Laboratory 1-Real time evolution: bacteria
Natural Selection in action—Evolution as we speak…
Introduction to BIO152 labs
1. Team work and academic honesty
Science is almost always a collaborative effort with teams working together to solve problems.
Likewise in this course, you will work in teams in most labs. You will be individually accountable
and expected to contribute equitably to the success of your team. If you truly collaborate in your
team, your assignments and projects will reflect a far greater depth of understanding (“the whole
is greater than the sum of the parts”). If you fail to learn how to collaborate in a team and
merely divide up the work between individuals, your product will resemble a “patch work quilt”
with no integration or synthesis—and you will not do as well.
Academic honesty is absolutely critical for scientists, so too is honesty essential for students.
Review the separate section on Academic Honesty and how to avoid plagiarism and other forms
of academic dishonesty. All work submitted in this and every course must be your own not
‘borrowed’ from another student or copied without appropriate referencing from an external
source.
2. Safe science in the lab WARNING
You MUST wear a lab coat for this lab. Read Safe Science and know the rules (found on the
Lab section of the course web page). The quiz will include questions about the rules of Safe
Science relevant to this lab.
In this laboratory exercise, you will learn the basic elements of scientific investigation and how
to apply this process to solving problems.
Before this lab
1. Read this lab chapter carefully; print a copy to bring to lab. You will have a quiz at the
beginning of lab based on Safe Science, the procedure for Lab 1, and the terms listed
below.
2. Read Freeman Chapter 1 Section 1.4 Doing Biology
3. Read and do the WEB Tutorials (also on your text CD):
a. 1.2 Introduction to Experimental Design
b. 23.1.Natural Selection for Antibiotic Resistance
4. Read about spices and their possible antimicrobial activity: Short summaryhttp://www.news.cornell.edu/releases/March98/spice.hrs.html.
Read before writing up the lab due in LAB 3: Original article: Sherman, and Billings,
1998, Darwinian Gastronomy: Why we use spice [link]
5. Read the separate article on Safe Science. (Safety issues related to this lab are covered
on the quiz)
6. Read the separate article on Academic Honesty and be able to explain how to avoid
academic dishonesty.
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Lab 1 Evaluation (50 points worth 5% )
2% (20 points) Prelab quiz in Lab 1
3% (30 points) Results and discussion of the experiments due in Lab 3
Objectives of Lab 1
1. Learn safe lab techniques working with bacteria.
2. Learn how effective certain chemical and physical agents are at controlling bacterial
growth.
3. Learn how natural selection affects bacterial sensitivity and resistance.
4. Learn the basics of the scientific method—including the similarities and difference
between a controlled and a comparative experiment.
5. Start the design for your major project due in Lab 5.
Terms
1) Related to experimental design and the scientific process:
data
evidence
experimental method (procedure, protocol)
hypothesis and prediction also null hypothesis
hypothesis testing by controlled experiments and comparative method
observation
replication
sample size
variables: dependent, independent, standardized
2) Related to the bacteria lab:
resistance versus sensitivity
antimicrobial—antibiotic, disinfectant, antiseptic, spice
bactericidal versus bacteriostatic
bacteria-- prokaryote
clonal reproduction
pathogen
sterilization
zone of inhibition
Lab Timeline: (3 hours)
0:10 – 0:20
(10 minutes) Introductions
0:20 -- 0:50 (30 minutes) Quiz on safe science, procedure & terms for Exercises 1-2 & 1-3
0:50 -- 1:20 (30 minutes) Exercice 1.1 The Mystery Box
1:10 – 2:10
(50 minutes) Exercise 1.2 Setup plates for bacteria experiment
2:10 – 3:00 (50 minutes) Exercise 1.3 Experimental design, controlled experiments, and the
“scientific method”; design your Major Project.
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Exercise 1.1 Practicing the scientific process: The Mystery Box
Your team (3-4 students) has been given a sealed plastic container containing two or three
objects. You have also been given a container with an assortment of objects that may have
been used in the mystery box. Determine the contents of the ‘mystery box’ without opening it.
1. Start with observations. Use any means you like and have available to you (except
opening the container!) to investigate. Record your observations and answers to the
following questions in your lab notebook. Why is it important to begin solving this problem by
making observations?
When you think you have an idea of what might be in your container, make a guess about
its contents. Record your guess in your lab notebook and on the board.
