Lab 5a ENUMERATION OF THE MICROBIAL POPULATION OF SOIL
Life on this planet could not be sustained in the absence of the microorganisms
that inhabit the soil. These flora are essential for degradation of organic matter
deposited in the soil as animal wastes and dead plant and animal tissue.
Soil contains many different microorganisms, including bacteria, fungi, protozoa,
algae and viruses. Bacteria and fungi are the most prevalent. It is essential to bear in
mind that the soil environment differs from one location to another and from one period
of time to another. Therefore factors such as moisture, pH, temperature, gaseous
oxygen content and organic and inorganic composition of soil will be crucial in
determining the specific microbial flora of a particular sample.
Microbial methods used to analyze soil vary as there is no single technique that
can be used to count all the different types of microorganisms present in a given soil
sample. Different microorganisms have different physical and nutritional requirements
necessary for the growth and there will be some that are unculturable.
The purpose of this laboratory exercise is to study the diversity in types and
numbers of the bacterial populations present in various types of soils.
Materials: (per pair)
1 g samples of soil
1 bottle with 9 ml sterile water
8 dilution tubes of 4.5 ml of sterile water
5 Agar Deep tubes with 1/10 strength Nutrient Agar
8 Sterile Petri Plates
4 Plates of Rose Bengal Agar
1 bottle of 9 ml Ethanol
3 Agar Deep tubes with ½ strength Tryptic Soy Broth Agar (TSBA)
Method:
1.
Add 1 g of soil (note soil type) to the bottle of 9 ml of water (10 -1 dilution) and
shake well. Also add 1 g of the same soil type to 9 ml of ethanol and place at
70C for 20 minutes in order to kill all vegetative cells.
2.
Prepare a dilution series by pipetting 0.5 ml from the 10-1 bottle to a tube of 4.5
ml of water and mix well. This will be the 10-2 dilution. Repeat the procedure by
drawing with a fresh pipette tip and adding it to a new tube of sterile water until
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you have a 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6 dilution.
3.
To enumerate bacteria label sterile petri dishes 10-2 to 10-6.
4.
Remove a molten agar deep of 1/10 strength nutrient agar from the waterbath
and add 1.0 ml of the 10-2 dilution. Immediately pour this into the appropriate
petri dish and swirl to mix.
5.
Repeat step 4 for dilutions 10-3 to 10-6.
6.
To enumerate fungi label the Rose Bengal Agar plates 10-1 to 10-4.
7.
Remove 0.1ml from the 10-1 tube and spread it (using sterile glass beads) on the
appropriate Rose Bengal plate.
8.
Repeat step 7 for the 10-2 to 10-4 dilutions.
9.
To enumerate the spores present in the soil remove the soil ethanol mix from the
waterbath. Pipette 0.5 ml of the ethanol/soil mixture to 4.5 ml of sterile water to
make a 10-2 dilution. Make a dilution series of 10-2 to 10-4 in the manner
described above.
10.
Label spore plates 10-2 to 10-4.
11.
Remove a tube of molten ½ strength TSBA from the waterbath, and add 1 ml
from the 10-2 tube. Immediately pour this into the appropriate petri dish and swirl
to mix. Repeat for the 10-3 and 10-4 dilutions.
12.
Allow all molten plates to solidify for 20 minutes before inverting. Incubate all
plates at room temperature for 14 days.
13.
Count the plates that are the appropriate dilution in the next laboratory period.
Compare counts from different soil types.
Notes About the Soil Lab:
1.
It is important to note that while the count data can easily be shared between lab
members, the qualitative results cannot. It is important, in order to assess the
diversity of microorganisms, to examine the different colours, morphologies and
other properties of the colonies that grow. Numbers alone cannot help answer
questions regarding diversity. There needs to be a qualitative assessment. So,
EVERY GROUP MEMBER SHOULD COME IN TO DO THIS. If you cannot
make it on Day 7 then come to the lab on Day 6 or Day 8.
2.
Both selective media and selective conditions were used to examine bacteria,
fungi and bacterial spores.
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a)
Low concentrations of nutrients in agar (1/10 and 1/2 strength) were used
in order to limit the growth of 'fast-growing' organisms. This gives all
organisms a chance to grow over the course of the 1 week incubation
period and helps reduce overgrowth. This will give a better indication of
the diversity present.
b)
Since fungi require a lot of oxygen, their growth is limited by the
microaerophilic conditions which exist within agar. Hence the use of pour
plates. So the first set of plates will contain bacteria, both the vegetative
cells as well as some spores.
c)
The Rose Bengal Agar plates contain chloramphenicol, which as we
remember from previous labs, inhibits bacteria growth, but not fungal.
