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Studying Living Organisms

Selecting Soil Organisms in Compost

Barbara J. Errington
Woodrow Wilson Biology Institute
1995

Target age or
ability group:
1st year college prep biology or honors biology.
Class time
required:
One 40-50 minute period and 20 minutes of the next day for observations. Various alterations would require additional class time, e.g., advanced prep of sterile water.
Materials and equipment: For each group:
Compost columns as designed in the Bottle Biology program
One Petri dish containing nutrient agar with 0.5% soluble starch
10% Lugol's solution
One sterile foil-wrapped test tubes containing 9 ml of water
One sterile foil-wrapped Ehrlenmeyer flask containing 99 ml of water
Sterile water
10 ml graduated cylinder
Triple beam balance

General supplies:
Sterile 1 ml pipettes or micropipette, if available
Inoculating loops
95% ethanol, or other alcohol

Summary of activity: The purpose of this lab is to demonstrate that many of the enzyme systems needed to break down--and therefore clean up-society's wastes already exist in nature among the decomposers, in fact, in your school's compost pile. I used the soil organisms lab in my microbiology unit shortly after completing an ecology unit in which students carried out long-term research projects/controlled experiments with compost piles created using procedures developed by the University of Wisconsin-Madison. During the ecology unit on nutrient cycling, students built the columns, planned a controlled experiment among two or three compost columns, varying either the type of soil or the type of vegetative matter to be decomposed. Students made weekly observations for up to two to three months.

In the soil organisms lab, samples are taken from the compost, diluted, inoculated onto agar plates containing starch, and incubated for 24 hours. The plates are then flooded with 10% Lugol's solution. Clear rings will appear around those bacteria capable of secreting enzymes that digest starch. Depending on the type of soil and type of organic matter originally put into the columns, the results can be quite dramatic.

The data obtained from the soils lab can be related to student data on the rate of decomposition; the correlation to type of bacteria becomes quite clear. Students who place sand in their columns have relatively little decomposition and also very few bacteria on their plates, not to mention starch-digesting bacteria. Students who use loamy soil interspersed with potato and pumpkin waste, for example, will obtain greater numbers of bacteria and more starch-digesting bacteria than those students who use loamy soil interspersed with leaves. Students learn that how natural selection of bacteria occurs depends on the environmental conditions in which they are situated.

Prior knowledge, concepts or vocabulary necessary to complete activity:
Serial dilutions
Sterile techniques for culturing bacteria
Function of enzymes
Chemical structure of carbohydrates and proteins (enzymes)
Information on bacteria, including spore formation

Vocabulary: bacteria, enzyme, starch, extracellular digestion, cellular respiration, decomposition

Teacher Instructions

Information:

1. The agar plates need to be prepared a day or two in advance. Mix the soluble starch in a small amount of cold water before adding to the boiling agar.

2 .It is advantageous to have the students measure out their 99 ml sample of water into an Ehrlenmeyer flask and their 9 ml sample of water into a test tube a day or two before the lab is to be performed. These can then be covered with aluminum foil and then autoclaved, or sterilized in a pressure cooker. This takes about 10-15 minutes.

3. The students may measure out their own 0.1 ml of the final dilution to be put in the Petri dish if enough micropipettes are available. I chose not to do this because of the expense of the available micropipettes.

Suggestions:

1. The students benefit from performing this experiment after having cultured bacteria from environmental samples taken around the classroom or school. We find that about half the students already have had this experience, but they seem to enjoy it and it provides a good background for the starch-digesting aspect of the activity. This also provides an excellent way to introduce/reinforce the various uses of controls in an experiment, and what multiple controls (e.g., Petri dish unopened, opened for 10 seconds, opened and inoculated with sterile water, etc.) can tell about your experiment.

2. A time-saving idea that has worked well is for the teacher to prepare either one or both dilutions of the compost in advance. This reduces contamination a great deal, although it further removes the student from participating in all steps of the experiment.

Options for Extension:

1. Compare the number of bacteria from the same column if grown in nutrient agar with starch vs. number of bacteria grown in regular agar supplemented with starch.

2. As stated in the student sheet, colonies of starch-digesting bacteria can be isolated.

Resources:

Sourcebook of Biotechnology Activities, published by NABT.

Bottle Biology Program, Department of Plant Pathology, University of Wisconsin-Madison, 1630 Linden Drive, Madison, WI 53706, (608) 263-5645.

Selecting Soil Organisms Lab

Introduction/Background Information:The purpose of this lab is to show that many of the enzyme systems needed to break down--and therefore clean up-society's wastes already exist in nature among the decomposers, and in fact, in your own compost. It is possible to "select" for these starch-munching organisms by altering the environment in which bacteria are grown. You have already done this by determining what type of organic material went into your compost. You will further select the best bacteria to digest starch in this lab.

Using bacteria (and fungi) to biodegrade waste is called bioremediation. Humans have used bacteria in sewage treatment plants and in composting for many years. On a more experimental basis, microbes have been used to decompose unwanted oil from oil spills and contaminants in ground water. In this lab you will simulate the process of selecting a bacterium from nature that can degrade a particular substance. We will use starch instead of a toxic substance such as oil. You will look for bacteria that have the enzyme amylase, which makes them capable of digesting starch. Starch is a large multi-branched molecule made up of many small sugar (glucose) units. Glucose is the most readily available compound for the process of cellular respiration and obtaining energy from food. Starch must be broken down into glucose before it can provide a bacterium (or you, for that matter!) with energy.

