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

T o fully understand the workings of the biolog-

ical world, it is important that students have a fun-
damental sense of the natural cycles that provide the
ogy to help meet the many demands and standards
that are part of todays science classes.
The Winogradsky column is a glass or clear
nutrients and energy that power life, as well as a
plastic column, filled with enriched soil or sedi-
sense of how these systems evolved. Many teachers
ment. When developed, it has an anaerobic lower
cover carbon cycles and emphasize microbial
zone and aerobic upper zone that allow growth of
processes when reviewing the complexities of nitro-
microorganisms under conditions similar to those
gen cycling, but often the sulfur cycle, if covered, is
found in sediments and water rich in nutrients
done so briefly. There may be many reasons for this:
(Sylvia et al., 1998). Often teachers simply convey
time limitations, the element is less prevalent than
the message that different microorganisms exist in
others as a biological constituent, or the topic is
different strata of the column and that some live in
thought to be too complex. However, teaching the
the aerobic and some in anaerobic zones. However,
sulfur cycle in conjunction with the classic
this is really where the discovery begins rather than
Winogradsky column exercise presents the opportu-
ends! Explaining the complexity that lies within the
nity to cover several important topics simultaneous-
depths of the ecosystem allows deeper insights into
ly. The exercise links microbial processes, concepts
the microbial world.
of biodiversity, inorganic chemistry, biogeochemical
cycling, evolution, microbiology, and microbial ecol- In the laboratory, the Winogradsky column
demonstrates how the metabolic diversity of
prokaryotes transforms sulfur, an essential con-
stituent of living matter and an abundant element in
BRIAN ROGAN teaches at The New Jewish High School,
Waltham, MA 02453. MICHAEL LEMKE is in the Biology
the Earths crust (Stanier et al., 1976), to different
Department, University of Illinois at Springfield, Springfield, forms with varying redox states, thus supplying
IL 62703-5407. MICHAEL LEVANDOWSKY and THOMAS GORRELL nutrients and/or energy to the organism. The micro-
work at Haskins Laboratory, Pace University, New York, NY bial assemblage that develops in the column spatial-
10038-1598. ly separates organisms into distinct layers several

348 THE AMERICAN BIOLOGY TEACHER, VOLUME 67, NO. 6, AUGUST 2005
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 sam-
ples 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 mul-
ticolored column of soil and water, each color linked to
a chemical or biological process. The defined zones of
microbes develop form according to concentration gra-
dients of oxygen, sulfur, nutrients, and light. Each func-
tional 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 inter-
actions between environmental conditions and micro-
bial activities using soil enrichment to isolate pure bac-
terial 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 cer-
tain 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 ener-
gy source. In a sense, by creating a Winogradsky col-
umn, we are modeling ancient environments, though
perhaps at lower temperatures.
Constructing a Winogradsky column from simple
homemade materials is only one of the exercises dis-
cussed 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 smooth-
sided, 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 con-
tainers 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 sedi-
ment 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 disinte-
gration of the cellulose (paper), but will probably not
fully form and stabilize for four weeks or more. The
SULFUR NUTRIENT CYCLE 349
ideal situation would be for students to investigate the
column over the course of the year studying ecology,
microbiology, biodiversity, evolution, and other biologi-
cal themes.
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 bacte-
ria and alter the composition of the microbial
community.
Acidity
Acid affects the biotic component of our environ-
ment 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 commu-
nity 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 ther-
mo-tolerant or even thermophilic bacteria. If you
put the column close to a light source that pro-
duces heat, these may grow. This could lead to a
classroom discussion about thermal vent biolog-
ical 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 stress-
ing 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 col-
umn 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.
350 THE AMERICAN BIOLOGY TEACHER, VOLUME 67, NO. 6, AUGUST 2005
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 organ-
isms. 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 water-
sediment interface. Flagellated organisms from the col-
umn, 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 micro-
scope. The green sulfur (Figure 2C) and sulfate-reduc-
ing (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 magnifi-
cation (Anderson & Hairston, 2000).
Growing microorganisms isolated from the diverse
conditions of the Winogradsky column will be chal-
lenging, yet students interested in the complexity of the
mechanisms underlying the fundamental processes of
nature will want to explore this process. Several meth-
ods 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, con-
structing a Winogradsky column out of a clear, smooth-
sided 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 anaer-
obic culture, we present here a method for culturing
anaerobic organisms that can work well under class-
room 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
 BIOLOGY

ORGANISMS R E P R E S E N TAT I V E M E TA B O L I S M MORPHOLOGY GRAM

Winogradsky Column
GENUS S TA I N
Chemistry
Diatoms various photosynthetic silica frustule N/A
Low Cyanobacteria various photosynthetic singular or filament N/A
Air Protists various photosynthetic or singular or simple

