H O W- T O - D O - I T

Exploring the Sulfur Nutrient Cycle Using the Winogradsky Column

BRIAN ROGAN MICHAEL LEMKE M I C H A E L L E VA N D O W S K Y THOMAS GORRELL

o fully understand the workings of the biological world, it is important that students have a fundamental sense of the natural cycles that provide the nutrients and energy that power life, as well as a sense of how these systems evolved. Many teachers cover carbon cycles and emphasize microbial processes when reviewing the complexities of nitrogen cycling, but often the sulfur cycle, if covered, is done so briefly. There may be many reasons for this: time limitations, the element is less prevalent than others as a biological constituent, or the topic is thought to be too complex. However, teaching the sulfur cycle in conjunction with the classic Winogradsky column exercise presents the opportunity to cover several important topics simultaneously. The exercise links microbial processes, concepts of biodiversity, inorganic chemistry, biogeochemical cycling, evolution, microbiology, and microbial ecol-

ogy to help meet the many demands and standards that are part of todays science classes. The Winogradsky column is a glass or clear plastic column, filled with enriched soil or sediment. When developed, it has an anaerobic lower zone and aerobic upper zone that allow growth of microorganisms under conditions similar to those found in sediments and water rich in nutrients (Sylvia et al., 1998). Often teachers simply convey the message that different microorganisms exist in different strata of the column and that some live in the aerobic and some in anaerobic zones. However, this is really where the discovery begins rather than ends! Explaining the complexity that lies within the depths of the ecosystem allows deeper insights into the microbial world. In the laboratory, the Winogradsky column demonstrates how the metabolic diversity of prokaryotes transforms sulfur, an essential constituent of living matter and an abundant element in the Earths crust (Stanier et al., 1976), to different forms with varying redox states, thus supplying nutrients and/or energy to the organism. The microbial assemblage that develops in the column spatially separates organisms into distinct layers several

BRIAN ROGAN teaches at The New Jewish High School, Waltham, MA 02453. MICHAEL LEMKE is in the Biology Department, University of Illinois at Springfield, Springfield, IL 62703-5407. MICHAEL LEVANDOWSKY and THOMAS GORRELL work at Haskins Laboratory, Pace University, New York, NY 10038-1598.

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centimeters thick even though in the environment establishment of similar layers of different organisms would typically exist in a few millimeters of sediment. The Winogradsky column creates conditions that expand the volume of natural processes, allowing a clear view of naturally-occurring phenomena. Soil samples are collected from wetland habitats, amended with simple inorganic and organic materials, then exposed to light as an external energy source. The results are a multicolored column of soil and water, each color linked to a chemical or biological process. The defined zones of microbes develop form according to concentration gradients of oxygen, sulfur, nutrients, and light. Each functional microorganism group is dependent on other functional microbial groups for development. The Winogradsky column was developed and named after Sergei Winogradsky (1856-1953), a Russian microbiologist. He studied the complex interactions between environmental conditions and microbial activities using soil enrichment to isolate pure bacterial cultures (Madigan et. al, 2000). Louis Pasteur, Robert Koch, and other scientists isolated cultures for study, but Winogradskys work was one of the first to study microorganisms in mixed enrichment cultures. The fact that the exercise works under a wide range of circumstances is a testament to the near ubiquity of certain functional groups of microbes. The Winogradsky column may also be used to demonstrate aspects of the earliest, sulfur-based life forms found on Earth. In an article in Nature, Nisbet (2000) paints a picture of an environment of early organisms in the Archean period 2.5 to 4 billion years ago that are analogous to those found in hydrothermal vents. Hydrothermal vents were first observed by the submersible vehicle Alvin that explored the Mid-Atlantic spreading ridge where the North American and European plates are inexorably moving apart. This observation marked the discovery of a system that may have remained intact since its formation as an ancient biotic system utilizing simple nutrient cycles as an energy source. In a sense, by creating a Winogradsky column, we are modeling ancient environments, though perhaps at lower temperatures. Constructing a Winogradsky column from simple homemade materials is only one of the exercises discussed here. We also present a technique that uses the respiration of seed germination to allow the reciprocal process, anaerobic metabolism, to occur in a simple glass baking dish. The objectives of this article are to: 1. describe the microbial ecosystem of the Winogradsky column as a tool for studying the cycling of sulfur 2. explain how use of the column may illustrate some features of development of early life on Earth

3. discuss how organisms may be isolated and grown in a homemade anaerobic chamber. Useful laboratory and educational extensions of the exercise are also discussed.

Materials & Methods

Materials needed for the construction of the Winogradsky column are simple and common. They include: a clear glass or plastic container (e.g., a smoothsided, quart plastic water bottle at least 15 cm in height and 5 cm in diameter. Plastic bottles are flexible and can be manipulated to allow for extraction of species for culturing. Very tall containers require longer development periods and the bacteria may be more difficult to extract.) plastic film and a rubber band a wooden dowel a sulfur source (e.g., calcium sulfate, magnesium sulfate, or egg yolk added at about 1-2% of the soil weight) an inorganic carbon source (calcium carbonate [e.g., chalk or limestone] or baking soda may be added to 1-2% of soil weight) hydric soil (e.g., pond mud or shallow river sediment collected near the surface) cellulose (e.g., shredded paper towels) a 60-75 watt light source water from the same source as the sediment Break up soil clumps and sieve out larger debris so the column can be packed evenly. The muddy mixture should be stirred to gain a uniform consistency and should include the sulfur and inorganic carbon sources. Place a 2-3 cm layer of the mud mixture in the column, add the source of cellulose, and stir and pack with the dowel. Add as much of the mixture as needed, 2-3 cm at a time, with gentle tamping with the dowel to force out trapped air, until the tower of mud is about 5 cm from the top of the container. Your last layer should be 2-3 cm of water. Cover the opening with plastic film and secure with a rubber band. Place the column next to a continuously-lit, low heat, moderate intensity light source, making sure the column does not overheat. Examine the columns weekly for at least a month, recording changes in color as they occur. For the Winogradsky column to be successful, enough time must be allowed for the cultures to develop. The columns may show growth in a week, as indicated by formation of a black color near the bottom and disintegration of the cellulose (paper), but will probably not fully form and stabilize for four weeks or more. The
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ideal situation would be for students to investigate the column over the course of the year studying ecology, microbiology, biodiversity, evolution, and other biological themes.

Isolation & Culturing of Organisms from the Winogradsky Column

When pigmented patches are visible in the column (Figure 1), one can attempt to isolate some of the organisms. Sampling can occur at weekly intervals to check succession or can be done at the end of the project to see the final flora of bacteria that develops. Sampling may be done using a standard bacteriological nichrome wire loop or hypodermic needle (pierced through the side of the plastic container), however distinction between the microbes and mud is often difficult. Look for Beggiatoa (Figure 2A) or Thiobacillus in the watersediment interface. Flagellated organisms from the column, such as Rhodopseudomonas (Figure 2B) or those with sulfur inclusion bodies, like Chromatium and Thiospirillium (Figure 2D) are more easily identified if you carefully adjust the contrast on a standard light compound microscope or have a phase-contrast microscope. The green sulfur (Figure 2C) and sulfate-reducing (Figure 2E) bacteria are more difficult to see. Another method is to plunge a microscope slide into the soil and allow growth of adherent biofilms to form on the glass, which is then easily examined under magnification (Anderson & Hairston, 2000). Growing microorganisms isolated from the diverse conditions of the Winogradsky column will be challenging, yet students interested in the complexity of the mechanisms underlying the fundamental processes of nature will want to explore this process. Several methods for culturing microorganisms from the column have been described and include pipetting mud from each colored zone to individual tubes for incubation with light (Benson, 2002) or separating the mud, layer by layer, and drawing off the liquid just above the layer for culturing or microscope observation (Atlas & Bartha, 1998). If the latter method is part of your plan, constructing a Winogradsky column out of a clear, smoothsided cylinder is very helpful. After removing the plastic wrap or plugs from the ends, the mud can be pushed out in a single unit and sampling can be easily done. In addition, specific microbiological media recipes (e.g., Atlas, 1995; DIFCO Laboratories, 1984) are available to establish enrichment cultures of some of the organisms. Although one can buy jars (e.g., GasPak system) and other materials (e.g., Brewers Petri plate, Wrights Tube; Harley & Prescott, 1999) designed specifically for anaerobic culture, we present here a method for culturing anaerobic organisms that can work well under classroom conditions using simple household items. The method is also intrinsically interesting for students because of the irony of pitting one biological process (i.e., seed respiration) to provide conditions for another

Variations
Construction and development of the Winogradsky column incorporates several variables. With just a few changes, different columns can be created to compare growth rates, microbial populations, and ecological diversity. Sulfur Source To illustrate the importance of the type of sulfur as a substrate, sodium sulfide or elemental sulfur can be added in place of a sulfate. This should reduce the growth of the sulfate-reducing bacteria and alter the composition of the microbial community. Acidity Acid affects the biotic component of our environment and alters its function. Changing the pH can affect which species grow and dominate. Many of the standard sulfur reducers are adapted to pH 6-8 (Madigan et al., 2000). Creating a more acidic or alkaline environment shifts the community composition and alters sulfur cycling. Heat Thermophilic bacteria are adapted to higher heat than most (i.e., mesophilic) bacteria. Mud from some sources (e.g., hot springs) may harbor thermo-tolerant or even thermophilic bacteria. If you put the column close to a light source that produces heat, these may grow. This could lead to a classroom discussion about thermal vent biological communities. Osmotic Stress or Marine Stimulation Columns with different salt concentration can illustrate several principles. If you begin with a freshwater or wetland source, salt can be a stressing agent favoring halophilic and halotolerant bacteria. On the other hand, you can show that nutrient cycles also occur normally in marine environments by simply collecting muds and water from a marine source and letting the column develop as described. Type of Light (Wavelength) Fluorescent lights, incandescent lights, or light filters (i.e., colored cellophane) that remove part of the spectrum from a light source could be used to select for organisms with different absorptive pigments.
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BIOLOGY ORGANISMS R E P R E S E N TAT I V E GENUS M E TA B O L I S M MORPHOLOGY various various various GRAM S TA I N N/A N/A N/A negative rods vibrio-spiral rods ovals or rods negative negative negative negative

Winogradsky Column

Chemistry

Low Air

Aerobic

Diatoms Cyanobacteria Protists

photosynthetic photosynthetic photosynthetic or heterotrophic filamentous

silica frustule singular or filament singular or simple colonies

Water Water-Soil Interface

Sulfur-oxidizing Bacteria Beggiatoa Thiobacillus Purple non-sulfur Bacteria Rhodospirilium Rhodopseudomonas Chromatium photoheterotrophic photoheterotrophic photoheterotrophic

Microaerophilic Micro-Aero Phillic

non-photosynthetic chemolithotrophic chemolithotrophic

Red-Violet Zone

Purple sulfur Bacteria Green sulfur Bacteria Chlorobium

Hydrogen Sulfide Concentration

photoheterotrophic

straight or curved rods chemoheterotrophic vibrio

negative

Green Zone

High Black Zone

Anaerobic

Sulfur-reducing Bacteria Non-sulfur Obligate Anaerobic Bacteria

Desulfovibrio

negative

Clostridium

fermentative

rods with endospores

positive

Figure 1.

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Diagram of a typical Winogradsky column showing zones of growth that correspond to oxygen and sulfide gradients. Organisms frequently found in the different microhabitats and their metabolic and other characteristics are shown in register with column zones.

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A.

B.

process (i.e., anaerobic respiration). This gives the teacher yet another chance to test the critical thinking of the student. The medium has the following components: 0.01% NaS9H2O (i.e., 0.01 g/100 ml water), 0.05% yeast extract, 0.05% sodium malate, 0.05% L-cysteine, and 1.5% agar in solution. This is most easily prepared by adding 0.01 g NaS9H2O, 0.05 g yeast extract, 0.05 g sodium malate, 0.05 g L-cysteine, and 1.5 g agar to 100 ml water. Adjust the pH to 7.3 using dilute acid or base (i.e., < 0.1 M NaOH or HCl) and bring to boil, then dispense ingredients in clean Petri dishes. Isolate mud from the red or green pigmented anaerobic or microaerophilic areas of the column and innoculate each to different plates by streaking. Place 1-2 cm layer of live oats or other seeds, such as grass seed, in a glass casserole dish, add enough water to moisten well, and cover with moist paper towels. Place the inoculated (streaked) Petri dishes on this layer. Cover the whole tray with a glass or plastic plate sealed around the edges with Vaseline to make an oxygen seal. Add a light source above the chamber to give light for photosynthesis (Figure 3). As the seeds germinate, they respire, and oxygen is depleted in the sealed incubation tray, and carbon dioxide is produced, making an appropriate atmosphere for anaerobic photosynthesizers. The media and chamber work as follows: the presence of sodium sulfide and cysteine (an amino acid with an exposed sulfhydryl group) helps maintain reducing conditions; it is also a key component for anoxygenic photo-

C.

D.

E.

Figure 2.
Drawings and photomicrographs of some representative organisms found in a typical Winogradsky column.Photomicrographs shown in E used with permission from N. Pfennig. A. Drawings by S.Winogradsky of the sulfur-oxidizing bacteria Beggiatoa. Fig. 1 shows drawings of the tip of a Beggiatoa alba filament that becomes depleted in sulfur globules (dots inside filament) over time: a) B. alba grown with sulfides, b) same species grown in low-sulfide water for 24 h, c) B. alba in low sulfide after 48 hours.Fig. 2: Beggiatoa media bacterial filament. Fig. 3: tip of B. minima. Fig. 4: degenerated B. alba lacking sulfur (Winogradsky, 1949). B. Non-sulfur purple bacteria Rhodopseudomonas (left) and Rhodospirilum (right) (Stanier et al., 1976). C. Green sulfur bacteria Chlorobium limicola (Stanier et al., 1976). D. Purple sulfur bacteria Chromatium (left) and Thiospirillium (right) (Stanier et al., 1976). E. Sulfate-reducing bacteria Desulfovibrio desulfuricans (left) and Desulfonema limicola (right) (Madigan et al., 2000).

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synthesis in these microbes (i.e., a source of reduced sulfur or electron donor, for some of the green sulfur bacteria). Certain red anaerobic photosynthesizers can use organic compounds, such as malic acid, as electron donors, so this medium serves double duty for this type of culture enrichment. The generalized anaerobic process is: CO + 2H X ____> (CH O) + 2X + H O
2 2 2 2

lished themselves as symbionts inside the cells of a primitive eukaryote. A little deeper in the column, hydrogen sulfide gas (H2S) is diffusing upward into the aerobic zone. Part of the sulfur cycle is evident here. The H2S gas has been produced by anaerobic microorganisms near the bottom of the column. These organisms reduced the sulfate originally mixed into the soil. Near the top of the column, the H2S can be oxidized back to sulfate by the sulfur-oxidizing bacteria, such as the genera Beggiatoa and Thiobacillus (Atlas & Bartha, 1998). These bacteria are chemoautotrophs and gain energy from the oxidation of reduced sulfur to elemental sulfur or to sulfate and they can also synthesize organic compounds autotrophically from CO2. Thiobacillus oxidizes sulfur while Beggiatoa is sulfide-oxidizing.

where X stands for some reducing agent (or none at allsome anaerobic bacteria can simply use H2 by itself). This metabolic reaction is thought to be ancestral in the sense of biochemical evolution to the familiar photosynthetic process in modern oxygenic photosynthesis, in which X has become oxygen.

Discussion
Relating Microbiology to Nutrient Cycling in the Column
After a month to six weeks, the column should stabilize into three distinct zones and develop communities of bacteria specific to their environmental requirements (Figure 1).

Microaerophilic Zone
The diffusion of H2S from the sediment below enables anaerobic photosynthetic bacteria (which typically appear in brightly colored bands) to grow. From bottom to top, green sulfur bacteria (GSB), such as Chlorobium, create an olive-green color zone. Purple non-sulfur bacteria (PNSB), such as Rhodospirilum and Rhodopseudomonas, usually require a small amount of oxygen and are located nearer to the top of column than are the GSB. Growth of these organisms results in a dark red-rust color. The metabolism of both GSB and PNSB provides an excellent opportunity to draw comparisons between oxygenic photosynthesis (oxygen producing, like green plants) and anoxygenic photosynthesis (non-oxygen producing organism that pre-dated green plants). GSB and PNSB gain energy from light reactions and metabolize CO2 in the same way as plants do. Yet, because they use H2S instead of water as the source of hydrogen (reducing power), they produce a more oxidized sulfur product (Atlas & Bartha, 1998). Consider plant photosynthesis (i.e., 6CO2 + 6H2O => C6H12O6 + 6O2) expressed as CO2 + H2O ___> [CH2O] + O2. Then it is easy show the parallel processes between photosynthesis and hydrogen sulfide oxidation (Atlas & Bartha, 1998) as: CO + H O __> [CH O] + O
2 2 2 2

Aerobic Zone
The top of the water column can contain large populations of diverse bacteria and protists. These are aerobic organisms found in organic-rich freshwater habitats such as shallow ponds and polluted streams. The bacteria are often flagellated, allowing them to migrate and establish themselves in new areas (Madigan et al., 2000). In addition, there may be larger protozoa and invertebrates from the original water and mud source. At the very top of the zone the mud is the most oxygen-rich part of the column, often colored a lightbrown from iron-oxide precipitate. Oxygen-producing organisms, such as the photosynthetic cyanobacteria, often grow above the mud, forming a green zone. These are the only bacteria that have photosynthesis like that of plants. In fact, there is very strong evidence that the chloroplasts of plants originated from ancestral cyanobacteria that estab-

Figure 3.
A simple, yet effective, anaerobic chamber can be constructed from common household items as depicted in this diagram.

(plant photosynthesis) CO + H S __> [CH O] + S

2 2 2

(bacterial anoxygenic photosynthesis)

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Anaerobic Zone
Organisms that grow in anaerobic conditions ferment organic matter or perform anaerobic respiration. Fermentation is a process in which organic compounds are degraded incompletely; for example, yeasts ferment sugars to alcohol. Anaerobic respiration is a process in which a substance other than oxygen is the terminal electron acceptor. Three primary strata form in the lower level of the column. The uppermost anaerobic layer often contains Gallionella and other iron-oxidizers. Enrich for these by adding a source of iron (i.e., a nail or piece of steel wool). If you isolate Gallionella and look after closing down the condenser iris (or using a phase-contrast scope), you will see cells and stalks that appear as twisted threads. These organisms oxidize iron and produce a rust-colored iron oxide layer. Moving deeper into the column, purple sulfur bacteria, such as Chromatium, may be found and these bacteria produce a red-to-purple layer in the soil. Purple sulfur bacteria reduce sulfates to sulfur; a type of metabolism that emerged on Earth early in the planets history. From an evolutionary standpoint, there is strong evidence that the mitochondria of present-day eukaryotes were derived from the purple bacteria (Margulis et al., 1986). In the deepest layers are obligate anaerobes that scavenge and metabolize sulfur and carbon in ways we do not often discuss in the science classroom. Sulfurreducing bacteria like Desulfovibrio utilize fermentation products (see below) in anaerobic respiration, using either sulfate or other partly-oxidized forms of sulfur (e.g., thiosulfate) to produce the H2S gas that diffuses through the column. The H2S spontaneously complexes with iron to form a black ferrous sulfide (FeS). This is why lake sediments (and our household drains) are frequently black. Some of these organisms are anaerobic cellulosedegraders, such as Clostridium, that grow when oxygen is depleted in the sediment. Though Clostridia cells are killed by exposure to oxygen, these organisms produce spores that can survive aerobic conditions (Madigan et al., 2000). They degrade the cellulose to glucose and then ferment the glucose to gain energy, producing a range of simple organic compounds (e.g., ethanol, acetic acid) as the fermentation end products. Sometimes, at the very bottom of the column, depending on the source of the mud, a pink layer will develop due to purple sulfur bacteria with gas vesicles. A characteristic species is Amoebobacter, which also photsynthesizes using H2S (Atlas & Bartha, 1998).

The Sulfur Cycle

Sulfur is an assimilable, nutritional requirement for most life, and yet represents a energy conduit, or dissimilatory pathway, for some microorganisms. As a nutrient, it is a component of several amino acids required for protein synthesis, and a number of other important biochemical components of the cell. Its chemical versatility, or ability to exist in several oxidation states (Figure 4), makes it a significant part of the energy cycles of many organisms. Because of this range of oxidation states, sulfur compounds can act as electron acceptors and donors. As electrons move from one molecule to another they also lose or gain energy. Thus the many microbial transformations of sulfur compounds form a basic part of energy metabolism. A number of species can use elemental sulfur anaerobically as a terminal electron acceptor (the usual role of oxygen in aerobic respiration), reducing sulfur to hydrogen sulfide (H2S). Others can use thiosulfate or sulfate as an electron receptor. A number of sulfur pathways exist in the column (Figure 4). There are numerous species in this environment beyond those mentioned, and each has its own unique contribution to the sulfur cycle.

Additional Questions
Other areas can be investigated as an activity with students. The column is, in fact, almost limitless as a source of questions and projects 1. If iron is increased, how might that affect growth of the organisms in the column? 2. Are there methanogens (i.e., microorganisms that produce combustible methane) in the column? How would they be detected? (Hint: Use a lighted match to check the head space or gas of an old column). 3. What would be the effects of manipulating the pH? Adding salt? Adding organic nutrients? Manipulating the temperature? Manipulating the spectral influx in the light source with filters?

Conclusions
The Winogradsky column is a complex system and an excellent example of an investigation that can span the level from guided inquiry all the way to open-ended projects that can occupy students imaginations and studies for months. It is also a window into the biodiversity of our world. The Winogradsky column is an excellent way to show students that not all bacteria are pathogens and they have an important role in the geochemical cycling of the biosphere, one they have been fulfilling since life first began nearly four billion years ago.

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Figure 4.
The sulfur cycle emphasizing the transition in oxidation state of the different sulfur compounds (after Fenchel et al., 1998) and the bacteriamediated processes that occur in aerobic or anaerobic habitats.

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Acknowledgments
We wish to extend a special thank you to Dr. Norbert Pfennig for his permission to use his photomicrographs in this publication and to Sherry Hutson (UIS) for graphic design assistance. We wish to thank the 2000 Woodrow Wilson National Fellowship Foundation Summer Leadership Institute for Teachers that focused on biodiversity for creating the forum for learning and interaction; one of the many end products being this article.

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References
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