MICROBIAL ECOLOGY (BLG3701)

PRACTICAL GUIDE (2021)

DEPARTMENT OF BIOCHEMISTRY,
MICROBIOLOGY AND BIOTECHNOLOGY
(2023)
INTRODUCTION

Microbes usually live in communities and rarely as individuals. They constitute a silent majority
on Earth, yet they have a mammoth effect on us and on our environment. Microbial ecology is the
science that examines explicitly the relationship between microorganisms and their biotic and
abiotic environment. Like plant, animal, and human ecology, microbial ecology applies ecological
principles to explain life functions of microorganisms in situ (directly in their natural environment)
rather than simulated under artificial laboratory conditions (ex situ or in vitro).
Although the in situ microbial processes are the ultimate goal in the majority of ecological studies,
it does not exclude laboratory experiments and mathematical modeling as efficient research tools
at intermediate stages aimed at the elucidation of underlying mechanisms and testing hypothesis.
The aim of this practical course is to apply a range of classical microbiological techniques that are
used in microbial ecology. Topics will include the roles of microorganisms the beneficial aspects
of microorganisms and their application in biotechnology.

1
CONTENTS

INTRODUCTION....................................................................................................................................... 1
LABORATORY SAFETY RULES ........................................................................................................... 3
EMERGENCY STAFF .............................................................................................................................. 9
LABORATORY SAFETY INDUCTION FORM .................................................................................. 10
ACADEMIC HONESTY.......................................................................................................................... 12
GROUND RULES .................................................................................................................................... 12
COURSE INFORMATION AND COURSE OUTLINE 2019................................................................................ 13
PRACTICAL REPORTS ......................................................................................................................... 16
PRACTICAL 1: EXAMINATION OF SOIL MICROORGANISMS USING CONTACT SLIDE
ASSAY ....................................................................................................................................................... 16
THEORY AND SIGNIFICANCE ....................................................................................................... 16
PROCEDURE ....................................................................................................................................... 20
First Period ........................................................................................................................................ 20
Second Period .................................................................................................................................... 21
Questions............................................................................................................................................ 22
PRACTICAL 2: PREPARATION OF A WINOGRADSKY COLUMN ............................................ 23
THEORY AND SIGNIFICANCE ........................................................................................................... 23
PROCEDURE ....................................................................................................................................... 25
PRACTICAL 3: ISOLATION OF BACTERIA FROM SOIL AND ................................................... 26
THEIR ENZYMATIC ACTIVITY ......................................................................................................... 26
THEORY AND SIGNIFICANCE ....................................................................................................... 26
First period ........................................................................................................................................ 26
PROCEDURE ....................................................................................................................................... 26
QUESTIONS ..................................................................................................................................... 27
PRACTICAL 4: ISOLATION OF PHOSPHATE SOLUBILIZING MICROBES (PSMS) FROM
SOIL ........................................................................................................................................................... 28
THEORY AND SIGNIFICANCE ....................................................................................................... 28
PROCEDURE ....................................................................................................................................... 29
PRACTICAL 5: NITROGEN FIXATION ............................................................................................. 30
THEORY AND SIGNIFICANCE ....................................................................................................... 30
5.1 The Process of Nitrification in Soil ...................................................................................... 31

2
LABORATORY SAFETY RULES
Accidents in a laboratory usually result from improper judgment on the part of the victim or one
of his/her neighbors. Learn and observe the laboratory safety rules listed below.

Safety precautions

1. Locate safety equipment.

During the first laboratory period familiarize yourself with the location and operation of the safety
features of the laboratory, including:

• Safety shower: Use if your clothing catches on fire or a corrosive chemical is spilled on you in

quantities that cannot be easily flushed away at laboratory faucets.

• Eye wash

• Fire extinguishers

• Laboratory first aid kit & Fire blanket (kept with the technologist in charge)

2. Always be on time for your practical sessions. Your instructor may deny you access to the
lab should you be late as your tardiness may pose a safety risk to yourself or others.

3. Perform only authorized experiments. Unless authorized to do so by the instructor, a student

will be subject to immediate and permanent expulsion from the lab if:

a) Attempting to conduct unauthorized experiments.

b) Attempting variations of the experiment in the lab manual.

Performing unauthorized experiments are dangerous. Students lack the experience to recognize
whether or not the chemicals and techniques are safe therefore these practices are strictly
prohibited.

4. Wear authorized clothing.

• Wear clothing that will protect you against spilled chemicals or flaming liquids.
• Therefore LAB COATS and hard-soled, covered footwear must be worn in the
laboratory at ALL times--no sandals or pumps allowed, NO EXCEPTIONS.

3
5. a) Eating, drinking, and smoking are strictly prohibited in the laboratory at all times
because of the possibility of chemicals getting into your mouth or lungs through contamination.
The chief hazard with smoking is fire.

b) Electronic devices (Cellphones, Laptops etc.). The use (making & receiving calls,
texting and social media) of electronic devices in the laboratory is strictly prohibited unless
permission to use such devices is granted by the instructor, in which case the device may only be
used for educational purposes as it pertains to that particular practical.

6. Keep your workspace orderly.

a) Place tall items, such as graduated cylinders, toward the back of the workbench so they
will not be overturned by reaching over them.

b) Clean up all chemical spills, scraps of paper, and glassware immediately.

c) Keep drawers closed while working and the aisles free of any obstructions, including
chairs.

d) Never place coats, books, and other belongings on the laboratory bench where they
will interfere with the experiment and are likely to be damaged. Instead place these
at the bottom in the cupboard stationed next to your seat or at your feet.

