EDVO-Kit #
958
Ecological
Equilibrium I:
Population Studies of
Unicellular Organisms
Storage:
Store all components
at room temperature.
EXPERIMENT OBJECTIVES:
The objective of this experiment is to determine the
impact of varying growth conditions on microbial
growth and measure the number of cells using
the colony count method.
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EDVO-Kit # 958
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
Table of Contents
Page
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Experiment Components
Experiment Requirements
3
3
Background Information
4
Experiment Procedures
Experiment Overview
Student Experimental Procedures
Study Questions
9
10
15
Instructor's Guidelines
Notes to the Instructor
PreLab Preparation
Expected Results
Study Questions and Answers
17
18
20
21
Material Safety Data Sheets
22
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EDVO-Kit # 958
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
Experiment Components
•
•
•
•
•
NaOH solution - Component A
HCl solution - Component B
Salt (NaCl) solution - Component C
Yeast Extract - Component D
Tryptone - Component E
•
•
•
•
•
•
•
•
•
•
•
E. coli slant
ReadyPour™ Luria Broth Agar
Nutrient Broth
Petri plates, small
Pipets
Inoculating loops
Sterile swabs
Microcentrifuge tube with attached caps (0.5 ml)
Conical tubes with screw caps (15 ml)
Calibrated transfer pipets.
pH paper
This experiment is
designed for 10 student
groups.
Store all components at
room temperature.
No human or animal
blood or blood
products are used
in this experiment.
Requirements
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Incubation oven
Microwave, hot plate or Bunsen burner (for heating ReadyPour agar)
Waterbath
UV transilluminator
Vortex (optional)
Automatic micropipets (5-50 µl) with tips
Lab glassware
Squeeze bottle
Foil, plastic wrap or parafilm
95% ethanol
Bleach or disinfectant
Sunscreen
Distilled water
Disposable vinyl or latex gloves
Goggles
All components are
intended for educational research only.
They are not to be
used for diagnostic
or drug purposes,
nor administered
to or consumed by
humans or animals.
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EDVO-Kit # 958
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
Background Information
Background Information
A unicellular organism is any life form that consists of just a single cell. Most
of life is unicellular, with bacteria serving as the majority. The main groups
of unicellular life are bacteria, archaea (prokaryotes), and the eukaryota
(eukaryotes). The differences between the prokaryota and eukaryota are
significant: eukaryotes possess a nucleus, while prokaryotes lack it, and
eukaryotes possess a range of subcellular organs called organelles, while
prokaryotes are very minimal.
Unicellular organisms are as diverse as they are ubiquitous. The oldest forms
of life, unicellular organisms existed 3.8 billion years ago, if not longer. They
pursue a variety of strategies for survival: photosynthesis (cyanobacteria),
chemotrophy (many archaea), and heterotrophy (amoeba). Some unicellular
organisms have flagella, little tails they use for locomotion, or lobopods,
extensions of the cellular skeleton (cytoskeleton), which appear as blob-like
arms. The flagellum of our unicellular ancestors is retained all the way up
into the animals, where it makes an appearance as flagellated sperm.
Of all the six-eukaryote supergroups, four are exclusively composed of
unicellular organisms. Only the opisthokonts, consisting of animals, fungi,
and close relatives, and the archaeplastids, consisting of both unicellular and
multicellular plants, are exceptions. Unicellular organisms vary in size, with
the smallest bacteria measuring only a third of a micron (300 nanometers)
across, ranging up to the titantic plasmodial slime molds, which can grow to
20 cm (8 in) across. The largest unicellular organisms may have millions of
nuclei scattered throughout the cellular envelope. To observe some of the
smallest unicellular organisms requires an expensive electron microscope,
while the very largest can be seen with the naked eye.
One can observe the larger unicellular organisms, such as amoebae, by using
the higher settings on a light microscope. Bacteria just appear as dots. To
gather unicellular organisms for observation, one can place a cover slip on
the surface of pond water, and leave it overnight. By the next morning, numerous unicellular organisms will have grown entire colonies on the bottom
of the slip. Unicellular organisms replicate fast: colonies can double their
size in between 30 minutes and a few hours.
EDVOTEK and
The Biotechnology
Education Company
are registered
trademarks of
EDVOTEK, Inc.
Practically every phrase of microbiology requires methods for measuring
microbial number. The most common method is the plate or colony count.
This method is based on the theoretical relationship of one bacterial cell,
or clump of cells, giving rise to one colony and on the assumption that the
number of colonies that develop on an agar plate corresponds to the original bacterial count. To obtain the bacterial count, the sample is diluted and
plated, and following the incubation the colonies that develop are counted.
To make a tenfold dilution of a sample, one will add 1 ml portion of the
original sample to a tube containing 9 ml of sterile media. By continuing
this dilution stepwise through additional dilution tubes, one can dilute the
sample 10-2, 10-3, 10-4, and more in a manner outlined in Figure 1.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/
laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose
without the written consent of EDVOTEK, Inc. Copyright © 2008, 2009 EDVOTEK, Inc., all rights reserved.