Mystery Box#
Observations
Contents ( best guess)
What other methods or materials that you don’t currently have available might be useful for
making observations about the contents of your container?
2. Discuss your guess(es) and the methods tried with your lab TA and classmates. After the
discussion, the TA will offer you some additional methods for investigating the contents of
your black box.
a. Using some of the additional methods that you, your classmates and TA suggested,
make and record further observations.
b. What new information leads you to conclude that your first guess was correct or to revise
your initial guess? Write your new guess in your lab notebook and on the board next to
your original guess.
Mystery Box#
Observations
Contents ( best guess #2)
3. When the entire class is finished making their second “guesses”, your instructor will have
each group open their container write the contents on the board. Please do not open
your container until your TA tells you. Consider the following questions after everyone
has written their box contents on the board.
•
Were any of the groups wrong for both guesses about the contents of their box?
What are some things that might have led groups to draw the wrong
conclusions?
•
Summarize the process that you used to solve the problem of what is in the
sealed plastic container.
4. Your investigation of the plastic container involved the same basic procedure followed in
many scientific investigations:
a. First a problem or question is posed by the investigator. What was the problem or
question you sought to solve or answer?
b. Next the investigator makes preliminary observations that lead to an educated guess or
hypothesis. What was your first hypothesis? And your second?
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c. Once a hypothesis is posed, the investigator designs and performs an experiment to
tests the hypothesis. What experiment(s) did your group choose to perform?
d. Based on the results of the experiment, the investigator draws conclusions that
support, refute, or lead to the revision of the initial hypothesis. How did the results of
your experiment compare with what you predicted in your initial hypothesis (i.e. first
guess)?
e. If the results are inconclusive, further observation, hypothesis formulation and
experimentation may follow until the investigator eventually reaches a point where (s)he
is confident of his/her conclusions.
Exercise 1.2 Genetic variation and selection: E. coli sensitivity to
antimicrobials (antibiotics, disinfectants, antiseptics, food spices)
Background
Bacteria are found almost everywhere. While most species are beneficial, some are harmful or
even pathogenic (cause disease). Chemical and physical agents may be used to control
bacterial growth, yet used inappropriately can by natural selection lead to bacterial species
which are resistant to our attempts to control them. In this exercise you will learn more about the
genetic diversity of bacteria and about different methods which may be more or less effective at
controlling bacterial growth.
The combined effects of fast growth rates, high concentrations of cells, genetic processes of
mutation and selection, and the ability to exchange genes, account for the extraordinary rates of
adaptation and evolution that can be observed in the bacteria. For these reasons bacterial
adaptation (resistance) to the antibiotic environment seems to take place very rapidly in
evolutionary time: bacteria evolve fast!
For example, the bacterium Escherichia coli (E. coli) used in this lab has about 5000 genes and
has a mutation rate of about 1 mutation in every 1 x106 (1 million) copies. The generation time
(time from parent to daughter cells) can be every 20 minutes under optimum conditions.
Therefore, one could expect to find 1 mutant gene in every 200 bacteria. A typical spoonful of
soil contains over a billion bacteria; within this population over 5 million of these bacteria would
contain a mutation.
Overview: Your team will be half the students at the bench (3-4 students). You will set up the
various bacterial plates in this lab, record the results next lab. Individually you will write up your
results and a brief discussion to hand in at the beginning of Lab 3.
Lab 1 (this week) Materials per team (3- 4 students)
4 agar plates one for each of the following
plate 1 antibiotics
plate 2 disinfectants
plate 3 antiseptics
plate 4 food spices
4 sterile swabs
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16 sterile disks—4 (controls); 4 disinfectants; 4 antiseptics; 4 food spices
1 marking pen
3 Parafilm strips
[2 small clear metric rulers—lab 2]
Bacteria Escherichia coli (strain #10)
For Plate 1 Bacteria + Antibiotics-commercially prepared disks
Control (plain sterile disk)
Neomycin (30 micrograms)
Penicillin (10 units)
Streptomycin (10 micrograms)
Ampicillin (10 micrograms)
Plate 2 Bacteria + Disinfectants
Control (sterile water)
Ammonia
Lysol
70% Ethanol
10% Bleach
Plate 3 Antiseptics
Control (sterile water)
Hydrogen Peroxide
Listerine mouthwash
Antibacterial soap
Regular liquid dish soap
Plate 4 Bacteria + food spices (select from a variety provided)
Control
______
______
______
______
This plate (# 4) will give you a chance to design an experiment to test some hypothesis about
the antimicrobial role of spices in food. Your team will need to select four spices. How are you
going to decide which spices to test? Before lab read the accompanying article “Darwinian
gastronomy: why we use spices” and be prepared to explain your choice of spices and include
your rationale in the write up of this lab due in Lab 3.