When dealing with chemicals

7. Read the label. Read the label carefully, read it twice, before taking anything from a bottle.
Many chemicals have similar names, such as sodium sulfate and sodium sulfite. Using the wrong
reagent can spoil an experiment or can cause a serious accident.

8. Wear safety goggles.

• Your eyes may be permanently damaged by spilled chemicals and flying broken
equipment, be sure to wear safety goggles or safety glasses whenever anyone is working
in the lab.
• If you get anything in your eye, use the eye wash immediately, and then report it to your
instructor. Use your hands to hold your eye open so that it can be rinsed thoroughly.

4
Note: Eye washing with a contact lens in place will not clear a splashed chemical from the eye.

The contact must be removed for effective cleansing. It is advisable for those wearing contacts to

switch to glasses for the lab period.

9. Use the fume hoods and face masks. Any experiment involving the use of or production of
poisonous or irritating gases must be performed in a hood, as far as is possible.

10. Assume that a particular reagent is hazardous unless you know for sure it is not.

a) Never taste a chemical.

b) If you are instructed to smell a chemical, point the vessel away from your face and
carefully fan the vapors toward your face with your hand and sniff gently.

11. Dilute concentrated acids and bases by pouring the reagent into water (room temperature
or lower) while stirring constantly. NEVER pour water into concentrated acids; the heat of
solution will cause the water to boil and the acid to splatter.

12. Always add a reagent slowly--never "dump" it in. Two reasons:

a) Some reactions give off a lot of heat, and unless added slowly, can become too vigorous
and thus potentially dangerous.

b) If you make a mistake and choose the wrong chemical, adding the chemical slowly
decreases the possibility of causing a serious accident.

13. Avoid using excessive amounts of reagent.

a) Never use more than what is called for in the experiment.

b) Do not return any excess chemical to the reagent bottle; share it with another student
or dispose of it in the appropriate waste container.

c) If you are uncertain how to dispose of an excess of a specific chemical, consult your

instructor

14. Treat chemical spills as follows:

a) Alert your lab neighbors and your instructor.

5
 b) Clean up the spill as directed by your lab instructor.

15. Wash chemicals from skin.

a) If you receive a chemical burn from a caustic material, i.e. acid or base, immediately
wash the burned area with large quantities of water. Ask another student to summon the
lab instructor.

b) Wash your hands and face quickly and thoroughly whenever they come into contact
with a chemical.

c) Always wash your hands, before leaving the lab since toxic chemicals may be transferred
to the mouth at a later time.

d) Chemicals spilled over a large part of the body require immediate action. Remove all
contaminated clothing and use the safety shower, flooding the burned area. Do not use
salves, creams, lotions, etc. Get medical attention.

16. Discard waste as follows:

a) All chemical waste must be properly labeled with the following:

• chemical name/ type of waste,

• concentration,
• hazard alert,
• date of preparation and name of person that prepared the solution (labels can also be
obtained from your technologist) and placed in the chemical waste containers as
provided by the technologist.
b) Paper products: Please place all paper products in the trash can/ bins provided in the
lab.

c) Bio hazardous waste (agar plates, tips, toothpicks, swabs etc.) must be autoclaved in
the departmental autoclave room and disposed of in the bins provided. Large specimens
(fish, hearts, lungs, kidneys etc.) should be disposed of as instructed by your technologist.

d) Glass tubing waste or broken glass should be placed in the designated Broken Glass
box/bin in the lab.

6
17. Never throw lighted matches or any chemicals into a sink. They may ignite a discarded
flammable liquid, contaminate our water sources and/or block the drainage system

Safety equipment and Fires

18. Be careful with flames. A lighted gas burner can be a major fire hazard.

a) General Precautions:

1) The burner should be burning only for the period of time in which it is actually utilized.

2) Before lighting your burner carefully position it on the desk away from flammable
materials.

b) Personal Precautions:

1) Keep long hair tied back so that it cannot fall forward into a flame.

2) Keep beards away from flames.

19. Never point a test tube toward a laboratory neighbor or yourself when:

a) Heating a test tube over a burner.

b) Carrying out a reaction in a test tube.

20. Know the ways to put out a fire.

a) If it is an open fire, such as a large chemical spill on a lab bench, the correct extinguisher
should be used as follows:

􀁮 Pull the pin.

􀁮 Point the extinguisher (of dry) or hose (if CO2) at the base of the fire.

􀁮 Squeeze the handle while moving the extinguisher back and forth.

7
NOTE: Be careful not to spread the fire by getting the nozzle of the extinguisher too close--

the material being emitted is under pressure.

b) If it is a small, contained fire, such as in a flask or beaker, cover the container cutting
off the supply of oxygen to the fire and thus putting it out.

21. Do not put hot objects on the bench tops. Place hot objects on a wire gauze or ceramic pad.

Glassware and cleanliness

22. Be careful when using glassware.

Cuts can be prevented by following a few simple rules:

a) Discard cracked or broken glassware in the designated container.

b) Never heat heavy glassware such as graduated cylinders, suction flasks, or reagent
bottles since they might shatter.

23. Clean up your workspace at the end of each laboratory period.

a) Wash and wipe off your bench top.

b) Be sure gas and water is turned off.

c) Return all special equipment to the stockroom.

24. Never work in the lab without the instructor present. This includes setting up equipment.

25. Be aware of your lab neighbors' activities; you may be a victim of their mistakes. If you
observe improper techniques or unsafe practices:

a) Advise your neighbor.

b) Advise your instructor if necessary.

26. Do not remove any chemicals from the lab.

8
EMERGENCY STAFF

First aid administrators in the Biological Sciences!!!