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EDVO-Kit # 958
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
5
Background Information
1 ml
1 ml
1 ml
1 ml
9 ml broth
in each tube
10-1
Dilutions
1 ml
10-2
1 ml
10-3
1 ml
10-4
1 ml
10-5
1 ml
Plating
10-1
10-2
10-3
10-4
10-5
Figure 1 – Diluting and plating sample for determination of microbial numbers.
Microbes, like all forms of life, are greatly influenced by their surroundings. Environmental effects fall into three general categories: physical – the
effects of temperature or extreme pressure; chemical- the need for food
and response to poison; and biological – the influences of coexisting species. There is a particular optimum set of environmental conditions for each
microbial species. The human body, for example, has a rigidly regulated normal temperature of 37°C that must remain constant despite of exposure to
extreme heat or cold. The bacterial cell has no regulated body temperature,
but assumes the temperature of its environment. Its response to prolonged
exposure to extreme cold or heat is merely the stopping of the enzymatic
activities; it does not necessarily die as would higher forms of life.
Temperature profoundly influences the growth of bacteria. Different types
of bacteria have distinct requirements as to the temperature at which they
will grow. Between the maximum temperature, above which a culture will
not develop, and a minimum temperature, below which a culture will not
develop, is a temperature range in which growth will occur. The best growth
takes place within a rather limited range called the optimum temperature.
The influence of temperature on growth is actually a measure of the influence of temperature on the enzyme actions of the cell. As the temperature
is lowered, the enzyme activity, thus the growth of the cell, is lowered. As
the temperature is raised above the optimum for growth, metabolic activity
is markedly increases, but at the same time, the rate of enzyme and protein
breakdown (due to protein denaturation) markedly increases, resulting in
damage and death of cell.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/
laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose
without the written consent of EDVOTEK, Inc. Copyright © 2008, 2009 EDVOTEK, Inc., all rights reserved.
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Background Information
Original
inoculum
1 ml
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EDVO-Kit # 958
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
Background Information
Background Information
Enzyme destruction
Point of diminishing returns
Increase
Enzyme activity
Total work done
0°C
20°C
37°C
50°C
Bacterial cells and the media in which they grow have
independent osmotic concentrations – a function and
relationship of substances in solution. When a bacterial cell is placed in a medium, an osmotic pressure
is exerted across the semipermeable membrane that
surrounds the cell. Osmotic pressure is an important
factor affecting cells. When the osmotic concentration of the medium is higher than that of the cell
interior, the medium is said to be hypertonic with
respect to the cell. In a hypertonic medium, water
leaves the cell, and the cytoplasmic membrane shrink
Figure 2 - Relationship between temperature and
enzyme destruction in a bacterial cell.
Neutrophiles
Acidophiles
Each species of microbe has its own characteristic
range of pH values in which it grows and reproduces
best. Most bacteria grow best around neutral pH
values (pH 5.5 - 8.5). Bacteria that grow best at this
pH range are referred to as neutrophiles. However,
some bacteria thrive in very acid conditions (pH 1
- 5.5). They are called acidophiles. Other bacteria
can live in very alkaline environment (pH 8.0 - 11.5).
Such alkaline loving microbes are called alkaliphiles.
Since pH limits the types of microorganisms capable
of growing under given conditions, its control has
considerable practical importance. For instance,
sulfur is sometimes added to soil to inhibit the
growth of Streptomyces scabies microbes causing
potato scab. Sulfur is converted to acid by certain soil
bacteria, and it is this resulting drop in the pH that
inhibits the potato pathogen.
Alkaliophiles
Growth
Rate
1
2
3
4
5
pH
6
7
8
9
10
11
Figure 3 - Growth rate vs. pH for three environmental
classes of prokaryotes.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/
laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose
without the written consent of EDVOTEK, Inc. Copyright © 2008, 2009 EDVOTEK, Inc., all rights reserved.
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EDVO-Kit # 958
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
7
Background Information
Hypertonic
H2O
Isotonic
H2O
Hypotonic
H2O
Figure 4 – Effect of osmotic pressure on blood cells.
A microorganism’s growth in a culture medium depends partly upon the
amount of available energy. Every organism must find in its environment all
of the substances required for energy generation and cellular biosynthesis.
The chemicals and elements of the environment that are utilized for bacterial
growth are referred to as nutritional requirements, a source of carbon and
other required nutrients. The nutritional requirements vary widely among
microorganisms. Autotrophic microorganisms, including those that are photosynthetic, can grow and synthesize their cell materials solely from inorganic
compounds; heterotrophic species requires one or more organic nutrients.
At an elementary level, the nutritional requirements of a bacterium such
as E. coli are revealed by the cell's elemental composition, which consists of
C, H, O, N, S. P, K, Mg, Fe, Ca, Mn, and traces of Zn, Co, Cu, and Mo. These
elements are found in the form of water, inorganic ions, and other small
molecules.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/
laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose
without the written consent of EDVOTEK, Inc. Copyright © 2008, 2009 EDVOTEK, Inc., all rights reserved.