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Exercise 1.3 Procedure
1. Wash your hands with soap and water.
2. Wash bench surface with 10% bleach.
3. Label around the side or near the edge of the bottom of the four agar plates (NOT the
top):
a. On EACH plate: Room# and the name of 1 student in your group and some
symbol to recognize your plate next week;
b. Number the plates: 1,2,3,4 (1=antibiotics; 2=disinfectants; 3=antiseptics;
4=spices)
c. On the antibiotic plate (1), label 4 areas of the plate with the first letter of an
antibiotic: N, P, S, A. Put the plain sterile disk (C) in the center.
1=Antibiotic plate
N
P
C
S
A
d. Plate #2 label each of the four quarters with the first letter of the specific
disinfectant.
e. Plate #3 “ label each of the four quarters with the first letter of each antiseptic.
f. Plate #4 label each of the four quarters with the first letter of the spice to be
tested.
g. CONTROL disk with just sterile water will be in the center of plates 2,3 and 4.
4. Prepare a bacterial lawn:
a. Wash bench surface with 10% bleach in squeeze bottle on bench.
b. Insert a sterile swab into the bacterial culture in liquid nutrient broth.
c. Allow the swab to drip for a moment before taking it out of the culture tube. (The
swab should be soaked but not dripping.)
d. Carefully lift the lid of one agar plate to about 45° and swab the entire surface of
the agar including right to the edges of the dish: VERY IMPORTANT
1. Apply bacteria evenly over the entire agar surface;
2. Rotate the plate and swab at right angles to the first application (Figure 1)
e. Cover the plate with the lid.
f. Repeat this procedure on the other agar plates.
g. Wash your hands with soap and water.
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Figure 1 Prepare a bacterial lawn by swabbing the entire surface of the agar plate
5. Plate #1 Antibiotics Carry one plate to the designated area to dispense the antibiotics.
a. Dip forceps in 70% alcohol
b. Flame using alcohol burner (TA will demonstrate)
c. Allow forceps to cool about 20 seconds.
d. Use dispenser to place correct antibiotic in each section of the plate; use
sterilized forceps to adjust position of disk to center of section.
e. Control disk: (sterilize forceps by repeating sets a and b above); transfer one
plain sterile disk to the center of the agar plate.
6. Plate #2 Disinfectants& plate #3 antiseptics (materials are on your bench)
a. Dip forceps in 70% alcohol
b. Flame forceps using alcohol burner.
c. Allow forceps to cool about 20 seconds.
d. Use sterilized forceps to add disk to each disinfectant and antiseptic liquid. Be
careful not to touch the liquid with the forceps. Soak in liquid for about 20-30
seconds.
e. Use sterile forceps to remove from liquid (allow excess liquid to drip off before
transferring disk to agar plate). Position appropriate disk in each labeled section.
f. Control disk: (sterilize forceps by repeating sets a-c above); transfer one plain
disk to soak in sterile water for about 30 seconds. With forceps hold disk over
liquid to drip off excess water before transferring disk to the center of the agar
plate.
7. Plate #3 Food spices follow the instructions for plates 2&3 to prepare disks for the four
to six spices your team is testing.
8. Seal each plate with a strip of Parafilm.
9. Put your plates in the designated tray with lid side up (so disks don’t come loose—
normally you store plates upside down so condensate doesn’t drip onto cultures). Your
plates will be incubated at 37°C for about 24 hours and then stored in the refrigerator
(about 4°C) until your next lab when you will record the results.
10. Wash bench surface with 10% bleach.
11. Wash your hands with soap and water.
12. Cleanup: put all used material in labeled containers. Leave area as you found it at the
beginning of lab.