Department
Ms Mildred Johnson Ms. Kaveire Kaitjizemine

Office number: W051 Office no: W097

Tel: 206 4943 Phone: 206 3110

Mr. Asser Katunahange

Office no: W068
Phone: 206 4577

In case of emergency
contact: 206 3760 or 3503
(UNAM Clinic)

9
LABORATORY SAFETY INDUCTION FORM
As per UNAM Occupational Safety and Health policy, all students in the department of Biological
sciences will be required to undergo the departmental Laboratory safety induction. Every student
must understand the safety procedures as outlined in this practical manual that will guide their
conduct and affirm their commitment to observe and obey all laboratory safety rules and
procedures. To this end, each student must complete and sign this Laboratory safety induction
form prior to being allowed to work in any of the Laboratories.

Student personal details:

Student Full Names

Student Number

Campus

Induction conducted by:

Date of Induction

Course of study
Name of Academic
research supervisor

Laboratory Safety induction check list to be completed by the Student, after induction course:
(Please tick in the right hand column to confirm that you agree)
Tick ()
1. INFORMATION
Laboratory safety guideline and procedures was provided and/or discussed
Contact details for Laboratory managers have been provided
Internal and External Emergency contact numbers provided

2. LABORATORY SAFETY RULES

Minimum personal protective equipment required for working in the Lab
Lab coat  Closed shoes  Gloves  Eye protection  Tied hair 
Hand washing/disinfectant: Usage:  Procedure: 
Eye wash station and Emergency shower: Location  Procedure for use
in the Laboratory 
Waste disposal procedures: General waste  Biological waste 
Chemical waste  Sharps  Micropipette tips  Broken glassware 
Safety equipment: Fire extinguishers  First aid kit  Chemical spills kit 
Electrical safety
Housekeeping safety procedures

10
 3. GENERAL LABORATORY RULES
Normal operating hours for Laboratories
After hours and Weekend work procedures for Laboratories
Working un-supervised (or in isolation) in the laboratories
Laboratory visitors
Safety guidelines for unattended laboratory experiments

4. LABORATORY EQUIPMENT SAFETY PROCEDURES

Centrifuges, Hot plates, freezers, refrigerators and cold store rooms
High pressure equipment i.e. the Autoclave
Fume hoods and Biosafety cabinets
Heating equipment i.e. hot plates, ovens. Etc.
Freezers, refrigerators and cold storage rooms: safety considerations

5. LABORATORY EMERGENCY PROCEDURES

First aid: Chemical splashes  Fire drill  Chemical splashes  Chemical
inhalation  Ingestion of toxin 
Chemical spills: Small scale spills  Large scale spills 
Accident/Incident reporting

6. CHEMICAL HANDLING AND STORAGE PROCEDURES

Transportation: In-house  Between Labs 
Handling & storage: liquid chemicals  solid chemicals  Flammable
chemicals  Corrosive chemicals  Explosive chemicals  Heavy metals 
Cryogenic & Carcinogenic substances 

Student Signature: ……………………………… Date: ……………………………..

Academic supervisor: ………………………….. Date: ……………………………..

11
ACADEMIC HONESTY
Academic honesty is a basic prerequisite for this module, including the lab sessions. Even though
most of the lab exercises will be done in groups, all practical and other assignments must be done
on an individual basis. There are NO group assignments.

UNDERTAKING

I ____________________________________student nr_______________
Hereby declare that I have read and fully understand the general instructions contained in the
Manual as well as the following regulation.
Practical’s are compulsory – students who miss any practical without a valid medical certificate
or other appropriate documentary proof handed in within 1 week missing the practical will
receive a 0 for that practical.

Signed in Windhoek on ______________________________2018

____________________________
Student Signature

GROUND RULES
Use of mobile phones, laptops and other electronic devices
For practicals with several sessions per week. Students sign up for one session and keep to it for
the entire semester. No changes allowed. If unable to attend your session, consult with
lecturer/technologist in charge, in advance

No friends or unauthorized persons are allowed to sit in the labs.

12
 COURSE INFORMATION, LECTURING AND PRACTICAL
PLAN 2019
GENERAL INFORMATION

Lecturer: Dr. Jean Damascene UZABAKIRIHO,

Tel: 061 206 3632, Office number: W095, Email: juzabakiriho@unam.na

Technologist: Mr. Monica AUALA, Tel: 061-206 3134, Email: ambangu@unam.na

Contact hours: 4 lecture periods (Wednesdays 8h30 and Fridays 9h30).

Venue X

PRESCRIBED TEXTBOOKS:

Atlas, R.M. & Bartha, R. (2013). Microbial ecology. Fundamentals and Applications (4th Edition).
Pearson Benjamin Cummings.

Madigan, M.T., Martinko, J.M., Stahl, D.A. & Clark, D.P. Brock Biology of Microorganisms (14th
Edition). Boston: Pearson Benjamin Cummings.

RECOMMENDED TEXTBOOKS: Tortora, G. J., Funke, B. R., & Case, C. L. (2010). Microbiology:
An introduction (11th ed.). San Francisco: Pearson Benjamin Cummings.

COURSE ASSESSMENT: 2 tests, Unannounced quizzes

1 Assignment

All practical reports

Continuous assessment (CA): 50% [tests (75%), assignment (20%), 5% UNANNOUNCED

QUIZES],

50% of PRACTICALS.

Examination: 60% (1 x 3h examination paper), and 40% CA marks

IMPORTANT DATES: See course outline provided

PRACTICALS: One practical session per week on Tuesdays and Wednesday (14h30 and 17h30 -
17h30).