EVT 090914AM
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Background Information
away from the rigid cell wall – a condition called plamolysis. In contrast, when
the osmotic concentration of the medium is lower than that of the cell interior
(a hypotonic medium), water flows across the cell membrane, causing the cell
to swell. In plant cells, the cell wall restricts the expansion, resulting in pressure
on the cell wall from within called turgor pressure. Isotonic is the presence of
a solution that produces no change in cell volume. Osmoregulation is the homeostasis mechanism of an organism to reach balance in osmotic pressure. The
effect of osmotic pressure is of great practical importance in the bacteriology
of food. The preservation of certain food (salted meat, jam, condensed milk) is
partially or completely due to their high osmotic concentrations.
8
EDVO-Kit # 958
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
Background Information
Background Information
G
C
A
T
G
C
A
T
Ultraviolet (UV) light kills cells by damaging their
DNA. The light initiates a reaction between two
molecules of thymine, one of the bases that make
up DNA. UV light at this short wavelength causes
adjacent thymine molecules on DNA to dimerize. The
resulting thymine dimer is very stable. If enough of
these defects accumulate on a microorganism's DNA
its replication is inhibited, thereby rendering it harmless. The longer the exposure to UV light, the more
thymine dimmers are formed in the DNA. If cellular
processes are disrupted because of DNA damage,
the cell cannot carry out its normal function. If the
damage is extensive and widespread, the irradiated
bacterial cell will die.
ht
UV
T
Dimer
A
A
Lig
The series of exercises in this experiment will illustrate
the response of E.coli to various environmental conditions – temperature, pH, osmotic potential, energy
source, and ultraviolet light.
T
G
C
A
T
Figure 5 – Lethal action of Ultraviolet light on DNA.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/
laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose
without the written consent of EDVOTEK, Inc. Copyright © 2008, 2009 EDVOTEK, Inc., all rights reserved.
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EDVO-Kit # 958
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
9
Experiment Overview
BEFORE YOU START THE EXPERIMENT:
Read all instructions before starting the experiment.
2.
Write a hypothesis that reflects the experiment and predict experimental
outcomes.
LABORATORY SAFETY:
1.
No human materials are used in this experiment. Gloves and safety goggles
should be worn as good laboratory practice.
2.
Exercise extreme caution when working with equipment which is used in conjunction with the heating and/or melting of reagents.
3.
DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS OR BULBS.
4.
The bacterium used in this experiment is not considered pathogenic, but
all microorganisms have the potential to cause disease in some individuals.
Although E. coli is rarely associated with illness in healthy individuals, it is
good laboratory practice to follow simple safety guidelines in handling and
disposal.
At the completion of the experiment:
A. Wipe down the lab bench with a 10% bleach solution, disinfectant or
soapy water.
B.
C.
All materials, including petri plates, pipets, transfer pipets, loops and
tubes , that come in contact with bacteria should be disinfected before
disposal in the garbage. Disinfect materials as soon as possible after use
in one of the following two ways:
•
Autoclave at 121° C for 20 minutes. Tape several petri plates together and close tube caps before disposal. Collect all contaminated
materials in a autoclavable, disposable bag. Seal the bag and place it
in a metal tray to prevent any possibility of liquid media or agar from
spilling into the sterilizer chamber.
•
Soak in a 10% bleach or disinfectant solution. Immerse petri plates,
open tubes and other contaminated materials into a tub containing a
10% bleach solution. Soak the materials overnight and then discard.
Wear gloves and goggles when working with bleach.
Always wash hands thoroughly with soap and water after handling contaminated materials.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/
laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose
without the written consent of EDVOTEK, Inc. Copyright © 2008, 2009 EDVOTEK, Inc., all rights reserved.
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Experiment Procedures
Wear gloves
and safety goggles
1.
10
EDVO-Kit # 958
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
Student Experimental Procedures
Experiment Procedures
HINT:
VARIOUS ECOLOGICAL CONDITIONS/VARIABLES:
Any dilution or plating procedure will be
simplified if dilution
tubes and petri plates
are arranged at the
beginning. Mark the
tubes and plates according to the conditions and dilutions.
Loops and other
materials used in this
experiment should
be properly disposed
in a designated
disinfectant tray.
Temperature:
This option requires 4-6 different chambers (incubators, refrigerators, etc.) for
incubation of cultures. Suggested temperatures are 4° C, 20° C, 37° C, and 65° C.
It is important to maintain accurate temperatures.
1.
Dispense 5.0 ml sterile nutrient broth into sterile tubes labeled with the
desired temperature growth conditions.
2.
Obtain the OD550 0.05 stock culture of E.coli from your lab instructor. Mix
the culture in between each aliquot and dispense 10 µl of this culture into
each of the tubes containing the nutrient broth from step 1.
3.
Cap loosely and place the tubes in the desired incubator.
4.
Grow the cultures for no longer than 19 hours.
5.
