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Microorganisms in Soil, Water, and Air

Chapter · January 2008

DOI: 10.1007/978-0-387-49493-7_22

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Experiment 22
Microorganisms in Soil, Water, and Air
Reference Chapters: 7, 11
Objectives
After performing the experiment, the student shall
be able to:
r Isolate bacteria, yeast, and fungi from soil, air, or
water samples with the aid of selective media.
r Observe the morphology of the colonies obtained
and identify their major physical characteristics.
r Stain when necessary and observe the results in
the microscope.
Introduction
Microorganisms have a considerable impact in the
environment as well as in our daily lives, as they
can gradually modify an ecosystem, cause disease
to plants, animals, and humans or they may be used
for the production of compounds such as antibiotics,
vaccines, enzymes added to detergents, or food such
as wine and yoghurt.
Thanks to the genetic manipulation of microor-
ganisms, they can be used to synthesize products
foreign to their natural metabolism (e.g., the hu-
man hormone insulin, bovine growth hormone, and
the factor VIII involved in coagulation). It is impor-
tant to isolate bacteria, yeasts, and filamentous fungi
from their natural habitats, and to be able to purify,
characterize and classify them.
An important distinction is their capacity to live
under various environmental conditions, sometimes
even in extreme conditions of temperature, pH, and
salinity that other organisms cannot tolerate.
In this experiment, students will isolate microor-
ganisms from different environments and observe
them through an optical microscope. The method
used here is the agar plate method using selective
media that allows for the growth of specific types
of microorganisms [i.e., nutrient agar is used for
the growth of bacteria, while potato dextrose agar
(PDA) is preferred for yeast growth, and Sabourad
medium for filamentous fungi].
Although this isolation method has several limita-
tions, it is simple and can give an idea of the diver-
sity of microorganisms in different environments.
For example:
r anaerobes in soil fail to grow on the surface of
agar exposed to air
r non-symbiotic, nitrogen-fixing organisms grow
under these conditions only to a limited extent
r many of the cellulose-digesting forms fail to grow
or grow poorly on nutrient agar
Therefore, the observed microorganisms represent
only a fraction of the total viable bacterial or yeast
population.
Experimental Procedure
Estimated time to complete the experiment: 3 h the
first day and 30 min after 24–48 h
When doing microbiological laboratory work,
the following rules must be followed:
1. Use a protective garment (e.g., a lab coat)
211
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212 22. Microorganisms in Soil, Water, and Air

2. Never eat or drink in the laboratory, and avoid
placing objects in your mouth
3. Always wash your hands before leaving the
lab
4. If you spill living organisms, cover the spilled
material with paper towels and pour a labora-
tory disinfectant over the towels and the entire
contaminated area. Wait 15 min before you
clean it up
6. Using a sterile pipet, transfer 0.1 mL of the 10−2
dilution to another test tube containing 9.9 mL of
the saline solution, and repeat the step in order to
have 10−4 and 10−6 dilutions. Mix thoroughly.
Use an aseptic technique when making serial di-
lutions and plating. Always use a clean, sterile
pipet for all transfers.