COURSE DESCRIPTION

This module aims to:

Make students aware of the interrelationships and roles of natural microbial communities among
themselves, with plants, animals and the environment

13
LEARNING OUTCOMES/SPECIFIC OUTCOMES:

On successful completion of the Course students should be able to:

1. Explain the underlying principles that drive microbial population structure

2. Demonstrate an understanding of the functioning and factors regulating microbial productivity

3. Distinguish microbial community function and dynamics and how these are influenced by both
abiotic and biotic components in the environment

4. Describe thermodynamic constraints on microbial community structure and provide examples

from various types of environments

5. Describe the basic metabolic requirements for microorganisms and how it relates to the Redox
cascade existing in natural environments

6. Apply culturing techniques as well as molecular and genomic tools for understanding the
physiology and ecology of microbial communities

7. Differentiate various microbial metabolisms and how these relate to biogeochemical cycling in
various ecosystems and the biosphere as a whole.

8. Demonstrate the adaptations of microorganisms to extreme environments

LECTURING & PRACTICAL PLAN

Weeks/dates Topics/units Laboratory Practicals

Week 1, 2 & 3 UNIT 1: WEEK 4 and 5: EXAMINATION OF

Principles of microbial ecology/Ecological SOIL MICROORGANISMS USING
concepts, microbial ecosystems and CONTACT SLIDE ASSAY (NO
biogeochemical cycling, Microbial habitats, REPORT. ANSWER QUESTIONS
Fresh water, soil, and plant microbial AT THE END OT THE PRACTICAL
ecosystems, Marine microbial ecosystems. GUIDE TO BE HANDED IN AT
THE BEGINNING OF THE
Week 4 UNIT 2: SECOND PERIOD OF THIS
PRACTICAL. Do not forget to
Nutrients cycles: Carbon cycle, Nitrogen cycle, mention your sources)
Phosphorus cycle, Sulfur cycle, Iron cycle.

Nutrients cycles: Carbon cycle, Nitrogen cycle,

Phosphorus cycle, Sulfur cycle, Iron cycle.

14
 Week 5 and 6 UNIT 3: WEEK 6: PRACTICAL 2:
PREPARATION OF A
Microbial bioremediation, Animal/ microbial WINOGRADSKY COLUMN
symbioses, Plant microbial symbioses.
(Results will be recorded weekly over
a period of 4 weeks. At the end a full
report should be submitted during
Week 7 and 8 UNIT 5:
week 10 Practical session)
Extremophiles: Definition of an extreme
environment; thermophiles (hydrothermal
vents, cold seeps and deserts); acidophiles and
alkalophiles (micro flora in the gut; peats and
bogs),

WEEK 7 and 8: PRACTICAL 3:

ISOLATION OF BACTERIA
FROM SOIL AND THEIR
ENZYMATIC ACTIVITY. (No
report required. Answer questions)

WEEK 9: PRACTICAL 4:
ISOLATION OF PHOSPHATE
SOLUBILIZING MICROBES
(PSMS) FROM SOIL. (No report
required. Answer questions)

Week 9 and 10 UNIT 6: WEEK 10: PRACTICAL 5:

NITROGEN FIXATION
Microbiological and molecular techniques in
Microbial Ecology: Quantitative ecology SUBMISSION REPORT FOR
(numbers, biomass, metabolic activity); method PRACT 2
for species identification; Metagenomic analysis
of communities.
WEEK11: SUBMISSION REPORT
PRACT5

Rules

15
 PRACTICAL REPORTS
You will be expected to write concise and detailed reports with the following sections:
1. Introduction/Background
2. Aims of Practical
3. Materials and Methods
4. Results (including tables, calculations and other illustrations)
5. Discussion
6. Conclusion
7. References
Each of the sections will be awarded Marks on the graded script.

PRACTICAL 1: EXAMINATION OF SOIL MICROORGANISMS

USING CONTACT SLIDE ASSAY
Objective: To utilize a microscope to view soil microbes and their relationship to each other and
soil particles.
• Adjust soil moisture to a value close to “field capacity” (value provided by your Lab
Technician)
• Insert glass slides into a beaker of moist soil
• Incubate for one week
• Remove slides, stain with phenolic Rose Bengal
• View under microscope

THEORY AND SIGNIFICANCE

The ability to view soil microbes in situ is important since it allows students to view the
interrelationships between soil microbes and their interactions with soil particles. However, it is
difficult to observe colloidal size microbes that exist within soil. A technique developed back in
the 1930s is still a valuable learning tool today. This is the contact slide or buried-slide technique
of Rossi et al. (1936), which is a simple technique for qualitatively assessing the spatial
relationships between soil microorganisms. Although it is not reliable enough to quantify soil
microorganisms as the original authors had intended, it is useful to illustrate the orientation of soil
organisms to one another and to soil particles. It also allows students to see bacteria, actinomycetes
and fungi, perhaps for the first time, through the use of a microscope (Maier et al., 2000). The
technique involves burying a glass slide in soil for a defined period of time (Figure 1-1). Nutrient
amendments, such as the carbon source glucose and the nitrogen source ammonium nitrate,
encourage the rapid proliferation of heterotrophic microorganisms.

After removing the slide from within the soil, the slide is fixed with acetic acid and stained to
provide contrast, as the often colorless organisms would otherwise not be visible under a
microscope. Viewed under a microscope, soil bacteria, actinomycetes, and fungi can be seen
growing on soil particles, in pure colonies on the slide, and in juxtaposition to each other, often

16
with bacteria lining the fungal hyphae. Spore formation by actinomycetes or fungi can also be
observed. Examples of what may be seen are shown in Figures1- 2 and 1-3.

