Winogradsky Column and Biofilm Study :

Teacher Information

Introduction

The Winogradsky column is a small ecosystem that will show many types of microorganisms and the importance of nutrient cycling. It is prepared by using a 3-liter soda bottle, soil, water, and a few chemicals. By also producing a biofilm in the Winogradsky column, the succession of microorganisms can be studied. The biofilm is made by placing a glass slide into the column that will serve as a surface for the organisms to attach to. If the biofilm is observed for several weeks in this study, the succession of organisms can be seen. The importance of nutrient cycling can be studied by noticing changes in the population of organisms that occur in low oxygen conditions as well as in good aerobic conditions. Students will notice changes in the color of the soil that reflect hydrogen sulfide production and the type of microbes that appear or disappear with changing levels of oxygen and hydrogen sulfide. This lab is intended to be a long-term study. It is recommended that the column be observed for at least 5 or 6 weeks.

This lab can be used to study populations of organisms; a survey of microorganisms; nutrient cycling; ecological succession; and chemical transformations.

Materials needed

For a group of 4 people

1 clear 3-liter soda bottle

Outdoor garden soil

Plastic wrap

5 g calcium carbonate

5 g calcium sulfate

10 g shredded newspaper

Balance

4 microscope slides

coverslips

Methylene blue

Gram stain reagents: crystal violet, iodine, ethyl alcohol, safranin (Optional)

Slide holders

Water squeeze bottle for Gram stain procedure

Microscope

Rubber band

Procedure

A. Construction of the Winogradsky Column and Biofilm

1. Using a utility knife cut the neck from a 3-liter soda bottle so that the bottle takes on the appearance of a column.

2. Soil from outside is ideal for this lab. Collect enough soil that can fill the soda bottle 2/3 full.

3. Lining a lab table with newspaper, clean the soil of debris. Remove any stones, pebbles, grass clippings, leaves, and moving organisms that you can see from the soil sample.

4. Mix the soil with 5 g of CaCO3, 5g of CaSO4, and 10 g of shredded newspaper.

5. Place the soil mixture into the soda bottle then add enough tap water to cover the surface of the soil by 3 or 4 cm.

6. Stir to mixture to release any air bubbles trapped in the soil. Let the soil sit for 5 minutes.

7. Add more water until the surface of the soil remains under water by a depth of 3 or 4 cm.

8. Mark a water line on the outside of the soda bottle to maintain the amount of water in the bottle.

9. Place 4 microscope slides in the bottle so that they are propped up vertically in the soil. One end of the slide should be resting on the surface of the soil and the other end of the slide should be leaning against the side of the bottle. In this position, the slide is partly submerged in the water. Angle the slides so that each is at least 70% under the surface of the water.

10. Cover the soda bottle with plastic wrap and fasten with a rubber band.

11. Let the Winogradsky column and biofilm stand at room temperature for as long as you can. It is recommended that the column be observed for at least a 6 weeks time period.

B. Observations of the Winogradsky Column and Biofilm

1. The column may be observed the next class period. Have students notice the level of water from where it was the day the column was set up. Other observations to notice while the cover is on are the following.

a. Odors

b. Color

c. Condensation on the plastic cover

d. Crust forming in the bottle

e. Film on the surface of the water

2. Observations to notice with the cover of the column off are the following.

a. Odors

b. Film over the surface of the water

c. Crust build up in the column

d. Macroscopic organisms

3. The biofilm may be observed by pulling out the microscope slide from the end that is not submerged in water. Each member of the group can observe a biofilm slide.

4. Clean off one side of the microscope slide so that it can lie on the stage of a microscope. Prepare a wet mount using low and high power objective lenses for observations. Have students be patient when making initial observations because the organisms that can be seen are very small. Most of what can be seen at this stage is bacteria. Have students notice the shape of the bacteria, and their colony formation. Have students record their data. You might have students log population counts of various organisms seen on this date.

5. When the observation study is completed for the day, rinse off the slide and place it back into the column. A new biofilm will develop for the next observation period.

6. It is recommended that observations of the biofilms be done as much as your time allows. This study has been successfully carried out on A/B block scheduling. Observations of the column and biofilm were done once a week for 30 minutes. Within 2 weeks, several protists will be able to be identified. Have posters or books available for students to identify what they are seeing.

7. If more characterization is appropriate for the type of Monerans seen, Gram staining may be used on the biofilm. Have students observe the slides for protists before the Gram staining procedure begins. When completed, have students replace the stained slides with fresh ones. Have a disinfectant bucket to place the slides in for future cleaning.

8. The problem of study written for this lab may be altered to suit the needs of ecology or classification studies.

Background Information to Chemical Transformations Occurring in the Winogradsky Column

Oxygen that is initially present in the Winogradsky column diffuses into the lower region of the column. The newspaper added to the column is a source of carbon. Anaerobic bacteria are responsible for the breakdown of cellulose. Products such as organic acids, alcohols, and hydrogen will be made. These fermentation products along with calcium sulfate will be used by sulfate-reducing organisms to produce hydrogen sulfide. The bacteria that help produce hydrogen sulfide will make the soil appear blackish in the lower part of the Winogradsky column. When cellulose is decomposed, its products are fermented and will diffuse upward. Hydrogen sulfide will also diffuse upward from where it is made in the soil. Metabolic compounds made by bacteria will cause various organisms to accumulate as a result of the required conditions for growth and survival.

Anaerobic photosynthetic bacteria, Chlorobium and Chromatium use the sulfides produced. Chlorobium bacteria have green pigmentation while Chromatium has a purple pigment. By responding to gradients of light and sulfides in the column, the 2 types of bacteria can have their location in the column identified. Six weeks is usually not enough time to physically observe locations of these Monerans within the column, but hydrogen sulfide will certainly be produced that will give evidence that these organisms are beginning to reduce the sulfides within the column. There are various tests that can be performed to verify the presence of these sulfide-reducing bacteria.

In the upper areas of the column, especially where mud and water meet, hydrogen sulfide and oxygen are present. It has been reported that aerobic, sulfide-oxidizing organisms will be present. These organisms are responsible for the cloudiness seen in the water. Algae usually are present in the upper most portion of the column producing oxygen. It is in this area where protozoans, aerobic bacteria, and invertebrate larvae are found.

Nutrient cycling

Nutrients are being recycled in the column by all organisms living in the Winogradsky column. Within the column, photosynthesis and respiration are recycling carbon, hydrogen, nitrogen, sulfur, and oxygen. Bacteria ferment many of the organic acids. The fermented products can be further metabolized with the nitrates and sulfates produced. Since the soil used in this lab will most likely contain nitrogen-fixing bacteria, nitrogen fixation will occur along with nitrification and denitrification. Both nitrates and sulfates can be assimilated into the heterotrophs. Certain bacteria to that form hydrogen sulfide will also reduce sulfates.

Look for more detailed information on the Winogradsky column and biofilm.

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