Continue with "General Plating Procedure" on page 13.
pH:
With this option, students will use NaOH (a base which raises pH) and HCl (an acid
which lowers pH) to adjust the pH of the bacterial growth medium used for growing the cultures. Suggested pHs are 4.0, 7.0, and 10.0. Use caution when working
with NaOH and HCl – wear gloves and safety goggles!!
1.
Aseptically dispense 5.0 ml sterile nutrient broth into tubes labeled with the
desired pH.
2.
Add diluted HCl or diluted NaOH (start by adding one drop at a time) to the
tubes containing nutrient broth. Cap, mix, and use the pH paper to check
the pH of each tube. Adjust by adding additional NaOH or HCl until the
desired pH is obtained.
3.
Use the OD550 0.05 stock culture of E.coli previously obtained from your lab
instructor.
4.
Mix the culture in between each aliquot and dispense 10 µl of this culture
into each of the tubes from steps 1 and 2.
5.
Cap loosely and place the tubes in a rack in a 37°C incubator.
6.
Grow the cultures for no longer than 19 hours.
7.
Continue with "General Plating Procedure" on page 13.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/
laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose
without the written consent of EDVOTEK, Inc. Copyright © 2008, 2009 EDVOTEK, Inc., all rights reserved.
EVT 090914AM
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EDVO-Kit # 958
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
11
Student Experimental Procedures
Osmotic Potential:
Invert plates (agar side
up) during incubation.
This prevents
condensation from
dropping onto the agar
surface.
1.
Use the following formula to calculate the salt concentration:
V (M) ÷ FV = FM
Example:
V=?
M = 5 M NaCl
FV = 6 ml
FM = 0.5 M NaCl
Use caution when
working with NaOH
and HCl – wear gloves
and safety goggles!!
V = volume of salt to be added
M = molarity of salt stock
FV = final volume of total solution
FM = final molarity of salt in total solution
V (5 M) ÷ 6 ml = 0.5 M
V = 0.6 ml of 5 M NaCl to a final volume of 6 ml (with sterile nutrient broth)
to yield 0.5 M NaCl. Label accordingly.
2.
Repeat Step 1, using the above formula to calculate the salt concentration
for the remaining growth medium tubes (i.e. 0.1M, 0.3M, 1.0M).
3.
Use the OD550 0.05 stock culture previously obtained from your lab instructor.
4.
Mix the culture in between each aliquot and dispense 10 µl of this culture
into each of the tubes from steps 1 and 2.
5.
Cap loosely and place the tubes in a rack in a 37°C incubator.
6.
Grow the cultures for no longer than 19 hours.
7.
Continue with "General Plating Procedure" on page 13.
Energy Source:
With this option, students can make adjustments to the nutrient source in the
bacterial growth medium used for growing the cultures and decide which combinations yield optimum growth.
1.
Aseptically dispense 5.0 ml sterile H2O into tubes.
2.
Add 100 µl of NaCl stock to each tube. Label tubes and add varying amounts
of tryptone and yeast extract concentrate to the desired concentration.
Suggestions include 1 ml yeast extract/1 ml tryptone, 2 ml yeast extract/1 ml
tryptone, 1 ml yeast extract/2 ml tryptone, 1 ml yeast extract/no tryptone, no
yeast extract/1 ml tryptone, etc.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/
laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose
without the written consent of EDVOTEK, Inc. Copyright © 2008, 2009 EDVOTEK, Inc., all rights reserved.
EVT 090914AM
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Experiment Procedures
By adjusting the salt concentration of the growth medium, students will determine the effect of changing the osmotic potential on the growth of the bacteria.
Suggested salt concentrations to try: 0.1 M, 0.3 M, 0.5 M, 1.0 M
12
EDVO-Kit # 958
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
Student Experimental Procedures
Experiment Procedures
Energy Source, continued...
3.
Use the OD550 0.05 stock culture previously obtained from your lab instructor. Mix the culture in between each aliquot and dispense 10 µl of this
culture into each tube from Steps 1 and 2.
4.
Cap loosely and place all the tubes in a rack in a 37°C incubator.
5.
Grow the cultures for no longer than 19 hours.
6.
Continue with "General Plating Procedure" on page 13.
Ultraviolet Light:
Students will observe the effects of ultraviolet rays and the effectiveness
of sunscreen in protecting the cells from the harmful light. By exposing the
cultures to UV light at different time intervals, students will also observe the
correlation between UV light exposure and cell death.
1.
Obtain the OD550 0.05 stock culture of E.coli. Mix and add 0.1 ml of the
culture to a fresh tube containing 5 ml of sterile nutrient broth.
2.
Mix well, cap the tube, and allow the culture to incubate for 2 hours (shaking or stationary) at 37°C.
3.
Set up 3 microcentrifuge tubes as follows:
•
Tube 1: Cover its body with aluminum foil and label it as "CP"
(Complete Protection).
•
Tube 2: Apply a thin layer of SPF sunscreen around the tube.
Label the tube as "SA" (Sunscreen Application).
•
Tube 3: Leave the tube as-is and label it "CE" (Complete Exposure).
4.
After the 2-hour incubation time, remove the culture tube from the incubator. Transfer 500 µl of the culture into each of the tubes in step 3.