Figure 1.1 Examples of soil microcosms with inserted buried glass slides (Photo courtesy
K.L. Josephson).

17
Figure 1-2 Contact slide images using the 100X objective lens (Photo courtesy W.H. Fuller).

Figure 1. 4 Position of the slides in the tumbler containing soil.

18
Figure 1-3 Contact slide images using the 100X objective lens (Photo courtesy W.H. Fuller).

19
PROCEDURE

First Period

Materials

300 g of each soil

1% glucose
NH4NO3
2 beakers for each soil type, volume (250ml)
Label tape and pens
Plastic wrap
4 microscope slides for each soil type
Rubber bands
Weighing paper
Deionized water in a wash bottle
Analytical balance, and benchtop balance (±0.01 g)
Graduated cylinder

1. Weigh out 150 g portions of each soil into two cups, recording the mass of the soil you
added to each cup. Label one cup as “treatment” and the other as “control.” A 100 g sample
of soil should be used for soils high in organic matter, as they are less dense than mineral
soils.

2. Calculate the amount of moisture necessary to alter the moisture content of the soil
samples to the moisture content as follow:

.
This soil moisture content is often close to field capacity. Measure out this much distilled
water with a graduated cylinder and add it to each of two vials. Label one vial “treatment”
and the other “control.”

3. Amend the water in the treatment vial with enough glucose for a final soil glucose
concentration of 1% (w/w) on a dry weight basis in the treatment soil above. Also add
200mg of NH4NO3 to the treatment vial. Stir to dissolve the amendments. Do not amend
the control vial.

4. Mix the contents of the treatment and control vials into their respective cups by adding the
liquid to the soil in small aliquots, and mixing with a spatula after each moisture addition.
For heavy textured clay soils avoid mixing as this will “puddle” the soil.

5. For each cup, label two clean microscope slides, designating the soil and treatment for that
slide. There will be two slides for each cup. Insert each slide vertically into its respective

20
 cup, leaving 2 cm of each slide projecting above the soil surface (see Figures 1-4). Do not
force the slides as they will break.

6. Cover the cups with plastic wrap, securing with a rubber band. Puncture the wrap or foil
several times with a probe to allow air in and yet preclude excessive evaporation of
moisture. Weigh each cup. Incubate the soil-filled cups at room temperature in a designated
incubator for one week.

Second Period

Materials

Incubated cups from Period 1

40% (v/v) acetic acid
Phenolic Rose Bengal stain
Staining racks with a pan to catch excess stain
Protective goggles
Microscopes
Immersion oil
Paper towels

1. Re-weigh the cup and calculate the soil moisture at the time of slide removal.

2. Remove the two slides from each cup after seven days by pressing each slide to an inclined
position and withdrawing in a manner such that the upper face of the slide is not disturbed.
Mark and identify the side to be stained (see Figures 5 and 6).

3. Gently tap the slide on the bench top to remove large soil particles from the slide surface.
Clean the lower face with a damp paper towel and dry the slide at room temperature.

4. Wearing protective goggles, immerse the slide in 40% (v/v) acetic acid for 1–3 min under
a fume hood, holding the slide with forceps.

5. Wash off the excess acid under a gentle stream of water, and cover the surface with
phenolic Rose Bengal from a dropper bottle, supporting the slide on a staining rack over a
container to catch the excess stain.

21
Figure 6 Example of how the slide plus accompanying soil should look following removal of
the slide from the soil (Photo courtesy K.L. Josephson).

Questions

1. Describe the size and shape of bacterial cells, filaments and spores of fungi and actinomycetes.
2. Note evidence of colony formation and the relationships of organisms to each other and to soil
particles. Make sketches showing typical fields for each soil/treatment combination. Label each
drawing with the soil and treatment it depicts.
3. Describe qualitative differences in the microbial populations between the different soils for each
treatment, i.e., unamended and amended with glucose and NH4NO3.
4. Were there quantitative differences in microbial population densities between the soils or
between the treatments? Speculate as to why there were or were not any differences between soils
and treatments.
5. How did you distinguish fungi from actinomycetes?
6. Discuss the usefulness of the method. Indicate how the results have influenced your concept of
the nature of the development of microorganisms in soil and the significance of numbers of cells
of microorganisms as found in published literature.
7. How were microorganisms oriented with respect to each other and soil particles?
8. Was there competition for space between microorganisms?
9. What appears to have been the limiting factor for microbial growth and activity in soil?

22
 PRACTICAL 2: PREPARATION OF A WINOGRADSKY
COLUMN

Objectives:
1. To create a microcosm (a model microbial ecosystem) in which complex microbial
communities are cultivated.
2. To gain an appreciation for the diversity of methods microorganisms use to gain energy
and how those metabolic processes affect the surrounding environment and, thus,
determine microbial community composition and succession.