Materials Reagents 7. Label each Petri dish (as 1–7) and add aliquots of
the dilutions and media according to the following
20 sterile Petri dishes, Dextrose potato agar
table:
60 × 15 mm Nutrient agar
10 1-mL pipets Sabourad agar
3 500-mL Erlenmeyer flasks Saline isotonic solution
1 mixing plate (NaCl 0.9%)
1 Bunsen burner Gram reagents, solutions of:
8 screw cap 13 × 100 mm – crystal violet stain
test tubes – Gram’s iodine solution
Test tube rack – acetone-alcohol mixture
Autoclave 70:30
2 Incubators (37◦ C and 30◦ C) – safranine stain
Inoculating loop
Glass slides and cover slips
Optical microscope
Immersion oil
Soil and water samples
Sterile spatula
Sterile mortar and pestle
8. Slowly, move each Petri dish so that the samples
become mixed with the culture medium.
1. Prepare 100 mL of an isotonic saline solution. 9. Let the agar solidify and incubate (at 37◦ C for nu-
Add 9.9 mL to each of six tubes and cap them. trient agar and 30◦ C for PDA and Sabourad media).
Sterilize for 15 minutes at 121◦ C. Water sample
2. Sterilize six 1-mL pipets.
10. Measure 0.1 mL of the liquid sample, dilute in
3. Prepare the necessary amount of nutrient agar, of 9.9 mL of saline solution and mix thoroughly. (This
Sabourad agar, and of potato dextrose agar, as indi- means a 1:100 dilution, or 10−2 ).
cated by the manufacturers (each Petri dish requires
11. Repeat steps 6–9.
10 mL of the medium). Mix thoroughly. Heat gently
and bring the mixture to boil. Autoclave for 15 min Air sample
at 15 psi and 121◦ C. Maintain at 45◦ C until they are
12. Prepare Petri dishes with each agar medium.
poured in sterile Petri dishes.
13. When the agar has solidified, open the dish for
Soil sample 20 min to allow airborne microorganisms contact
4. If necessary, place some soil in a sterile mortar the medium.
and break up the lumps with a pestle. 14. Close and incubate, as in step 9.
5. Weigh 0.1 g of the sample soil. Suspend in the 15. Observe and count the cultures after 24 and 48 h
9.9 mL of saline solution and mix thoroughly. (This for bacterial growth, and 48–72 h for yeast and fungi
means a 1:100 dilution, or 10−2 ). growth.
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Experimental Procedure
Bacteria
1. Observe the macroscopic characteristics of the
colonies of microorganisms that grew on each
plate.
2. Prepare separate smears of the unknown bacteria
in the following way:
r Place a drop of tap water at the center of a glass
slide.
r Using the inoculating loop, aseptically remove
half a loopful of bacteria from the culture.
r Mix the large amount of organisms on the loop
into the drop of water on the slide.
r Spread the mixture.
r Flame the loop to prevent contamination of the
worktable, your culture and yourself.
r Allow the smear to air dry.
r Heat-fix by passing the slide back and forth
through the flame of your Bunsen burner.
3. Perform the Gram stain as follows:
r Flood the slide with crystal violet and allow to
react for 1 min.
r Handle the slide with slide forceps. Tilt it
to an angle of approximately 45◦ and drain
the dye off the slide into a pan or staining
sink.
r Continue to hold the slide at 45◦ and immedi-
ately rinse it thoroughly with a gentle stream
of water from the wash bottle.
r Flush the slide with iodine solution. Allow the
iodine to react for 1 minute.
r Tilt the slide and allow it to drain.
r Immediately rinse the slide thoroughly with
water.
213
r With the slide still held at a 45◦ angle, decol-
orize it by allowing the acetone–alcohol mix-
ture to run over and off the smear.
r Rinse immediately with water.
r Flood the slide with the safranine counterstain.
Allow the countestain to react for 1 minute.
r Rinse the slide thoroughly with water.
r Carefully blot your stained slide and examine
each smear in the microscope under high power
and oil immersion objectives.
The Gram stain separates bacteria into two
groups, depending on whether the original stain
(violet crystal) is retained or lost when the stained
smear is treated with an iodine solution and then
washed with the acetone–alcohol mixture.
Organisms that retain the stain when washed
with alcohol are termed Gram positive; those that
fail to retain the original stain but take the coun-
terstain (safranine) are called Gram negative. The
Gram stain is of considerable value in identifying
and classifying bacteria.
Yeast and fungi
Yeast and fungi are observed directly under the
microscope by placing a loopful of each colony in
a drop of water between a slide and a cover slip.
Observe the form, size, and number of different
colonies under the microscope (at high power and
oil immersion objectives).
Autoclave all Petri dishes when the experiment
is concluded to kill the isolated microorganisms
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214 22. Microorganisms in Soil, Water, and Air

Name Section Date

Instructor Partner

PRELABORATORY REPORT SHEET—EXPERIMENT 22

Objectives:

Flow sheet of procedure

Waste containment procedure:

PRELABORATORY QUESTIONS AND Additional Related Projects:

PROBLEMS
1. Why are bacteria and fungi classified in different
kingdoms?
2. What is the difference between yeasts and fila-
mentous fungi?
3. What are the mean sizes of these microorganisms?
4. What is a selective medium?
5. Describe the shapes of bacteria and yeasts.
r The dilution factors may have to be changed as the
original soil or water sample used will have differ-
ent microorganism concentrations. There should
be fewer than 50 colonies per Petri dish with the
highest dilution aliquot.
r Dilutions can be plated by triplicate so that the
mean number of colony-forming units (CFU)/mL
present in the sample can be determined.
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Experimental Procedure 215

Name Section Date

Instructor Partner

LABORATORY REPORT SHEET—EXPERIMENT 22

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216 22. Microorganisms in Soil, Water, and Air

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Experimental Procedure 217

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218 22. Microorganisms in Soil, Water, and Air

POSTLABORATORY PROBLEMS Literature References

AND QUESTIONS
1. What are the morphologic characteristics of the
isolated bacterial colonies?
2. What type of microorganisms (bacteria, yeast
or fungi) predominate in the different samples
analyzed?
3. Why does one have to dilute the samples?
4. Why is the incubation time varied among the dif-
ferent types of microorganisms cultured?
Student Comments and Suggestions
Beishir, L. Microbiology in Practice: A Self Instructional
Laboratory Course; HarperCollins College Publishers:
New York, NY, 1996.
Brown, A. E. Benson’s Microbiological Applica-
tions: Laboratory Manual in General Microbiology;
McGraw-Hill Science/Engineering/Math: New York,
2006.
Cappuccino, J.; Sherman, N. Microbiology: A Labora-
tory Manual; Benjamin Cummings: Menlo Park, CA,
2004.
Pepper, I. L; Gerba, C. P.; Brendecke, J. W. Environmental
Microbiology: A Laboratory Manual; Academic Press:
San Diego, CA, 1995.
Sharma, P. D. Environmental Microbiology; Alpha Sci-
ence International: Harrow, U.K., 2005.

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