5.
Starting with the tube labeled "CP", mix and lay it on its side directly on a
transilluminator (254 - 302 nm).
6.
Set the timer for 20 seconds. Turn on the transilluminator to expose the cells
to the light for 20 seconds. Be sure to shield eyes and skin!
7.
Turn the light off and remove 50 µl to a tube labeled "20 sec" and place on
ice.
8.
Repeat the exposure for different time periods until you have collected
a sufficient number of data points. Remember to take samples from the
same culture tube that has been exposed to increasing time on the UV light
source.
9.
Repeat steps 6-8 for the "SA" and "CE" tubes.
10. After all the time points have been collected, follow steps 3 - 6 of the "General Plating Procedure" on page 13 to inoculate the plates.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/
laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose
without the written consent of EDVOTEK, Inc. Copyright © 2008, 2009 EDVOTEK, Inc., all rights reserved.
EVT 090914AM
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EDVO-Kit # 958
13
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
Student Experimental Procedures
GENERAL PLATING PROCEDURE
After the incubation period is complete, remove cultures from the 37°C incubator and proceed to diluting (if required) and plating.
Temperature: See Table I on page 14 for dilutions.
pH, Osmotic Potential, Energy source: See Table II on page 14 for dilutions.
2.
Aseptically dispense 5 µl of each culture onto a labeled agar plate. Using a
sterile loop, spread the culture evenly over the entire surface of the plate.
Discard the loop in the disinfectant tray.
To avoid contamination
when plating, do not set
the lid down on the lab
bench -- Lift the lid of the
plate only enough to allow
spreading. Be careful to
avoid gouging the loop
into the agar.
3.
Spread
in one
direction
Same plate spread again in
opposite direction.
Stack your group's set of plates on top of one another and
tape them together. Put your initials or group number on
the taped set of plates.
Group 4
The plates should be left in the upright position to allow the
samples to be absorbed by the agar.
4.
Place the set of plates in a safe place designated by your
instructor.
5.
After the samples are completely absorbed by the agar,
you or your instructor will place the plates in the inverted
position (agar side on top) in a 37°C bacterial incubation oven for overnight
incubation (19-24 hours).
If the samples have not been absorbed into the medium, it is best to incubate
the plates upright. The plates are inverted to prevent condensation on the lid,
which could drip onto the culture and interfere with experimental results.
continued
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/
laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose
without the written consent of EDVOTEK, Inc. Copyright © 2008, 2009 EDVOTEK, Inc., all rights reserved.
EVT 090914AM
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Experiment Procedures
1.
14
EDVO-Kit # 958
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
Student Experimental Procedures
Experiment Procedures
GENERAL PLATING PROCEDURE, CONTINUED
6.
Store all plates inverted at 37°C. After 19-24 hour incubation, remove plates
from incubator, count and record the number of colonies on each plate.
Calculate the CFU/ml based on the following formula.
CFU/ml original sample = CFU/plate x (1/ml aliquot plated) x dilution factor
For Example:
89 colonies counted on plate x 1/0.005 ml (aliquot) x 104 (dilution factor) =
1.78 x 108 CFU/ml
TABLE I: DILUTIONS FOR TEMPERATURE CONDITION
4° C incubated tube: Dilute the original culture to a 10-1 dilution.
20° C incubated tube: Dilute the original culture to a 10-3, 10-4, and 10-5 dilution.
37° C incubated tube: Dilute the original culture to a 10-3, 10-4, and 10-5 dilution.
65° C incubated tube: No dilution required.
TABLE II: DILUTIONS FOR PH, OSMOTIC POTENTIAL, ENERGY SOURCE CONDITIONS
Serially dilute the original culture to a 10-3, 10-4, and 10-5 dilution.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/
laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose
without the written consent of EDVOTEK, Inc. Copyright © 2008, 2009 EDVOTEK, Inc., all rights reserved.
EVT 090914AM
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EDVO-Kit # 958
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
15
Study Questions
Answer the following study questions in your laboratory notebook or on a
separate worksheet.
Name some of the environmental factors that profoundly influence the
growth of a microorganism.
2.
Compare and contrast acidophiles and alkaliphiles. Describe one example to illustrate practical applications of such microorganism in the
environment.
3.
What force does a bacterial cell experience as it is placed in a medium.
What determines whether a cell is hypertonic or hypotonic?
4.
In what specific way does ultraviolet light damage bacterial cell?
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/
laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose
without the written consent of EDVOTEK, Inc. Copyright © 2008, 2009 EDVOTEK, Inc., all rights reserved.
EVT 090914AM
!"
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Experiment Procedures
1.
16
EDVO-Kit # 958
EVT 090914AM
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
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EDVO-Kit # 958
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
17
Notes to the Instructor
Prelab preparations require approximately 45-60 minutes.
Agar plates can be prepared up to a week in advance.
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experiments for
biotechnology and
biology education.