THEORY AND SIGNIFICANCE

Prokaryotes (Bacteria and Archea) collectively encompass a taxonomic and a metabolic diversity
that far exceeds that of plants and animals (the so-called ‘higher animals’). This week we will set
up a model microbial ecosystem (a microcosm) called a Winogradsky column that will simply and
elegantly demonstrate a cross section of that diversity.
Sergei Winogradsky (1856-1953) who, along with Martinus Willem Beijerinck (1851-1931),
developed the technique, was a pioneering russian microbiologist who worked in the late 1800’s
and early 1900’s, when microbiology was still a fledgling science. At a time when microbiological
methods barely existed and most microbiologists focused on the ‘germ-theory’ of disease, the
isolation of individual microbes and the characterization of ‘domesticated’ laboratory strains,
Winogradsky focused on environmental microbiology and developed techniques for cultivating
and studying complex microbial communities (such as the Winogradsky column). In so doing, he
made great advances in our understanding of environmental microbiology, microbial diversity,
microbial physiology, microbial autotrophy (the ability to obtain energy from non-organic
substances) and environmental nutrient cycling (particularly sulfur and nitrogen cycling).
He is considered one of the founding fathers of microbial ecology. The Winogradsky column is a
miniature, self-contained ecosystem that in many ways models ecological conditions found in
most lakes and dams in late summer.
Spring thaw results in a mixing of lake water so that the water is more or less uniform at different
depths. As summer progresses gradients begin to form in the water column (light gradient,
temperature gradient, nutrient, O2 and H2S concentration gradients). These gradients result in a
complex interaction of microbes with their environment and with one another resulting in a series
of community successions and, ultimately, stratification of microbial populations in the water
column.
Similarly, the Winogradsky column begins as a uniform slurry of soil, mud or lake sediment and
water (preferably from some outdoor source) supplemented with nutrients (carbon and sulfur). The
ingredients are mixed into a uniform slurry, poured into a tall, transparant vessicle such as a test

23
tube, graduated cylinder or soda bottle and exposed to sunlight to provide energy for the system
and to enrich for both aerobic and anoxic photosynthetic bacteria.
The sediment, soil and water serve as inocula of diverse microbial populations with diverse
metabolic processes. A series of gradients (e.g. oxygen, H2S) soon develop in the column as a
result of bacterial metabolic processes. For example, aerobic photosynthetic organisms and
exposure to the air will produce O2 near the top of the column which can then be used by other
microbes for aereobic respiration. Similarly, sulfate reducers in the anoxic region near the bottom
of the column will produce H2S, which can be used by H2S utilizers and anoxic phototrophs.
Different organisms soon gain a competative advantage in different regions of the column causing
a stratification and continual regional succession of organisms. Because this culture system more
closely resembles a natural environment than do traditional culture methods, a wider variety of
physiological types can be observed than by the more standard culture methods. Also, different
microbes will be concentrated (enriched) in specific regions of the column where local conditions
are optimal for their particular growth (e.g areobes at the top in the oxic zone and anaerobes at the
bottom, anoxic zone).
Winogradsky columns demonstrate the interdependence of diverse microorganisms in complex
communities for survival and growth. As such, variations in Winogradsky column ingredients and
manipulations are as infinite as your imagination will allow. Virtually any microbial process could
be studied with the correct design and initial inocula.

Figure 2.1 Demonstration of Winogradsky technique.

24
PROCEDURE

Materials
Water sample, agriculture soil, forest soil, mixing container, grass cuttings, pine needles, calcium
sulfate, and calcium carbonate.
Protocol
1. Obtain a clean and clear container and label your initial, date, and soil type.
2. Place about 200 mL compost soils (agriculture or forest soil) to a mixing container.
3. Add pond water and stir until it is about the consistency of apple sauce.
4. Add 5 g grass cutting to the agriculture soil and 5 g pine needle cutting to the forest
soil as a carbon source (carbon source is in the form of cellulose).
5. Add an equal amount of calcium carbonate and calcium sulfate and mix until the
mixture become drier (source of carbon and sulfur).
6. Pour or spoon this mixture into the decomposition chamber to approximately 3–4 cm
in depth.
7. Mix it well with a spoon or stirring rod to remove any air pockets.
8. Add plain agriculture or forest soil to the respective chamber until the depth of the soil
mixture reaches between 6 and 8 cm. DO NOT STIR!
9. Add pond water, leaving about 4 cm of headspace.
10. Insert a thermometer into the soil and record the starting temperature in °C.
11. The soil column in each decomposition chamber should be covered with aluminum
foil, seal with parafilm, and place next to the window (Figure 2.1).

Results
1. Observe your column at least once a week and record any changes that occur (ie, formation of
gas pockets, blackening, development of coloration, etc.) in your laboratory notebook. It usually
takes 2 to 4 weeks for chromogenic, anaerobic photosynthetic bacteria to become obvious but
incubation of the columns can continue long beyond that to follow long-term community
development. [You may in future want to sample the overlying water and record microscopic
observations].
2. Compare your column to the columns of other teams and those that have been kept in the dark.
3. Include the observations you record in your lab notebooks with the carbons that you turn in
weekly.

25
 PRACTICAL 3: ISOLATION OF BACTERIA FROM SOIL AND
THEIR ENZYMATIC ACTIVITY

Objective: to determine the enzyme activity of soil microorganisms:

(a) Saccharolytic microorganisms

(b) Proteolytic microorganisms and
(c) Lipolytic microorganisms

THEORY AND SIGNIFICANCE

While enzymes in soil are sometimes associated with roots, many are of microbial origin. Soil
enzymes may retain their activity long after their release from microbial cells primarily because
the enzymes form complexes with humic and clay colloids. Complexing with these colloids
renders the enzymes highly resistant to denaturation and degradation. Thus, soil can have
significant enzymatic activity independent of a native microbial community.

Microorganisms are metabolically highly versatile and can utilize variety of complex organic
compounds. Some of these compounds are quite large in size and thus can't be transported inside
the cells in native state. Such compound necessitates the synthesis and secretion of extra cellular
enzyme(s) for the breakdown of complex substrate into simpler one, which can be easily
transported and assimilated inside the cell. Most of these enzymes are inducible enzymes and their
producers are usually Gram-positive organisms. Gram-negatives are highly conservative in this
regard. The complex compound(s) is often supplemented to the medium and the medium is
inoculated with the sample to be screened for microbes capable of utilizing the added substrate.
The expression of enzyme or substrate utilization can be quantified with the conversion of
substrate.