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Lab 2: Record the results
In the next lab you will determine the bacterial sensitivity: Use a small clear metric ruler to
measure the diameter of the zone of inhibition (clear area) around each disk. This zone is the
area where bacteria growth has been inhibited. For examples see
http://gold.aecom.yu.edu/id/micro/directsensi.htm
In addition to the measurements, use the following arbitrary criteria to rank the relative bacterial
sensitivity:
NS= not sensitive = no zone of inhibition S= sensitive= zone < 1cm VS= very sensitive= >1cm
Table 1 Escherichia coli sensitivity to antibiotics, disinfectants, antiseptics, and food spices.
Zone of inhibition Relative sensitivity
#f colonies growing
(cm)
NS
S VS
in the Zone
Plate 1 Antibiotics
Neomycin
Penicillin
Streptomycin
Ampicillin
Control (water)
Plate 2 Disinfectants
Ammonia
Lysol
70% Ethanol
10% Bleach
Control (water)
Plate 3 Antiseptics
Hydrogen Peroxide
Listerine
Antibacterial soap
Control (water)
Plate 4 Spices
Analysis of the results (due in Lab 3):
1. Graphs summarizing the data in Table 1
2. Describe and briefly discuss the results (2 pages maximum) In your discussion:
• State your original hypothesis and prediction of bacterial sensitivity to antimicrobials in
general and then each of the four types of antimicrobials (antibiotic, antiseptic, disinfectant,
food spices).
• Based on your results, describe which antibiotic, disinfectant, antiseptic, and food spice are
the most effective and which are the least effective antimicrobial to kill E. coli?
• Discuss the meaning of any colonies growing in a zone of inhibition—did your plates have
any? How did the number of colonies vary for the different antimicrobial substances?
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Exercise 1.3 More on the Process of Science (“Doing biology”)
Objectives
1.
2.
3.
4.
5.
6.
Identify questions that can be answered through scientific investigations.
Generate hypotheses from observations.
Formulate predictions from hypotheses.
Understand the difference between controlled and comparative experiments.
Recognize the components of an experiment: variables, controls, samples, & replication.
Design an experiment and critique other designs.
Background
A. Hypotheses
A hypothesis (plural, hypotheses) is an “educated guess” to the answer of a question being
investigated based on observations or previous research (the scientist’s or others’).
When you were given your mystery box, you were given a scientific problem to investigate: its
contents. Thus, the question was already defined for you. Recall the steps you went through to
scientifically investigate the contents of your mystery box (Exercise 1.1). These steps began
with observations that led to testable hypotheses.
How do you test a hypothesis? Generally, a hypothesis leads to very specific predictions that
are testable. For example, based on your observations (e.g. sound, feel, weight), you may have
hypothesized that your mystery box contained a roll of tape and a paper clip. You could have
tested this hypothesis directly by opening the sealed box, but this kind of direct observation is
not always possible. Instead, based on this hypothesis, you could predict what these contents
might sound like or weigh in another similar box. These predictions are then what is testable.
This is what we mean by saying that a hypothesis is testable.
B. Predictions
Do not begin an experiment without predicting the outcome. Your prediction should be based
on the particular experiment designed to test a specific hypothesis. It is easiest to phase your
prediction as an “if …then” statement:
“If the experiment [hypothesis] is true, then the results of the experiment will be …
Example 1: Hypothesis: Regular interaction with pets improves the health of the elderly.
Prediction: If regular interaction with pets improves the health of the elderly [notice this is a
restatement of the hypothesis], then the heart rate will be lower after exercise and return to
normal faster in elderly people who spend 20 minutes daily with their pet cat [predicted results
from the experiment].
Example 2: Hypothesis: Music lessons cause a greater increase in children’s IQ.
Prediction: If music lessons increase children’s IQ , then students having weekly piano lessons
for a year should have a higher increase in IQ than students who do not have lessons.
Example 3: Hypothesis: Giraffes have long necks to out compete other animals for food.
Prediction: If the main function of the giraffe’s long neck is for feeding, then giraffes should
regularly extend their necks when feeding. (See Freeman, Chapter 1 for a discussion on
hypothesis testing and why giraffes have long necks.)
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Example 4: Hypothesis: The presence of capsaicin (molecule which makes chili peppers hot)
will deter some predators but not others. (See Freeman, Chapter 1.)
What is the null hypothesis? Prediction:
What is the dispersal hypothesis? Prediction:
C. The Nature of Scientific Knowledge
Scientific knowledge based on generalizations and conclusions drawn from specific
observations and experiments, is a process known as inductive reasoning. An alternative way
of reasoning that we also use in science is when we begin with general principles and predict
their consequences (deductive reasoning). While deductive reasoning is an important part of
science and is an essential part of the application of scientific principles, it is not a means for
gaining new scientific knowledge. All new scientific knowledge depends on inductive reasoning.