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-TE
C H S E RV I C E
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email:
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Please have the following information:
• The experiment number and title
• Kit Lot number on box or tube
• The literature version number
(in lower right corner)
• Approximate purchase date
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/
laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose
without the written consent of EDVOTEK, Inc. Copyright © 2008, 2009 EDVOTEK, Inc., all rights reserved.
EVT 090914AM
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
Instructor's Guide
This experiment module contains biologicals and reagents for 10 Lab Groups.
18
EDVO-Kit # 958
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
Pre-Lab Preparations
Instructor's Guide
PREPARING INITIAL CULTURE (INOCULUM)
To be done the afternoon before the lab.
Wear Hot Gloves
and Goggles during all
steps involving heating.
Quick Reference:
Pouring Agar Plates
• Use a sterile 10 ml pipet with
a pipet pump to transfer the
designated volume of medium
to each petri plate. Pipet carefully to avoid forming bubbles.
1.
Use a sterile loop to inoculate 35 ml sterile nutrient broth with a small
loopful of cells scraped from the E.coli cell slant.
2.
Add the cells to the nutrient broth and cap – vortex or shake well to
resuspend cells.
3.
Discard the loop in the disinfectant tray.
4.
Grow cells @ 37°C overnight (~19 hours). The next day, check OD550 and
dilute the cells to 0.05 with additional sterile nutrient broth.
5.
Dispense 2 ml of the culture into tubes for each of the 10 groups.
6.
The inoculum is now ready for the experiment.
PLATING
Pouring LB plates (Prior to the Lab experiment)
Heat to Melt the ReadyPour™ Medium
1.
Equilibrate a water bath at 60°C for step 5 below.
2.
Loosen, but do not remove, the cap on the ReadyPour™ medium
bottle to allow for the venting of steam during heating.
Caution: Failure to loosen the cap prior to heating or microwaving
may cause the ReadyPour™ medium bottle to break or explode.
• Rock the petri plate back and
forth to obtain full coverage.
• If the molten medium contains
bubbles, they can be removed
by passing a flame across the
surface of the medium.
• Cover the petri plate and allow the medium to solidify.
3.
Squeeze and vigorously shake the plastic bottle to break up the
solid agar into chunks
4.
Heat the bottle of ReadyPour™ medium by one of the methods
outlined below. When completely melted, the amber-colored solution should appear free of small particles.
A.
•
•
•
Microwave method:
Heat the bottle on High for two 30 second intervals.
Using a hot glove, swirl and heat on High for an additional 25
seconds, or until all the ReadyPour™ medium is dissolved.
Using a hot glove, occasionally swirl to expedite melting.
B.
•
•
•
Hot plate or burner method:
Place the bottle in a beaker partially filled with water.
Heat the beaker to boiling over a hot plate or burner.
Using a hot glove, occasionally swirl to expedite melting.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/
laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose
without the written consent of EDVOTEK, Inc. Copyright © 2008, 2009 EDVOTEK, Inc., all rights reserved.
EVT 090914AM
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
EDVO-Kit # 958
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
19
Pre-Lab Preparations
Allow the melted ReadyPour™ medium to cool slightly. Placing the
bottle in a 60°C water bath will allow the agar to cool, while preventing
it from prematurely solidifying.
When the ReadyPour™ reaches approximately 60°C, the bottle will be
warm to the touch but not burning hot.
6.
Pour small LB plates
•
Use a pipet pump and 10 ml pipet to dispense 5 ml of ReadyPour™
medium to each plate. You will need a minimum of 50 plates.
•
Replace the lid onto each plate after the medium has been poured.
•
Stack the plates and allow them to cool & solidify
•
Refrigerate plates (wrapped in plastic sleeves)
inverted.
PREPARING COMMON SOLUTIONS FOR LAB STATIONS
Instructor will set up various lab stations that will allow the students to study
different growth conditions. Student groups are encouraged to explore
themselves to these lab stations based on their own interests to determine
the impact of varying growth conditions on microbial growth.
1.
2.
3.
pH-based Lab Station
a.
NaOH Solution: Add all of the concentrated NaOH solution (Component A) to 45 ml of distilled water. Mix. Label with "diluted
NaOH solution".
b.
HCl Solution: Add all of the concentrated HCl solution (component
B) to 45 ml of distilled water. Mix Label this "diluted HCl solution".
Osmotic Potential - based Lab Station
a.
Remove 3 ml of the provided Salt Solution (Component C) into a
tube. Save this tube for the Energy Source-based Lab Station.
b.
Use the remaining Salt Solution to provide to this lab station. Students will use this for their experiment.
Energy Source-based Lab Station
a.
Salt Solution: provide this lab station the tube containing 3 ml of
the 5M Salt Solution. Students will use this for their experiment.
b.
Yeast Extract and Tryptone Solutions: provide this lab station the
Yeast Extract (Component D) and Tryptone (Component E) solutions
that are included in this kit.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/
laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose
without the written consent of EDVOTEK, Inc. Copyright © 2008, 2009 EDVOTEK, Inc., all rights reserved.
EVT 090914AM
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
Instructor's Guide
5.
20
EDVO-Kit # 958
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
Expected Results
Instructor's Guide
1.