First period

Requirements

c. Starch agar
d. Milk agar
e. Tributyrin agar
f. Sample from garden soil, sterile petri plates, pipettes.

PROCEDURE
1. Weigh 10g of garden soil. Suspend it in 100 ml sterile distilled water and mix
thoroughly.

2. Now, transfer 0.1 ml of this suspension to starch agar, milk agar and tributyrin agar.

26
 3. Spread the soil sample with spreader on the surface of each plate.

4. Invert the plates and incubate at 37°Cfor 24 h.

5. Saccharolytic or amylase producer: At the end of incubation period flood the starch
agar plate with Gram's iodine for 30 sec. Decant off iodine and observe the plate for
colorless zones around the colonies in blue background. These are amylase enzyme
producer's colonies.

6. Proteolytic organisms: Observe the milk agar plates for clear transparent zone
appearing around the proteolytic organisms.

7. Lipolytic organisms: Observe a clearing zone around the colonies of each Itpolytic
organism.

QUESTIONS

1. Design an experiment to isolate pectin-degrading organism from forest soil.

2. What are enrichment media? Why are these so important for isolation of organism of interest
from samples containing large number of native flora as well?
3. How would you isolate bacteria that produce phospholipase?
4. What is the role of microbial enzymes in soil fertility?
5 What is rancidity? What kinds of organisms are responsible for it?

27
 PRACTICAL 4: ISOLATION OF PHOSPHATE SOLUBILIZING
MICROBES (PSMS) FROM SOIL

THEORY AND SIGNIFICANCE

Phosphorus is one of the essential inorganic nutrients needed for variety of plant metabolic
activities and is applied to soil in the form of phosphatic fertilizers. . By and large, Namibian soils
are poor in phosphorus. However, a large portion of soluble inorganic phosphate applied to the
soil as chemical fertilizer is immobilized rapidly and becomes unavailable to plants.
Microorganisms are involved in a range of processes that affect the transformation of soil P and
are thus an integral part of the soil P cycle. Soil bacteria are effective in releasing P from inorganic
and organic pools of total soil P through solubilization. Genus Bacillus, Rhodococcus, and
Arthrobacter are some important genus of bacteria, which are actively involved in phosphate
solubilization (Figures 4-1 and 4-2). Such microbes are being used as Biofertilizers.

Figure 4-1 Phosphorus cycle

P is one of the vital nutrients for microorganism next to nitrogen. Several species of bacteria such
as Bacillus sp. and Pseudomonas sp. degrade and solubilize the insoluble phosphates into soluble
forms through the mechanism of secretion of organic acids such as glycolic acid, acetic acid, etc.
This acid decreases pH and causes dissolution of bound form of phosphate. The source of
phosphates in soil is both minerals and organic phosphates. For isolation of these bacteria, a special
type of Pikovskaya’s medium is used.

28
Figure4-2 Schematic illustration of phosphate mineralization in soil by different bacterial genus
and the phosphate-solubilizing zone formed on assay medium containing (a) Ca3(PO4)2 and (b)
lecithin.

Requirements

a. Agricultural soil
b. Pikovskaya's medium.
PROCEDURE

1. Aseptically collect 5-10 g of rhizospheric soil from the agricultural land.

2. Transfer aseptically 1-2 g of soil in a tube containing 10 ml of sterilized water.
3. Shake the soi I vigorously for 2-3 min. Let it stand for 5-10 min. for the soil particles to
settle down.
4. Take 1.0 ml of the liquid suspension and centrifuge at 2000-3000 rpm at room
temperature. Discard the debris and transfer the supernatant in a fresh sterile tube.
5. Place 0.1 ml of original and 1:10 and 1:100 dilution of the supernatant on Pikovskaya's
medium contained in petri plates.
6. Spread out the samples using sterile spreader.
7. Incubate the plates at 25-300C for 24-72h. Colonies showing zone of clearance are of
phosphorous solubilizers.

Questions:

I. What is the morphology and Gram character of phosphorous solubilizers?

2. Describe the mechanism of phosphorous solubility by microbes when tricalcium
phosphate is added to the nutrient medium.
3. What are the advantages of PSMs over chemical phosphorous fertilizers?

29
 PRACTICAL 5: NITROGEN FIXATION
THEORY AND SIGNIFICANCE

The activity of the organisms involved in the series of processes that constitute the nitrogen cycle
results in nitrogen being made available to plants in an assimilable form. These organisms,
therefore, act in an analogous way to those involved in the carbon cycle and which make CO2
available as a result of oxidation of carbon-containing compounds.

An important difference between the two cyclic systems is that nitrogen turnover determines
productivity in most agricultural situations, hence the activity of those soil organisms metabolizing
nitrogen compounds in such a way as to remove nitrogen in a form assimilable by higher plants
from the soil, may be very important with respect to loss of soil fertility. The various
transformations involving nitrogen can conveniently be summarized in diagrammatic form (Figure
5-1). The process of the production of ammonia from organic compounds is called
ammonification. The illustration only includes the main biological processes involved in nitrogen
conversion. Nitrogen is essential for the synthesis of structural and functional protein for all living
organisms. Protein is decomposed to amino acids that, in turn, are deaminated to liberate ammonia
when the organisms die. The process of production of ammonia from organic compounds is called
as ammonification. Majority of bacteria in soil participate in the process and is a very important
step for bacteria and plants to assimilate it.
The process of the production of ammonia from organic compounds is called ammonification
(Figure 5-2). In addition to the ammonification of amino acids, other compounds such as nucleic
acids, urea, and uric acid go through the ammonification process. The bacteria that accomplish it
(Bacillus, Clostridium, Proteus, Pseudomonas, and Streptomyces) are called ammonifying
bacteria. Ammonification of organic compounds is a very important step in the cycling of nitrogen
in soils, since most autotrophs are unable to assimilate amino acids, nucleic acids, urea, and uric
acid and use them for their own enzyme and proto -plasm construction.