(Why?)
However, information gathered through inductive reasoning has a level of uncertainty. In the
inductive process, generalizations are made based on specific observations. Since it is never
possible to observe every possible case or scenario in an investigation, we must rely on
observations of a sample of all possible observations. For this reason, scientific “facts” are
always regarded with a certain level of skepticism rather than as absolute truth. In fact,
statistics are a formal way of quantifying an investigator’s uncertainty when experimentally
testing a scientific hypothesis. We will use statistics to test hypotheses in the next lab.
New knowledge is actually an accumulation of evidence which support hypotheses. When we
accept a hypothesis as “true”, we accept the hypothesis on a conditional basis: evidence may
support the hypothesis, evidence does NOT prove the hypothesis. Some future technology,
experiment or other information may falsify it.
Examine the scientific inquiry method closely: a single experiment can prove a hypothesis false,
but it takes many types of investigations before a hypothesis appears to be true. When we
prove a hypothesis false, we say that we falsify or refute it. Scientists DO NOT SAY that an
investigation proves a hypothesis true, recognizing the level of uncertainty involved in inductive
reasoning. Instead, we say that the data support the hypothesis.
New scientific knowledge seems tentative and it is only after much data has been gathered from
many experiments and observations that the knowledge is generally accepted as the “facts” you
read in your text books. Many of these “facts” were once very controversial. An excellent
example was the discovery of DNA as the hereditary material by Avery in 1944. Until then, DNA
was a weak candidate for the genetic material and most geneticists favored protein as the likely
molecule. It wasn’t until the unique structure of DNA was clearly elucidated by Watson, Crick
and Franklin in 1953 that the last of the skeptics was convinced.
We have focused here on the scientific method using hypotheses that can be proven false
through controlled experiments. Experiments may also be based on the predictive power of
observation and comparison (comparative experiment), which cannot be tested with controlled
experiments. Hypotheses in the comparative method are tested by making predictions about
patterns that should exist in nature if the hypothesis is correct; data are gathered to determine if
the patterns exist. Examples: paleontologists who study the fossil record and astronomers who
study the stars.
Both a controlled experiment and an observation-based comparative experiment depend on
observable phenomena and inductive reasoning.
By its nature, scientific knowledge is
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knowledge that can be proven false. This is not a requirement for other forms of knowledge
(e.g. aesthetic, philosophical, ethical, religious, etc.)
Understanding this is critical to
understanding the limitations of scientific inquiry. There are certain things it is simply not
possible to learn through science. For example, consider the following hypothesis: “The best
music of the century was written in the 1960s”. There is no experiment that can be performed
or observations made to test and potentially falsify the hypothesis.
D. Controlled experiments: components and design
(1) Variables
(2) Controls
(3) Replication and Sample Size
Once a question or problem has been identified and a hypothesis formulated, the next step in a
scientific investigation is to conduct an experiment to test the hypothesis. There are several
important factors to keep in mind when designing a suitable experiment. These include:
identifying variables to be tested, measured and held constant, controls to be run and how
many times to replicate the experiment.
(1) Variables
Variables are things that might be expected to vary in an experiment. There are those factors
the investigator wishes to manipulate in order to test their effect. These are known as
independent variables. As a result of changing something about an independent variable,
there may be some effect. Variables that are expected to change in response to independent
variables are called dependent variables. In order to be certain that any change is actually
due to changes in the independent variables and not to other factors, it is important to keep
standardized variables constant.
a. The independent variable is the variable the investigator wishes to test and is
deliberately varied.
For example, in today’s lab you were looking at the effectiveness of different antibiotics
on limiting bacterial growth. The independent variable are the four antibiotics (the
resulting zone of inhibition of E. coli is the dependent variable)
What is the independent variable in today’s lab testing E. coli sensitivity to the
1. Disinfectants
2. Food spices
It is not always possible or necessary for an investigator to directly manipulate an
independent variable in order to test its effect. So, for example, perhaps you thought
that the phase of the moon might affect mouse reproduction and could be an important
independent variable in a thorough study of their reproductive biology. Even if the phase
of the moon can not be experimentally manipulated, it can still vary and be recorded as
an independent variable.
