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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 In this experiment, students will isolate microor-

ganisms from different environments and observe
After performing the experiment, the student shall them through an optical microscope. The method
be able to: used here is the agar plate method using selective
media that allows for the growth of specific types
r Isolate bacteria, yeast, and fungi from soil, air, or of microorganisms [i.e., nutrient agar is used for
water samples with the aid of selective media. the growth of bacteria, while potato dextrose agar
r Observe the morphology of the colonies obtained (PDA) is preferred for yeast growth, and Sabourad
and identify their major physical characteristics. medium for filamentous fungi].
r Stain when necessary and observe the results in Although this isolation method has several limita-
the microscope. 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
Introduction
agar exposed to air
r non-symbiotic, nitrogen-fixing organisms grow
Microorganisms have a considerable impact in the
environment as well as in our daily lives, as they under these conditions only to a limited extent
r many of the cellulose-digesting forms fail to grow
can gradually modify an ecosystem, cause disease
to plants, animals, and humans or they may be used or grow poorly on nutrient agar
for the production of compounds such as antibiotics, Therefore, the observed microorganisms represent
vaccines, enzymes added to detergents, or food such only a fraction of the total viable bacterial or yeast
as wine and yoghurt. population.
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
Experimental Procedure
the factor VIII involved in coagulation). It is impor-
Estimated time to complete the experiment: 3 h the
tant to isolate bacteria, yeasts, and filamentous fungi
first day and 30 min after 24–48 h
from their natural habitats, and to be able to purify,
characterize and classify them.
An important distinction is their capacity to live When doing microbiological laboratory work,
under various environmental conditions, sometimes the following rules must be followed:
even in extreme conditions of temperature, pH, and
salinity that other organisms cannot tolerate. 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 6. Using a sterile pipet, transfer 0.1 mL of the 10−2
placing objects in your mouth dilution to another test tube containing 9.9 mL of
3. Always wash your hands before leaving the the saline solution, and repeat the step in order to
lab have 10−4 and 10−6 dilutions. Mix thoroughly.
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 Use an aseptic technique when making serial di-
clean it up 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 213

Bacteria r With the slide still held at a 45◦ angle, decol-

orize it by allowing the acetone–alcohol mix-
1. Observe the macroscopic characteristics of the
ture to run over and off the smear.
colonies of microorganisms that grew on each r Rinse immediately with water.
plate. r Flood the slide with the safranine counterstain.
2. Prepare separate smears of the unknown bacteria
Allow the countestain to react for 1 minute.
in the following way: r Rinse the slide thoroughly with water.
r Place a drop of tap water at the center of a glass
r Carefully blot your stained slide and examine
slide.
r Using the inoculating loop, aseptically remove each smear in the microscope under high power
and oil immersion objectives.
half a loopful of bacteria from the culture.
r Mix the large amount of organisms on the loop The Gram stain separates bacteria into two
groups, depending on whether the original stain
into the drop of water on the slide.
r Spread the mixture. (violet crystal) is retained or lost when the stained
r Flame the loop to prevent contamination of the smear is treated with an iodine solution and then
washed with the acetone–alcohol mixture.
worktable, your culture and yourself.
r Allow the smear to air dry. Organisms that retain the stain when washed
r Heat-fix by passing the slide back and forth with alcohol are termed Gram positive; those that
fail to retain the original stain but take the coun-
through the flame of your Bunsen burner.
terstain (safranine) are called Gram negative. The
3. Perform the Gram stain as follows:
r Flood the slide with crystal violet and allow to Gram stain is of considerable value in identifying
and classifying bacteria.
react for 1 min.
r Handle the slide with slide forceps. Tilt it
Yeast and fungi
to an angle of approximately 45◦ and drain
the dye off the slide into a pan or staining Yeast and fungi are observed directly under the
sink. microscope by placing a loopful of each colony in
r Continue to hold the slide at 45◦ and immedi- a drop of water between a slide and a cover slip.
ately rinse it thoroughly with a gentle stream Observe the form, size, and number of different
of water from the wash bottle. colonies under the microscope (at high power and
r Flush the slide with iodine solution. Allow the oil immersion objectives).
iodine to react for 1 minute.
r Tilt the slide and allow it to drain.
Autoclave all Petri dishes when the experiment
r Immediately rinse the slide thoroughly with
is concluded to kill the isolated microorganisms
water.
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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 r The dilution factors may have to be changed as the
1. Why are bacteria and fungi classified in different original soil or water sample used will have differ-
kingdoms? ent microorganism concentrations. There should
2. What is the difference between yeasts and fila- be fewer than 50 colonies per Petri dish with the
mentous fungi? highest dilution aliquot.
r Dilutions can be plated by triplicate so that the
3. What are the mean sizes of these microorganisms? mean number of colony-forming units (CFU)/mL
4. What is a selective medium? present in the sample can be determined.
5. Describe the shapes of bacteria and yeasts.
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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 Beishir, L. Microbiology in Practice: A Self Instructional
1. What are the morphologic characteristics of the Laboratory Course; HarperCollins College Publishers:
isolated bacterial colonies? New York, NY, 1996.
Brown, A. E. Benson’s Microbiological Applica-
2. What type of microorganisms (bacteria, yeast
tions: Laboratory Manual in General Microbiology;
or fungi) predominate in the different samples McGraw-Hill Science/Engineering/Math: New York,
analyzed? 2006.
3. Why does one have to dilute the samples? Cappuccino, J.; Sherman, N. Microbiology: A Labora-
4. Why is the incubation time varied among the dif- tory Manual; Benjamin Cummings: Menlo Park, CA,
2004.
ferent types of microorganisms cultured?
Pepper, I. L; Gerba, C. P.; Brendecke, J. W. Environmental
Microbiology: A Laboratory Manual; Academic Press:
San Diego, CA, 1995.
Student Comments and Suggestions Sharma, P. D. Environmental Microbiology; Alpha Sci-
ence International: Harrow, U.K., 2005.

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