30
Figure 5-1 The Nitrogen Cycle

Figure 5-2 Ammonification

5.1 The Process of Nitrification in Soil

Some microbes can utilize nitrates as electron acceptors and metabolize organic substances
without the need for oxygen. This process reduces nitrates to nitrites and free nitrous oxide and
nitrogen gases (NH4+ →NO2 → NO3−). The nitrate released into the soil is highly soluble and
easily assimilated by photoautotrophic bacteria, algae, and plants that convert it into the amino

31
acids needed for their own enzyme and protoplasm construction. This process is called nitrification
(Figure 16.9). The chemoautotrophic bacteria that accomplish it (Nitrobacter, Nitrococcus,
Nitrosococcus, and Nitrosomonas) are called nitrifying bacteria. These chemoautotrophs use the
energy of reduced nitrogen compounds, such as ammonium and nitrite, as an energy source for the
autotrophic production of organic compounds. Nitrification requires oxygen as a reactant and
occurs in aerobic soils.
Nitrosomonas and Nitrobacter are autotrophic and appear to be the principal organisms in soil that
can perform these reactions. There are some heterotrophs that can produce nitrites and nitrates, but
the amount is insignificant. These organisms are very small Gram-negative rods and do best in a
completely inorganic medium using CO2 as their carbon source. Many kinds of organic matter are
toxic to them, especially the substances that have free amino groups.
To demonstrate the existence of nitrification, inoculate two broths with a soil sample, incubate
them for a week, and test for nitrite and nitrate production. Ammonium sulfate broth (nitrite
forming broth) will be observed to see if ammonium has been converted to nitrite (NH4+→NO2−)
in the tube. Nitrate forming broth will be observed to see if nitrite has been converted to nitrate
(NO2 − →NO3− ) in the tube (Figure 5-3). After a total of 7 day incubation, the tubes will be
observed to see if nitrite and nitrate has been released.

Figure 5-3 Nitrification process by bacteria in soil.

32
Figure 5-4 Biological conversion of ammonia to nitrate by bacteria.

Materials

33
REAGENTS
Trommsdorf’s solution: Add slowly, with constant stirring, a boiling solution of 20 g of zinc
chloride in 100 mL of dH2O to a mixture of 4 g of starch in water. Continue heating until the starch
is dissolved as much as possible and the solution is nearly clear.
Dilute with water and add 2 g of zinc iodide (potassium iodide will do).
Dilute to 1 L and filter. [Alternatively, the reagent is commercially available].
Nessler’s reagent (for ammonia): Dissolve 50 g of potassium iodide (KI) in the smallest possible
quantity of cold water (50 mL). Add a saturated solution of mercuric chloride (about 22 g in 350
mL of water will be needed) until an excess is indicated by the formation of a precipitate. Then
add 200 mL of 5 N sodium hydroxide (NaOH) and dilute to 1 L. Let settle and draw off the clear
liquid. [Alternatively, the reagent is commercially available].
Diphenylamine: Dissolve 0.2 g in 100 mL of concentrated sulfuric acid. Ammonium sulfate and
sodium nitrate is separately sterilized by bacterial filter to protect the chemical nature of salt and
added to maintain the required concentration.
Ammonium ion containing medium as a nitrogen source is to be used to detect the presence of
nitrite producing (Nitrosomonas) bacteria, and a medium containing nitrate is to be used for
isolating nitrate producing (Nitrobacter) bacteria.

Figure 5.5 Test for nitrification (nitrite production by Nitrosomonas).

Protocol

Test for Nitrosomonas

Take 25 mL broth of the medium in 50mL flat bottle separately for Nitrosomonas and Nitrobacter
bacterial isolation, inoculate with 100–200 μL soil suspension and incubate at room temperature
for 1 week, and measure the nitrite production in ammonium containing medium and nitrate
production in nitrite medium (Figure 5-6).

Measurement of Nitrite Production.

Mix 3–5 drops of Trommsdorf’s reagent with 1 drop of dilute sulfuric acid (1:3 concentration) on
a spot plate and transfer 2–3 drops of the culture from the ammonium medium to this reagent, mix
to avoid false result.

34
Appearance of a blue black color confirms the production of conversion of ammonium to nitrite.
Subsequently perform the test of the residual ammonium ions in the medium by Nessler’s reagent.
Negative test for absence of ammonium ions confirms the complete conversion of ammonium to
nitrite.
Perform the Gram stain test each time Gram-negative cocci confirm for the presence of
Nitrosomonas.

Test for Nitrobacter

Diphenylamine test is performed for the production of nitrate by Nitrobacter bacteria. This reagent
produces a blue–black color in the presence of nitrate or nitrites. It is, therefore, necessary to make
sure that no nitrite is present when it is used as a test for nitrates (Figure 5.6).

First, test the nitrite medium culture with Trommsdorf’s reagent to establish the absence of nitrites.
Mix one drop of diphenylamine reagent, two drops of concentrated sulfuric acid, and one drop of
culture on the plate. The blue–black color will be evidence of nitrate production. Perform the Gram
stain test each time Gram-negative cocci confirm for the presence of Nitrobacter.

RESULTS

35