The more diluted the culture is, the fewer colonies are seen when culture is plated.
(i.e. 10-5 dilution yield fewer colonies than 10-3 dilution does).
2.
When a microorganism is placed out of its optimal growth conditions
(temperature, pH, etc.), it will perform less effectively.
(i.e. Higher temperature or higher pH cultures yield fewer colonies than
37°C culture and pH 7.0 colonies.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/
laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose
without the written consent of EDVOTEK, Inc. Copyright © 2008, 2009 EDVOTEK, Inc., all rights reserved.
EVT 090914AM
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
EDVO-Kit # 958
Ecological Equilibrium I:
Population Studies of Unicellular Organisms
21
Study Questions and Answers
Name some of the environmental factors that profoundly influence the
growth of a microorganism.
Factors influence bacterial growth are temperature, pH, osmotic pressure, energy source and ultraviolet light.
2.
Compare and contrast acidophiles and alkaliphiles. Describe one example to illustrate practical applications of such microorganism in the
environment.
Both acidophiles and alkaliphiles greatly depend on the pH of their
external environment to live and reproduce. While acidophiles live at pH
around 1-5.5, alkaliphiles prefer pH around 8.0 - 11.5.
The S. scabies, a microorganism that can survive indefinitely in slightly
alkaline soil but is relatively scarce in highly acid soils, is known to cause
potato scab. Sulfur is added to the soil and then converted to acid by
certain soil bacteria, and it is this resulting drop in the pH that inhibits
the potato pathogen.
3.
What force does a bacterial cell experience as it is placed in a medium.
What determines whether a cell is hypertonic or hypotonic?
Once a bacterial cell is placed in a medium, it experiences osmotic pressure, the movement of water molecules from an area of high concentration to an area of low concentration.
The movement of water into or out of a cell determines whether the
cell is hypertonic or hypotonic to its medium. If a cell is hypertonic to its
environment (water is in higher concentration inside the cell), water will
move by osmosis, to the outside in an attempt to equalize the concentrations. Water leaving the cell will cause shrinkage of the cell. If a cell is
hypotonic to its environment (water is in higher concentration outside
of the cell), water will move, by osmosis, to the inside of the cell, causing
the cell to swell.
4.
In what specific way does ultraviolet light damage bacterial cell?
Ultraviolet light damages bacterial cell by create chemical bonding
between adjacent thymine bases on the same DNA strand, or sometimes
between DNA strands, that interferes with the proper replication and
function of the DNA molecule. As a result, the irradiated bacterial cell
cannot reproduce and will die.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/
laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose
without the written consent of EDVOTEK, Inc. Copyright © 2008, 2009 EDVOTEK, Inc., all rights reserved.
EVT 090914AM
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
Instructor's Guide
1.
Section V - Reactivity Data
Material Safety Data Sheet
EDVOTEK
IDENTITY (As Used on Label and List)
Nutrient Broth
Emergency Telephone Number
Manufacturer's Name
EDVOTEK, Inc.
Incompatibility
Date Prepared
14676 Rothgeb Drive
Rockville, MD 20850
Hazardous Components [Specific
Chemical Identity; Common Name(s)]
OSHA PEL
Inhalation?
Vapor Pressure (mm Hg.)
Vapor Density (AIR = 1)
Other Limits
Recommended
% (Optional)
Specific Gravity (H 0 = 1)
2
No sata
No data
Melting Point
No data
No data
Evaporation Rate
(Butyl Acetate = 1)
No data
IARC Monographs?
NTP?
OSHA Regulation?
Medical Conditions Generally Aggravated by Exposure
Emergency First Aid Procedures
Flash Point (Method Used)
LEL
Flammable Limits
NA
Waste Disposal Method
Smaller quantities can be disposed of with household waste. Disposal must by made according to
official regulations. Recommended cleansing agent: water, if necessary, with cleansing agents.
Precautions to be Taken in Handling and Storing
gloves, safety goggles
Section VIII - Control Measures
UEL
Respiratory Protection (Specify Type)
Extinguishing Media
Protective Gloves
Use NIOSH approved SCBA and full protective equipment.
Eye Protection
Yes
Yes
Work/Hygienic Practices
Product is not Flammable. Product does not present an explosion hazard.
Section V - Reactivity Data
Material Safety Data Sheet
Section I
Emergency Telephone Number
Manufacturer's Name
EDVOTEK, Inc.
Telephone Number for information
Address (Number, Street, City, State, Zip Code)
Date Prepared
14676 Rothgeb Drive
Rockville, MD 20850
Incompatibility
Hazardous Decomposition or Byproducts
Emits toxic fumes of chloride and sodium oxide when heated to 801°C.
Hazardous
Polymerization
OSHA PEL
11/13/08
ACGIH TLV
N/A
Carcinogenicity:
Other Limits
Recommended
% (Optional)
N/A
Inhalation?
Incompatible materials
Skin?
Yes
Yes
Ingestion?
Yes
Irritant. Irratating to eyes, reapiratory system and skin.
IARC Monographs?
NTP?
OSHA Regulation?
Irritant to skin and mucous membranes. Irritating to eyes.
Medical Conditions Generally Aggravated by Exposure
No sensitizing effects known. If large doses, can cause vomiting diarrhea, and prostration.
Emergency First Aid Procedures
If swallowed, seek medical attention. If inhaled, supply fresh air or oxygen. Seek medical attention.
If eye contact, rinse open eye for 15 min. Seek medical attention. If Skin, wash with soap and water.
Section VII - Precautions for Safe Handling and Use
Section III - Physical/Chemical Characteristics
3
Boiling Point
1413°C
Specific Gravity (H 0 = 1)
2
2.16 g/cm
Vapor Pressure (mm Hg.)
1.0 @ 865°C
Melting Point
804°C
N/A
Evaporation Rate
(Butyl Acetate = 1)
N/A
Steps to be Taken in case Material is Released for Spilled
Ventilate spill area. Wear suitable protective clothing. Wipe up with damp sponge or mop.
Waste Disposal Method
Keep container tightly closed. Protect from moisture. Suitable for any
general chemical storage area. Store away from oxidizing agents.
Other Precautions
Colorless crystals or white powder. Characteristic odor.
N.D. = No data
Flammable Limits
N/A
LEL
N/A
UEL
Suitable extinguishing agents CO , extinguishing powder or water spray.
2
Special Fire Fighting Procedures
Fight larger fires with water or alcohol resistant foam. Firefighters
should wear protective equipment and self-contained breathing apparatus.
Dispose of in accordance with all applicable federal, state and local
environmental regulations.
Precautions to be Taken in Handling and Storing
35.7 g/100g at 0ºC
Section IV - Physical/Chemical Characteristics
X
Health Hazards (Acute and Chronic)
Signs and Symptoms of Exposure
Hazardous Components [Specific
Chemical Identity; Common Name(s)]
Sodium Chloride, NaCl, Common salt
Conditions to Avoid
May Occur
Will Not Occur
Route(s) of Entry:
(301) 251-5990
Stable under ordinary conditions of use and storage.
Reacts with acids, alkalis, oxidizing agents, Lithium and bromine trifluoride.
Section VI - Health Hazard Data
(301) 251-5990
Signature of Preparer (optional)
Section II - Hazardous Ingredients/Identify Information
Conditions to Avoid
X
Stable
Note: Blank spaces are not permitted. If any item is not
applicable, or no information is available, the space must
be marked to indicate that.
Ready Pour Agar
Unstable
Stability
May be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted for
specific requirements.
IDENTITY (As Used on Label and List)
Unusual Fire and Explosion Hazards
N/A
N/A
Other
Other Protective Clothing or Equipment
Unusual Fire and Explosion Hazards
N/A
Special
N/A
N/A
Mechanical (General)
Special Fire Fighting Procedures
Vapor Density (AIR = 1)
NIOSH/MSHA approved respirator
Local Exhaust
Ventilation
CO2, extinguishing powder or water spray.
®
No special safety precautions are required.
Other Precautions
light golden colour
Section IV - Physical/Chemical Characteristics
Extinguishing Media
Ingestion?
Yes
Steps to be Taken in case Material is Released for Spilled
No data
Appearance and Odor
Flash Point (Method Used)
Yes
Section VII - Precautions for Safe Handling and Use
soluble
Appearance and Odor
Skin?
Yes
Swallowed - wash out mouth with water.
Skin/eye contact - flush with water Inhalation - remove to fresh air
Boiling Point
Solubility in Water
Conditions to Avoid
Signs and Symptoms of Exposure
Section III - Physical/Chemical Characteristics
EDVOTEK
None known
Will Not Occur
Carcinogenicity:
Non Hazardous ingredients
Solubility in Water
May Occur
Health Hazards (Acute and Chronic) Causes no irritation, no sensitizing effects.
11/13/08
ACGIH TLV
Hazardous
Polymerization
Route(s) of Entry:
(301) 251-5990
Signature of Preparer (optional)
Section II - Hazardous Ingredients/Identify Information
None known
Hazardous Decomposition or Byproducts
Section VI - Health Hazard Data
(301) 251-5990
Telephone Number for information
Address (Number, Street, City, State, Zip Code)
Conditions to Avoid
X
Stable
Note: Blank spaces are not permitted. If any item is not
applicable, or no information is available, the space must
be marked to indicate that.
Section I
Unstable
Stability
May be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted for
specific requirements.
®
Prevent formation of dust. Ensure good ventilation. Avoid prolonged exposure.
Section VIII - Control Measures
Respiratory Protection (Specify Type)
None required where adequate ventilation conditions exist.
Local Exhaust
Ventilation
50-100 CFM
Mechanical (General) N/A
Protective Gloves
Proper disposable gloves
Special
Other
Eye Protection
Chem. resistant goggles
Other Protective Clothing or Equipment
During heating or in case of fire, poisonous gases are produced.
Impervious clothing to prevent skin contact
Work/Hygienic Practices
Keep away from food and beverages. Immediately remove all soiled or contaminated cloting.