Therefore these plates are selective for fungi only.
d)
Ethanol and heat were used to kill vegetative cells (bacteria and fungi)
leaving the more resilient spores. This process may not have killed all the
vegetative cells, and similarly may have killed some spores and therefore
could contribute to errors. Ideally what was left to grow are only bacterial
spores.
3.
As for other sources of error: by reporting the data as CFU/g soil it assumes that
all of the microorganisms were released from the soil in the initial step. Is this
reasonable?
4.
Be sure that you share data with, and examine the plates of, a group that did the
opposite soil type (i.e., garden vs. contaminated) as BOTH sets are needed to
answer the questions.
5.
The contaminant was phenol.
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Student #: _________________
CHEE 229 Lab 5 Questions (Hand in)
Read pp.1064-1066 of Prescott’s Microbiology, 8th Edition on “Biodegradation and
bioremediation”
1. Draw the structure of phenol.
2. What are the end products if phenol is mineralized by microorganisms in the soil?
3. Tabulate all data including dilutions values. Express calculated data as number
of cells per g of soil.
Dilution
No. of colonies
CFU/g soil
Garden soil
Bacteria (vegetative cells and spores)
Bacterial Spores only
Fungi
Contaminated soil
Bacteria (vegetative cells and spores)
Bacterial Spores only
Fungi
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4. Give 2 reasons why these results may only represent 10% of the total population
of bacteria and fungi in the soil samples.
5. Describe an enrichment technique to isolate a pure culture of a phenol-degrading
bacteria from the phenol-contaminated soil.
6. Sketch a graph to show the change in diversity, total cell number and phenol
concentration (on y-axis) over time (on x-axis) after garden soil is contaminated
with phenol.
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Lab 5b DEMONSTRATION: HISTOLOGICAL EXAMINATION OF CELLS,
TISSUES, AND ORGANS
Clinicians and researchers commonly examine prepared slides to characterize
cells, tissues, and organs. Slides can be prepared from thin sections of tissues of
interest, as well as from intact single or multi cell cultures. In general, the samples must
be fixed in a medium, such as formalin, that preserves the biological material in its
native state. The sample is often embedded in paraffin or plastic, and a microtome is
used to cut the resultant block into thin sections (typically 4 – 10 m thick), which can be
mounted on glass slides. The embedding material is then removed by solvent
extraction, and the samples can be stained to highlight structural or compositional
features of interest. The sample is then covered in a mounting medium, such as
permount, and a glass cover slip is added to ensure the long-term stability of the
sections. This laboratory demonstration involves the microscopic examination of two
different sets of histological slides: (i) the basic zoology (BZ) set and (ii) the introductory
biology (IB) sets. Demonstration microscopes have been set up with the slides at the
appropriate magnification (10X or 40X) for comparative purposes. Changing to a higher
magnification can result in accidental slide breakage. DO NOT CHANGE THE
OBJECTIVE. If you are having difficulty, please ask for help.
Use your time during the demonstration to examine the slides, make drawings
and notes but answer the questions later. However, the questions are a guide to the
observation you should make.
Suggested reference: www.microscopyu.com
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Student #: _________________
Questions (3 to Hand in)
1. Examine and make a sketch of the whole mount slides of lichen (IB2) and moss
(IB3). Describe the similarities and differences between the two organisms.
Based on the histology, identify which organism is a plant, and which is a
composite, symbiotic association between a fungus and either algae or
cyanobacteria.
Organs
2. The skin of mammals can generally be divided into two different regions: the
epidermis and the dermis. Examine the transverse section (BZ1) and draw the
architecture of the skin and label each region. Note the hair follicles extending
from the dermal region. Each hair follicle is lined with epidermal cells. Following
injury to the skin, rapid re-epithelialization is facilitated by the outgrowth of the
epidermal cells from the wound edges, as well as from the hair follicles. Discuss
the implications of this in terms of wound healing in humans versus animals.
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3.
Examine the lung section (BZ8) and make a sketch. What is the function of the
lungs? In terms of engineering design, describe how the cellular organization in
the lungs is tailored to ensure maximum lung efficiency.
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