►Can you think of other independent variables like this that can’t be experimentally
manipulated?
b. Since more than one factor can be an independent variable that might affect bacteria
resistanc/sensitivity, it is typical to test only one independent variable at a time.
►Why is testing one variable at a time important?
►What must be done with the other possible independent variables you mentioned while
you investigate the effects on bacterial growth?
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c. The dependent variable is what is expected to vary in response to the experimenter’s
manipulations, it is what will be measured or counted during the investigation.
For example, as stated above in the study of the effect different antibiotics on bacterial
growth, next lab you will be measure the zone of inhibition (killed bacteria or bacteria that
did not grow in the presence of the specific antibiotic).
What is the dependent variable in the experiment today with E. coli and various food spices?
(2) Controls
Control treatments are another necessary part of a well designed experiment. A control
treatment is a treatment in which the independent variable is either eliminated or set at a
standard value. The results of the control treatment are compared to the results of the
experimental treatments.
For example, in the experiment where bean plant seed production is measured after spraying
pesticides at various frequencies, it is important to include unsprayed plants as a control
treatment. However, for an experiment to test activity level of lizards at various temperatures, it
would not be possible to include a “no temperature” control treatment. Instead, the investigator
chooses a standard temperature (perhaps the average field temperature) as a basis of running
speed.
► Indicate an appropriate control treatment for each of the following examples:
a.
An investigator wants to determine the dose of penicillin that is most effective at
combating strep throat infections.
b. Antibacterial soap is investigated to determine its effect during hand washing.
(3) Replication: repetition and sample size
Replication is another important part of good experimental design. A scientist must repeat
(replicate) an experiment many times, keeping the conditions as identical as possible, in order
to draw conclusions from the experimental results. Each time an experiment is done, the results
may be slightly different, because biological systems are inherently variable. (For example,
lizards don’t always run at exactly the same speed (do you?), so that any experiment comparing
lizard running speed may give slightly different running speed values from earlier experiments
even if the independent and standardized variables are exactly the same. Thus, when we
replicate an experiment, we can get a measure of the average value and also an idea of how
much variation there is among replicates. These values (average = mean, variation = variance)
are important statistical parameters that can be useful in hypothesis testing.
Sample size is another aspect of replication. One way to replicate an experiment, is to repeat it
over and over. Another way is to perform it on a large sample size simultaneously. In the
investigation of insecticide effect on bean seed production, it would be risky to conclude much
from a sample of six plants: two each with each of three levels of insecticide. If one plant died in
any of the groups, it would be hard to know if it was due to the insecticide level or to some other
factor. The most convincing results come from experiments done with both replication and with
adequate sample size.
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Observation-based comparative experiments
Some experiments do not have a control, but rather an investigator compares two or more
groups to look for similarities and differences.
Example: You are a chicken farmer and wanted to know the ideal number of chickens per room
so that the chickens laid the most number of eggs. You could set up an experiment with
different numbers of chickens in identical rooms (same light, temperature, air circulation, etc.)
and compare the resulting number of eggs.
Notice in this example the farmer does not have a control unless she set up the experiment to
compare the density greater and less than current standard number of chickens kept in each
room.
Appendix Background on controlling microbial growth
"Control of growth" means to prevent growth of bacteria in two basic ways: (1) by killing or (2) by
inhibiting growth. Control of growth usually involves the use of physical or chemical agents.
Agents which kill cells are called cidal agents; agents which inhibit the growth of cells (without
killing them) are referred to as static agents. Thus the term bactericidal refers to killing
bacteria and bacteriostatic refers to inhibiting the growth of bacterial cells. A bactericide kills
bacteria; a fungicide kills fungi; and so on.
A. Physical means
1. Heat The lethal temperature varies for different microorganisms. The time required to kill
depends on the number of organisms, species, nature of the product being heated, pH, and
temperature. Whenever heat is used to control microbial growth inevitably both time and
temperature are considered.
Sterilization (boiling, autoclaving, hot air oven) kills all microorganisms with heat;
commonly employed in canning, bottling, and other sterile packaging procedures.
Pasteurization is the use of mild heat to reduce the number of microorganisms in a
product or food.
2. Low temperature (refrigeration and freezing): Most organisms grow very little or not at all
at 0º C. Store perishable foods at low temperatures to slow rate of growth and consequent
spoilage (e.g. milk). Low temperatures are not bactericidal.
3. