Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Cut the tops off the plastic bottles with a scissors or other cutting device. Make the cut close to the top of the bottle so as much of the length of the bottle is preserved as possible.
2. Mud samples need to be collected prior to the laboratory. Be sure to clear any required permission at the collection sites you select. Collecting samples from more than one site will create a diversity of initial nutrients and microbe populations. You can collect samples for the entire class, have students collect samples and bring them to class or take a field trip to collect the samples. Your local resources, course goals and school location will dictate how you do the collecting.

Safety Precautions

Students should wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. The mud mixture materials should be handled carefully. Provide facilities for students to wash thoroughly upon completion of laboratory work.

Disposal

All Winogradsky mud components can be disposed of following Flinn Suggested Disposal Method #26a or they can be returned to the original collection site. Columns can be thoroughly washed and reused a number of times.

Teacher Tips

• Enough materials are provided in this kit to set up 10 Winogradsky columns. Students can work in groups as appropriate to your class size. Initial setup of the column can be completed in one 50-minute class period. You will need to allow class time on successive days to observe the columns. You can schedule specific time frames for the observations or you can monitor the columns and schedule observation time when interesting changes have occurred. Recommended observation times are after approximately 2 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months and 3 months.

• Soil (mud) samples must be collected. Samples taken from several different places will increase the probability of columns producing a variety of organisms and interesting color changes. You can collect the samples and mix them all together and then have students take samples from the entire mixture or students can collect samples and make their own “brew.”
• Columns should be placed in a well-lighted area. The columns should get good consistent light but not be in direct sunlight if the activity is done during very warm weather. The temperature inside the bottle might get high enough to kill most organisms.
• Each Winogradsky column is unique and will produce its own results. The columns are ideal for inquiry-based lab investigations. Students can be imaginative in where samples are collected and what is placed in columns as a cellulose source. The variety of organisms and pigmentation in the column varies depending upon the nature of the soil and the nutrients contained in the sediments. Hence, there is no predetermined result or right answer for a Winogradsky column.
• Some columns may develop a strong odor over a period of time. This should not go unnoticed in student observations. What is occurring differently in the columns that smell? If the smell is too strong, the columns may have to be housed in a more ventilated area outdoors. Disposal should also be done outdoors rather than in the school garbage.

Sample Data

Discussion

The Winogradsky column is a miniature ecosystem in which microorganisms and nutrients interact over time. The mud in the column develops several gradients:

1. An oxygen gradient—Air dissolves into the water at the top of the column and supplies oxygen to the top layer of mud. There is less oxygen deeper within the column. Ideally, the bottom of the column will be oxygen-depleted, providing an environment for anaerobic bacteria.
2. A sulfur gradient—Some bacteria use hydrogen sulfide gas as part of their photosynthetic process and some hydrogen sulfide is released naturally as organic matter decays. The sulfur concentration will be greatest at the bottom of the column and decrease further up the column.
3. Light gradient—Some bacteria grow in response to light, while other bacteria grow best in dark environments. The storage position of the column relative to the predominant light source will likely determine the position of the color bands noted in the column.

After the column is made and placed near a light source, microbes begin to flourish in their compatible niches. Cyanobacteria and algae grow in the water above the column and, by producing oxygen, help to keep this zone aerobic. This watery top layer will contain a diversity of algae, cyanobacteria, aerobic bacteria, fungi and protozoa. Degradation of the cellulose source in the mud by anaerobic bacteria will fairly quickly lead to the production of organic acids, alcohols, and hydrogen gas. These compounds are suitable substitutes for growth of sulfate-reducing bacteria, like Desulfovibrio, which produces hydrogen sulfide. The appearance of blackened areas in the lower portion of the column signals the presence of these bacteria.

Along with the production of sulfide, notice green and purple patches appearing on the deep outer layer of the mud exposed to light. The green sulfur bacterium Chlorobium grows in the lower part of the column, and above that grows the purple sulfur bacterium Chromatium. Higher in the column, where there are fewer sulfides purple non-sulfur bacteria Rhodopseudomonas and Rhodospirillum can grow. These organisms produce rust-colored patches in the transition zone between aerobic and anaerobic conditions. All the patches of color are best observed with intense sunlight or a bright light shining at various angles onto the column.

Because mud sources vary, the time it takes for the column to flourish will vary as well. Mud from the edge of wetlands works best. Color starts to appear in a week, but it may take three to four weeks for really interesting bands of color to appear.

In addition to watching microbial colonies grow, students will smell the results of the microbial action. They may smell methane and nitrous oxide. Hydrogen sulfide gas produces a very unpleasant odor.

Introduction

Ever wade through a swampy wetland area? You may not have known it at the time, but were right in the middle of one of the most productive ecosystems in the world! Conduct this experiment and discover a biosphere in a bottle.

Concepts

• Aerobic vs. anaerobic

• Bacterial photosynthesis
• Succession
• Biofilms

Background

Microorganisms can be found everywhere. But just like plants and animals, different types of microorganisms can survive in many different places. Though difficult to see, pigmented photosynthetic bacteria are found in the soil. They are especially abundant in muddy, wetland areas where conditions are ideal for their rapid growth and reproduction. Different photosynthetic bacteria have different pigments, each absorbing different wavelengths of light. These soil microbes also have varying requirements for oxygen. Some soil microbes are aerobic (requiring oxygen for survival) and some are anaerobic (not requiring oxygen). The top layer of soil where oxygen (O₂) can diffuse is called the aerobic zone. Lower layers in the soil, where hydrogen sulfide (H₂S) is prevalent, are called the anaerobic zone.

Photoautotrophs use light as a source of energy and carbon dioxide as their chief source of carbon. The process by which photoautotrophs transform CO₂ into carbohydrates is called photosynthesis. Photosynthesis is often described as occurring in two phases: a light reaction and a dark reaction. In the light reaction, light energy is converted into chemical energy (ATP) using light-trapping pigments. Carbon dioxide is then reduced to a carbohydrate during the dark reaction. Carbon dioxide reduction (also called carbon dioxide fixation) requires an electron donor and energy.

Chlorophyll a is used in green plants, algae and cyanobacteria to generate ATPs. The resulting oxygen is produced by oxidation of the electron donor, water. The reaction can be summarized as:

Most photosynthetic bacteria use bacteriochlorophylls to generate electrons for ATP synthesis and use sulfur-containing compounds, hydrogen gas, or organic molecules as electron donors. This reaction is often summarized as:

Quite often the sulfide ions donate electrons during photosynthesis and produce sulfates. Bacterial photosynthesis differs from green plant photosynthesis in that bacterial photosynthesis occurs in an anaerobic environment and does not produce oxygen.

Photosynthetic bacteria contain bacteriochlorophylls and may appear to be brown due to the presence of red accessory pigments called carotenoids. Purple photosynthetic bacteria also contain large amounts of carotenoids. In a column of soil, conditions vary from aerobic at the top to anaerobic at the bottom. If the column is created in a clear container, the various conditions and resulting colored organisms can be observed. The soil column created in this activity is modeled after a study method first created by a Russian microbiologist, Sergei Winogradsky. Winogradsky created long clear soil columns in order to study soil microorganisms. His method became known as the Winogradsky column.

In natural aquatic environments one may encounter a phenomenon known as biofilms. Biofilms form rapidly in fluid environments and increase over time, especially in nutrient rich areas. The development of biofilms is initiated by microorganisms forming a monolayer attachment on any submerged surface. Over time, this film becomes more complex as layers of different types of organisms colonize the surface. Depending upon nutrients, a tremendous variety of organisms can grow in the film area. Biofilms have been rigorously studied recently because of their huge economic impact on water systems, medical equipment, transport systems, and other key environments. Controlling microorganisms and eliminating biofilms is a significant problem and often a big challenge.

Materials

Safety Precautions

Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Handle the soil samples carefully since they contain many microbes. Wash thoroughly when the lab work is completed.

Procedure

1. Estimate an amount of soil or mud needed to fill the Winogradsky column about ⅔ full. Remove rocks, twigs and any other non-soil items. Place the collected soil into a bucket and mix thoroughly with a wooden stir stick. Add 5 g of calcium carbonate, 5 g of calcium sulfate and a handful of shredded newspaper or other cellulose materials and stir completely.
2. Slowly add water to the mixture, while stirring, until it gets to be a mixture that can be poured like thick concrete.
3. Pack the semi-liquid mud into the column until it is about ⅔ full. Leave at least 3" above the mud at the top of the column.
4. Pour additional water slowly down the side of the column on top of the mud so that there is about a 2-inch layer of water above the mud column. Gently tap the entire column on the table to remove as many air bubbles as possible. Do not tap too hard and create a mess!
5. Add six clean microscope slides standing on end to the top of the chamber. Set them vertically on top of the mud but submerged in the water. Make sure that the slides are submerged about ⅔ of their length.
6. Mark the water level of the column and cover the top with aluminum foil to help prevent evaporation. You may need to replace water to the original water level, over a period of time.
7. Place the completed Winogradsky column in an environmental chamber where a light will shine on it 24 hours a day. If this is not possible, place it in a greenhouse, on a window sill, or any place that receives good light on a regular basis. Your instructor will guide you in placing your column.
8. Observe the Winogradsky column for an extended period of time as directed by your instructor. Each time an observation is made:

1. Remove a microscope slide. Wipe one side clean (since microbes will colonize both sides). Place a coverslip on the slide and observe under a microscope. High power magnification will be required to observe the bacterial microbes. Slides can be stained with Gram stain or methylene blue to make bacteria more visible. If slides are not stained, they can be returned to the column after observation.
2. Record all observations on the Winogradsky Column Worksheet. Use additional paper to draw sketches of all observed organisms. Record general observations of the column over a long period of time. Note the appearance of the column. Draw and label pictures of your observations noting any color changes or growth that occurs at various places in the column. Some bacteria will bloom and then disappear over time.

9. Consult with your instructor for appropriate disposal procedures.

Student Worksheet PDF

10342_Student1.pdf