The Winogradsky Column and Biofilms

The Winogradsky Column and Biofilms:

Student Sheet

Purpose:

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. You 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.

You will need to keep a journal of your observations and data. Every entry you make should include the date of study, qualitative, and quantitative data.

For this lab, you will set up a Winogradsky column and biofilm slides. You will then interpret the general chemical transformations that occur in the column as a result of color changes in the soil, identify common microorganisms, and determine the microbial composition of the column over specified intervals of time.

Problem: What types of microorganisms will be seen in the process of ecological succession as chemical transformations occur in a Winogradsky column over a six week time period?

Hypothesis:

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

Slide holders

Microscope

Rubber band

Procedure:

A.  Construction of the Winogradsky Column

1.  Have your teacher cut the neck from a 3-liter soda bottle.

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.  Using a large spoon, 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 the 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 throughout the study.

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 slides stand at room temperature for 6 weeks.

B. Observations of the Winogradsky Column and Biofilm

Keeping the cover on the column, note the appearance of the column and draw any changes in color patterns or growth that occur in the soil and water over the incubation period. Keep these in a journal. 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 of the soil

c.  Condensation on the plastic cover

d.  Crust forming in the bottle

e.  Film on the surface of the water

Observations to notice with the cover of the column off are the following. (Do not put your nose into the column for detecting odors. Use your hand to bring the air from the bottle up to your nose.)

a. Odors

b.  Film over the surface of the water

c.  Crust build up in the column

d.  Macroscopic organisms

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. If the slide has fallen into the column, use forceps or a slide holder to remove it.

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. 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. Notice the shape of the bacteria, and their colony formation. Record and illustrate what you see.

Log the population count of each type of organism that is seen on this date.

For algae and protozoans, note the shape of the organisms, their color, and structures for movement and be able to describe any behaviors they may have to light or to other organisms.

If bacteria shapes are hard to identify, use a drop of Methylene blue stain to help distinguish the shapes. If this procedure is used, do not place the stained slides back into the column. Replace them with fresh slides. Place the stained slides into the disinfectant bucket for cleaning.

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.

After one week has passed, observe the biofilm for protozoans first. Afterwards, stain the biofilm with Methylene blue to help in the identification of Monerans seen. When you finish with the slide, replace the stained slide with a clean one. Give your teacher the stained slide.

Your teacher may give directions for Gram stain procedures if more classification of bacteria is necessary. Use Gram stain procedures to determine if the Monerans seen are Gram + or Gram -. Do not return these slides in the column. Place the slides in the disinfectant bucket for cleaning. Replace the 4 slides with new slides. Position the slides into the column the same way as they were.

Continue microscopic and visual observation of the column and biofilms for 4 more weeks.

More Information on the Winogradsky Column

As oxygen diffuses downward from the surface of the water, fermentation products from the breakdown of cellulose and hydrogen sulfide move up to the top of the column. Oxygen conditions remain very poor at the bottom of the column. Bacteria that are present at the bottom of the column are mainly responsible for the decomposition of the cellulose found in the shredded paper. As the chemical products (organic acids, alcohols, hydrogen, and sulfates) are released from bacteria, they diffuse upward within the column and are responsible for groups of organisms to accumulate in response to their required conditions for survival. This will create a vertical distribution of organisms within the Winogradsky column.

Nutrient Cycling

Cycling of elements does occur as macro and microorganisms break down different organic substances found in the soil and the cellulose in newspaper. No extra newspaper to supply organic carbon will be added to the ecosystem like it is added in nature. In the column carbon, hydrogen, and oxygen are cycled mainly by photosynthesis and cell respiration.

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