The Search for Microbial Biodiversity: Using a Winogradsky Column to Isolate Bacteria from Aquatic Sediment
 

An inquiry project on Biodiversity
Grades 9 through 12
Submitted by Ann-Marie McCoy, James Perlmutter, Cynthia Shorter, and Glenn Zwanzig

1999 Woodrow Wilson Biology Institute
 

Abstract

Aquatic sediment from ponds and rivers was collected, mixed with paper clippings and calcium sulfate, and transferred to Winogradsky columns. The mixture of sediment was placed under lighted conditions to accelerate growth of phototrophic aerobic and anaerobic microorganisms. Organisms were extracted, isolated, and identified using biochemical and staining techniques to determine the genus of unknown isolates.
 

Introduction

Biodiversity, a concept used to describe the range of living organisms in a given area, takes into consideration the variety of life forms, the genes they contain, and the ecosystems they form. Life forms within an ecosystem vary in their size and shape from the simplest unicellular prokaryote, the bacteria, to the more complex multicellular eukaryotic organisms, such as elephants or bottlenose dolphins. Each organism plays an important role and contributes to ecosystem stability. Each type of environment, aquatic or terrestrial, will have its own characteristic type of microorganisms or microbes.
 
 

In an aquatic environment such as a river, pond, or lake, several habitats exist. One type of habitat is the bottom, or benthic zone, of an aquatic environment. The sediment contains a variety of microscopic and macroscopic organisms dwelling on or within the substrate. Microbes of the benthic substrate vary in the roles they play within the aquatic community. Some of those roles include the recycling of materials through nitrification, proteolysis, nitrogen fixation, and sulfur reduction, all of which provide the vital elements for other organisms.

During the 1880s, Sergei Winogradsky developed a device that allowed the study of microbial communities in aquatic and soil environments (Tortora, Funke and Case, 1998). This cylinder-like device, now known as the Winogradsky column, provides an environment where anaerobic and aerobic organisms can be grown, isolated, and studied while exposed to varying amounts of light and oxygen. Biodiversity within the column becomes apparent as the column ages and many microbial colonies become visible, forming various regions of color. Aerobic organisms gradually reproduce and colonize the upper regions of the Winogradsky column. Algae, aerobic sulfur bacteria, and cyanobacteria are found in the upper layer of the column where oxygen is available. Descending down the column, the quantity of oxygen gradually decreases changing from an aerobic to an anaerobic environment. Organisms that require environments low or without oxygen colonize the lower regions of the column. In the upper regions of the anaerobic zone of the column, purple non-sulfur bacteria, which appear reddish purple to rust, and purple-sulfur producing bacteria, which appear in violet patches, can be seen. In the lower anaerobic regions, where the highest concentration of hydrogen sulfide is located, green sulfur bacteria appear as green patches. Green sulfur bacteria are resistant to high hydrogen sulfide concentrations typically found in these regions of the Winogradsky column.

This inquiry-based laboratory activity investigates the biodiversity found in microbial systems taken from a large urban pond, a small urban pond, and a large river using the Winogradsky column as an instrument to grow phototrophic aerobic and anaerobic microorganisms. Bacteria will be isolated, cultured, and identified using a variety of morphological and staining tests.
 

Methodology
 

Making a Winogradsky Column
Any type of tall, clear container can be used for a Winogradsky column (Figure 1).
 

After collecting the mud samples from the three sites using a Ekman dredger, each column was prepared by adding cellulose and sulfur. In this investigation, paper towels were cut into small strips. Two grams of paper per 500-milliliter container was mixed with the mud. One gram of calcium sulfate was added to the mud as the sulfur source. An alternative to calcium sulfate that works well is an egg yolk from a hard-boiled egg. After mixing, the materials were placed into clear plastic containers. Two liter soft drink bottles work perfectly for this. Water was added to the top of the containers to keep the soil moist and decrease air exchange. The containers were sealed using plastic wrap, secured with rubber bands, and placed in front of a light source as illustrated in the picture above.

After a period of several weeks patches of red, green, and purple appeared at different depths of the column indicating the enriched media of cellulose and sulfur provided a suitable environment to colonization of aerobic and anaerobic bacteria (Figure 2).

Photo courtesy of Mudnet, University of Texas

 

                                                            Figure 2: Example of Winogradsky column.
                                                            Green patches are green sulfur bacteria.  The
                                                            rust patches are colonies of purple non sulfur
                                                            bacteria.  The red patches are purple sulfur
                                                            bacteria.

Using the aseptic technique, slides were prepared for Gram staining and endospore staining.
 