Remember: Bacteria do not have a digestive system. Bacteria secrete, or give off, the enzymes needed to digest their food. The food is broken down extracellularly. Then the bacterium can absorb the pre-digested food. Fungi also digest their food in the same way.

Purpose: The purpose of this lab is contained within the Introduction section. Determine what it is and write it in your logbook.

Materials:

For each team to gather at your lab desk:

    One gram of compost from the compost column
    One Petri dish containing nutrient agar with 0.5% soluble starch
    Lugol's solution
    One sterile capped bottle or foil-wrapped test tube containing 9 ml of water
    One sterile foil-wrapped Erlenmeyer flask containing 99 ml of water
    sterile water
    10 ml graduated cylinder
    Triple beam balance

Available at the teacher's desk to be used there:

    Sterile 1 ml pipette
    Inoculating loop
    95% ethanol, or other alcohol

Procedure:

1. At your own lab station, mass out 1 gram of your own compost soil and add this to 99 ml of sterile distilled water that you premeasured and put in a flask for sterilization previously.

2. At the teacher's desk: Dilute this further by removing 1 ml of your compost mixture with a pipette and adding it to another labeled test tube that contains 9 ml of sterile distilled water you have previously prepared. Mix by swirling at your lab desk. Focus on the sterility of the pipettes you are using: Did they come out of a sealed package? Were they rinsed in alcohol? etc. On lab day, make notes on pipette sterility in logbook at the beginning of your observations section.

3. Label the bottom of your Petri dish with the name(s) of the people in your group, your teacher's name, and your class period.

4. At the teacher's desk, he or she will remove 0.1 ml of your mixture and put it in your Petri dish for you.

5. Using sterile technique as demonstrated previously by your teacher, dip an inoculating loop in alcohol, hold it in a flame briefly and cool it on the inside edge of the Petri dish. Use this to spread the 0.1 sample evenly around the plate. If your inoculating loop is not cool enough, it will kill the bacteria as you spread them around!

6. Turn the plates over and leave overnight at room temperature in the area designated by your teacher.

7. The next day, examine the plates. Look to see if clear areas are developing in the starch agar. Make observations as to the quantity, types, and sizes of different bacterial colonies in your logbook.

8. Flood the plate with the Lugol's solution. After one minute, pour off the excess iodine into the sink with running water. Bacterial colonies capable of digesting starch will be surrounded by a brown "halo," as opposed to the blue-black color typical of a reaction of Lugol's solution to starch.

9. Draw in the colonies on your plates into the observations section in your logbook. Do not forget to write additional notes and observations that are more difficult to convey with a drawing.

10. Compare your plate with several other groups' plates in terms of degree of starch digestion. Note what the other groups had put into their compost columns in terms of type of soil and vegetable material. Summarize these observations in your logbook.

Optional Extension:

11. If you have some good, clear areas around some bacteria, you may isolate these bacteria and try to grow a pure culture of these starch-digesting bacteria. In order to do this, the colony must be taken off with a sterilized inoculating loop immediately after the Lugol's is poured to prevent killing the colonies. These can then be spread on a new sterile Petri dish containing nutrient agar with starch.

Selecting Soil Organisms Lab -- Questions

Bacteria do not have a digestive tract like higher organisms (like you and me!). In order to "feed," they must secrete enzymes to break down their food, and then the bacteria absorb the digested food.

Directions: On a separate piece of paper, answer the following questions in complete sentences. Use information gained from the lab and your own prior knowledge.

1. The agar in the Petri dishes looked cloudy because starch was suspended in it. What process caused the clear areas on the plates?

2. What enzyme do starch-digesting bacteria contain?

3. How could these bacteria survive in soil or water which did not contain starch?

4. Suppose you or your teacher took some of the bacteria which had a brown halo around them after adding the Lugol's solution and spread them onto a new sterile Petri dish containing starch agar. Then you repeat the experiment. What would you expect to see when you observed the Petri dish 24 hours later, after adding the Lugol's solution again?

5. Pumpkin contains a large amount of starch. Bacteria containing amylase are needed to digest it for efficient decomposition. Which would decompose faster -- a compost column containing pumpkin and humus from a forest floor, or a column containing pumpkin and sand from the beach?

6. What possible uses can you think of in the "real world" for these starch-digesting bacteria? In your answer, take into account all of our great mountains of excess waste in landfills, and then make your answer more detailed.

7. Now you are the CEO of a large petrochemical company which has a long history of oil spills in Alaska and the Middle East. In order to reduce your pollution fines by several billion dollars, you have agreed to develop a new strain of bacteria to degrade, or decompose oil from spills in marine environments. How would you go about developing these oil degrading bacteria? Be specific about the role of the environment in your answer. Be as specific as possible in your methods.

Extra Credit

8. When Exxon tried to clean up the oil spill in Prince William Sound in Alaska, why did they add fertilizer to the oil?

On to The Value of Behavioral Variation
in Homo sapiens

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