Aerobic
heterotrophic colonies N/A

Sulfur-oxidizing Beggiatoa non-photosynthetic filamentous negative

Water
Bacteria chemolithotrophic

Phillic
Thiobacillus chemolithotrophic rods negative
Water-Soil Interface
Purple non-sulfur Rhodospirilium photoheterotrophic vibrio-spiral negative
Bacteria Rhodopseudomonas photoheterotrophic rods negative

Microaerophilic
Micro-Aero
Red-Violet Zone
Purple sulfur Chromatium photoheterotrophic ovals or rods negative
Bacteria

Green sulfur Chlorobium photoheterotrophic straight or curved negative

Hydrogen Sulfide Concentration

Bacteria rods
Green Zone
Sulfur-reducing Desulfovibrio chemoheterotrophic vibrio negative
Bacteria

Anaerobic
High Black Zone Non-sulfur Clostridium fermentative rods with positive
Obligate Anaerobic endospores
Bacteria

Figure 1.
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.

SULFUR NUTRIENT CYCLE

351
 process (i.e., anaerobic respira-
A. B. tion). This gives the teacher yet
another chance to test the crit-
ical thinking of the student.
The medium has the fol-
lowing 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,
C. D. 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
E. 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 plas-
tic plate sealed around the
Figure 2. edges with Vaseline to make an
Drawings and photomicrographs of some representative organisms found in a typical Winogradsky oxygen seal. Add a light source
column.Photomicrographs shown in E used with permission from N. Pfennig. above the chamber to give light
for photosynthesis (Figure 3).
A. Drawings by S.Winogradsky of the sulfur-oxidizing bacteria Beggiatoa.
As the seeds germinate, they
Fig. 1 shows drawings of the tip of a Beggiatoa alba filament that becomes depleted in sulfur
respire, and oxygen is depleted
globules (dots inside filament) over time: a) B. alba grown with sulfides, b) same species in the sealed incubation tray,
grown in low-sulfide water for 24 h, c) B. alba in low sulfide after 48 hours.Fig. 2: Beggiatoa and carbon dioxide is pro-
media bacterial filament. Fig. 3: tip of B. minima. Fig. 4: degenerated B. alba lacking sulfur duced, making an appropriate
(Winogradsky, 1949). atmosphere for anaerobic pho-
B. Non-sulfur purple bacteria Rhodopseudomonas (left) and Rhodospirilum (right) (Stanier et al., tosynthesizers.
1976). The media and chamber
C. Green sulfur bacteria Chlorobium limicola (Stanier et al., 1976). work as follows: the presence
of sodium sulfide and cys-
D. Purple sulfur bacteria Chromatium (left) and Thiospirillium (right) (Stanier et al., 1976).
teine (an amino acid with an
E. Sulfate-reducing bacteria Desulfovibrio desulfuricans (left) and Desulfonema limicola (right) exposed sulfhydryl group)
(Madigan et al., 2000). helps maintain reducing con-
ditions; it is also a key com-
ponent for anoxygenic photo-

352 THE AMERICAN BIOLOGY TEACHER, VOLUME 67, NO. 6, AUGUST 2005
synthesis in these microbes (i.e., a source of reduced lished themselves as symbionts inside the cells of a
sulfur or electron donor, for some of the green sulfur primitive eukaryote.
bacteria). Certain red anaerobic photosynthesizers can
A little deeper in the column, hydrogen sulfide gas
use organic compounds, such as malic acid, as electron
(H2S) is diffusing upward into the aerobic zone. Part of
donors, so this medium serves double duty for this
the sulfur cycle is evident here. The H2S gas has been
type of culture enrichment. The generalized anaerobic
produced by anaerobic microorganisms near the bot-
process is:
tom of the column. These organisms reduced the sulfate
CO + 2H X ____> (CH O) + 2X + H O
2 2 2 2 originally mixed into the soil. Near the top of the col-
umn, the H2S can be oxidized back to sulfate by the sul-
where X stands for some reducing agent (or none at
fur-oxidizing bacteria, such as the genera Beggiatoa and
allsome anaerobic bacteria can simply use H2 by itself).
Thiobacillus (Atlas & Bartha, 1998). These bacteria are
This metabolic reaction is thought to be ancestral in the
chemoautotrophs and gain energy from the oxidation
sense of biochemical evolution to the familiar photo-
of reduced sulfur to elemental sulfur or to sulfate and
synthetic process in modern oxygenic photosynthesis,
they can also synthesize organic compounds autotroph-
in which X has become oxygen.
ically from CO2. Thiobacillus oxidizes sulfur while
Beggiatoa is sulfide-oxidizing.
Discussion
Microaerophilic Zone
Relating Microbiology to Nutrient The diffusion of H2S from the sediment below
Cycling in the Column enables anaerobic photosynthetic bacteria (which typ-
ically appear in brightly colored bands) to grow. From
After a month to six weeks, the column should sta- bottom to top, green sulfur bacteria (GSB), such as
bilize into three distinct zones and develop communi- Chlorobium, create an olive-green color zone. Purple
ties of bacteria specific to their environmental require- non-sulfur bacteria (PNSB), such as Rhodospirilum and
ments (Figure 1). Rhodopseudomonas, usually require a small amount of
oxygen and are located nearer to the top of column than
Aerobic Zone are the GSB. Growth of these organisms results in a dark
red-rust color.
The top of the water column can contain large pop-
ulations of diverse bacteria and protists. These are aer- The metabolism of both GSB and PNSB provides an
obic organisms found in organic-rich freshwater excellent opportunity to draw comparisons between oxy-
habitats such as shallow ponds and polluted streams. genic photosynthesis (oxygen producing, like green
The bacteria are often flagellated, allowing them to plants) and anoxygenic photosynthesis (non-oxygen pro-
migrate and establish themselves in new areas ducing organism that pre-dated green plants). GSB and
(Madigan et al., 2000). In addition, there may be larger PNSB gain energy from light reactions and metabolize
protozoa and invertebrates CO2 in the same way as plants
from the original water and do. Yet, because they use H2S
mud source. At the very top instead of water as the source
of the zone the mud is the of hydrogen (reducing power),
most oxygen-rich part of the they produce a more oxidized
column, often colored a light- sulfur product (Atlas &
brown from iron-oxide pre- Bartha, 1998). Consider plant
cipitate. photosynthesis (i.e., 6CO2 +
6H2O => C6H12O6 + 6O2)
Oxygen-producing expressed as CO2 + H2O ___>
organisms, such as the photo- [CH2O] + O2. Then it is easy
synthetic cyanobacteria, show the parallel processes
often grow above the mud, between photosynthesis and
forming a green zone. These hydrogen sulfide oxidation
are the only bacteria that (Atlas & Bartha, 1998) as:
have photosynthesis like that
of plants. In fact, there is very CO + H O __> [CH O] + O
2 2 2 2

strong evidence that the Figure 3. (plant photosynthesis)

chloroplasts of plants origi- A simple, yet effective, anaerobic chamber can be constructed CO + H S __> [CH O] + S
2 2 2
nated from ancestral (bacterial anoxygenic photo-
from common household items as depicted in this diagram.
cyanobacteria that estab- synthesis)

SULFUR NUTRIENT CYCLE 353

Anaerobic Zone
Organisms that grow in anaerobic conditions fer-
ment 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 twist-
ed threads. These organisms oxidize iron and produce a
rust-colored iron oxide layer.
Moving deeper into the column, purple sulfur bac-
teria, such as Chromatium, may be found and these bac-
teria produce a red-to-purple layer in the soil. Purple sul-
fur bacteria reduce sulfates to sulfur; a type of metabo-
lism that emerged on Earth early in the planets history.
From an evolutionary standpoint, there is strong evi-
dence 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. Sulfur-
reducing 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 fre-
quently black.
Some of these organisms are anaerobic cellulose-
degraders, 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 pur-
ple sulfur bacteria with gas vesicles. A characteristic
species is Amoebobacter, which also photsynthesizes
using H2S (Atlas & Bartha, 1998).
354 THE AMERICAN BIOLOGY TEACHER, VOLUME 67, NO. 6, AUGUST 2005
The Sulfur Cycle
Sulfur is an assimilable, nutritional requirement for
most life, and yet represents a energy conduit, or dis-
similatory 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 oxida-
tion 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 elec-
tron acceptors and donors. As electrons move from one
molecule to another they also lose or gain energy. Thus
the many microbial transformations of sulfur com-
pounds form a basic part of energy metabolism.
A number of species can use elemental sulfur anaer-
obically as a terminal electron acceptor (the usual role of
oxygen in aerobic respiration), reducing sulfur to hydro-
gen 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 light-
ed 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 biodiver-
sity of our world. The Winogradsky column is an excel-
lent way to show students that not all bacteria are
pathogens and they have an important role in the geo-
chemical cycling of the biosphere, one they have been ful-
filling since life first began nearly four billion years ago.
Figure 4.
The sulfur cycle emphasizing the transition in oxidation state of the different sulfur compounds (after Fenchel et al., 1998) and the bacteria-
mediated processes that occur in aerobic or anaerobic habitats.

SULFUR NUTRIENT CYCLE 355

Acknowledgments
We wish to extend a special thank you to Dr.
Norbert Pfennig for his permission to use his photomi-
crographs 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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