Gram Stain: The Gram technique involves the application of two dyes, crystal violet and safranin. If the cell wall is composed of peptidoglycan, crystal violet would adhere to the molecules and will appear violet purple in color. If the cell wall contains little or no peptidoglycan, the crystal violet will decolorize and a counterstain of safranin is used.
 
 

Endospore Stain (Schaeffer-Fulton stain): The unknown isolate underwent additional staining due to the question of the clear bodies that were present in the Gram stain. These clear bodies, believed to be endospores, are not uncommon in Gram positive rods. Endospores protect bacteria when conditions become unfavorable for growth. Following the staining technique indicated in the laboratory manual, it was determined that the unknown isolate did contain endospores. See figure 5.

Biochemical Testing to Determine Unknown Isolates

Upon isolating and studying the morphology of the unknown microorganism from the Winogradsky column, a battery of biochemical tests were performed to determine the type of metabolic processes the unknown bacteria underwent. These tests were used to help ascertain the identity of the unknown isolate.

These tests included:

Fermentation of carbohydrates: lactose, sucrose, and dextrose

Catalase Production

DNA Digestion

Hydrogen Sulfide Production

Indole Production

Urea Digestion

Lipid Digestion

Casein Digestion
 

Although the protocol for these biochemical tests follow the method of Alcamo (1997), they can be found in any microbiology laboratory manual (for example, Johnson and Case, 1995).

Fermentation of Carbohydrates: Three test tubes containing 0.5% solution of dextrose, sucrose or lactose, nutrient broth, and a Durham tube were inoculated with the unknown isolate and incubated at room temperature for 24 hours. Yellow color indicates acid production and air in the Durham indicates gas production.

Hydrogen Sulfide Production: A Sulfide Indole Motility medium containing the amino acid cysteine and iron ions was inoculated with a straight wire inoculating needle and the unknown isolate. The tube was incubated at room temperature for 24 hours. Colony production and growth indicates motility. An iron precipitate indicates hydrogen sulfide production.

Catalase: Unknown isolate was grown on a nutrient agar plate for 24 hours at room temperature. Several drops of hydrogen peroxide was placed onto the culture. Production of oxygen bubbles indicates a positive response.

Casein: A petri dish of skim milk agar was inoculated with isolate and incubated for 24 hours at room temperature. Clear regions around the colonies indicated casein digestion.

DNA Digestion: A DNAase agar plate was inoculated with isolate and incubated for 24 hours at room temperature. The agar plate was flooded with 1N hydrochloric acid. A cloudy precipitate indicates a negative digestion of DNA.

Urea Digestion: A test tube containing nutrient broth, urea, and phenol red was inoculated with the unknown isolate and incubated at room temperature for 24 hours. A fuchsia color indicates the digestion of urea.

Indole Production: A test tube containing tryptone, a tryptophan-rich medium, was inoculated with the unknown isolate and incubated at room temperature for 24 hours. Twenty drops of Kovac’s reagent was added following incubation. A red ring in the top of the media indicates a positive test.

Table 1 summarizes the results of biochemical tests performed on the unknown isolate. These tests helped to ascertain the identity of the unknown isolate.
 

Gram Stain and Endospore Stain: The unknown isolate was Gram stained and examined at 1000x using oil immersion. It was apparent that the isolate absorbed the crystal violet and did not decolorize, indicating a Gram positive organism. This technique also indicated that the cell wall of the organism is composed primarily of peptidoglycan and has a distinct rod shape (Figure 6). Because of the bipolar staining pattern in the unknown isolate, an endospore stain was performed. The unknown isolate’s endospores absorbed the malachite green and were easily observed (Figure 5).
 
 

Conclusion

From the morphology studies and the biochemical tests, it has been determined that the unknown isolate is from the Genus Bacillus. This genus has many species that are known for their rhizoid colonies, endospores, motility, and production of catalase. Bacillus cereus var. mycoides, a common soil bacterium, is well known for its unusual rhizoid colonies.

The Winogradsky column takes several weeks for microbial colonies to reach their full potential. Within one week of constructing the columns, reddish colonies were visible. By the end of the second week, purple sulfur bacterial colonies became apparent in the anaerobic regions of the columns. Several days later green patches of green sulfur bacteria appeared in the lowest region of the column. Unfortunately, there was a limited amount of time available to allow for the columns to reach their potential.
 
 

Bibliography:

Suggested laboratory manuals:

Alcamo, I. E., Laboratory Fundamentals of Microbiology. 5^th Ed. Addison Wesley

Publishers, 1997.

Johnson. Ted and Case, Christine. Laboratory Experiments in Microbiology,

Benjamin/Cummings Publishing Company, 1995.

Textbooks:

Tortora, G. B. Funke, and C. Case. Microbiology: An Introduction, 6^th ed. Menlo

Park:Benjamin/Cummings, 1998.

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