material ask your Instructor to verify what you think you are seeing.

PART 2: TOTAL SOIL COUNT

A single gram of soil may contain over a billion bacteria representing 1,000s of different species. Some microbiologists have even suggested that a gram of rich garden soil might contain one of almost every bacteria species that exists on earth. While this might be a bit of hyperbola, there are clearly a lot of different kinds of microbes, Pro- and Eukaryotes, in most soils. Determining the numbers and species of bacteria present in a given sample of soil is basically a hopeless task as we currently know how to cultivate only a tiny fraction of the earth's bacterial inhabitants. However, you have to be an optimist if you are a microbiologist so this means we will try (in this case "The 'Ol College Try").

As no one medium is capable of supporting the growth of more than a tiny fraction of all bacteria, quantifying the numbers of bacteria in a soil sample is fraught with problems from the very start. The general approach to enumerating the bacterial content in a mixed environment like soil, air or water, is to chose a rich medium and assume that a significant number of the species present in the sample will be able to grow on it, even if it isn't the optimal medium for many microbes. However, one is limited by time, money and facilities to choosing a limiting set of environmental conditions (e.g. oxygen, light, temperature etc.) for incubation which clearly limits which species will grow. For example, no obligate anaerobes will grow in the presence of air, nor will obligate phototrophs (which require light) grow in the absence of light (and it must be light of a particular wavelength for a particular species). Because of the multiplicity of these limitations and restrictions, it is likely that less that a fraction of a percent of the bacterial species present in an average sample of soil are capable of being cultivated under one particular set of conditions.

In this exercise we will use the plate-counting technique in which the soil is suspended in sterile water and various dilutions are spread on a plate of medium. Subsequently, the number of colonies that grow up after a suitable period of incubation are counted and the total number of bacteria (that grew) in the original quantity of soil are calculated from the size of the soil-sample, the volume of suspending water and the dilutions employed.

PURPOSE OF LABORATORY

1. To give students an idea of the vast numbers of bacteria that live in the soil.
2. To further student's understanding of the process of dilution and plate counting for the enumeration of bacteria.

MATERIALS

1. Scales and weighing paper.
2. Spatulas.
3. 100 ml of melted agar-medium at ~45^oC.
4. Two sterile 1.0 ml pipettes/pair.
5. Two sterile 99.0 ml water dilution bottles.
6. Four sterile petri dishes/pair.
7. One container of rich soil/scale.
8. Gram stain kits.
9. Dissecting scopes.

PROCEDURE

1. Each pair of students pick up 4 petri dishes and label them 1:100, 1:1000, 1:10,000, 1:100,000 along with your name and the date. See Atlas pg. 81-83.
2. Each pair pick up two sterile 99 ml water dilution blanks and label then 1 and 2.
3. Determine where the sterile pipettes are, but don't pick one up until you are ready to go.
4. Take bottle #1 to the side bench where the scales and soil samples are located.
5. Weigh out 1.0 gram of rich soil as demonstrated by your instructor and add it to bottle #1.
6. Close the cap tightly and shake the bottle vigorously for ~2 minutes. Allow the heavy particles to settle for ~1.0 minute.
7. Carefully remove a sterile pipette from the container, being very careful to touch only the large end while not allowing the pointed end to touch anything else except the contents of the bottle.
8. From the top of the solution in bottle #1, pipette 1.0 ml and 0.1 ml into the centers of the petri dishes labeled 1:100 and 1:1,000 respectively.
9. From the top of the solution in bottle #1, pipette 1.0 ml and add it to bottle #2. Shake well with the lid tight.
10. From the top of the solution in bottle #2, pipette 1.0 ml and 0.1 ml into the centers of the petri dishes labeled 1:10,000 and 1:100,000 respectively.
11. As demonstrated by the Instructor, aseptically add enough melted and cooled agar medium to each plate to cover the bottom of the plate (~15-20 ml). As one partner adds the agar the other should immediately mix the medium and the bacterial sample by picking up the plate and tilting it in a circle as demonstrated by the instructor.
12. Following mixing, place the plates on the desk top until the agar solidifies (~10-15 min) and them incubate upside down at 37^oC.
13. Incubate for 24 hours and refrigerate the plates until they can be counted.
14. Observe the plates by eye and under the dissecting microscope so that you can distinguish the colonies growing embedded in the medium.
15. Count the colonies on the plates and calculate the number of viable cells/gram of soil (Atlas pg. 82).
16. Place this information on the chart on the board by your names and be prepared to discuss any disagreements in the counts.
17. Each student carry out a Gram stain on 4 colonies that look different and record their appearance below.

SAMPLE QUESTIONS: You should be able to answer these questions at the conclusion of this laboratory.

• What is a nitrogen-fixing organism?
• What is Rhizobium and where would you go to isolate them?
• Why doesn't every living organism fix nitrogen?
• What is the "ELECTIVE CULTURE" technique and how does it work?
• How do leguminous plants improve the fertility of the soil?
• You have carefully dug up a leguminous plant from your very productive garden. When you carefully wash away the soil from the roots you observe that there are no root nodules. Why?
• In the nitrogen-free media used in this exercise, what is the purpose of each of the following components?
  • Mannitol
  • K₂HPO₄
  • MgSO₄.7H₂O
• Why would you expect to isolate a nitrogen-fixing organism from this medium?
• What does the cultivation of peas in the Palouse have to do with this exercise?
• What is "Green Fertilizer".

Copyright © Dr. R. E. Hurlbert, 1999.
This material may be used for educational purposes only and may not be duplicated for commercial purposes.
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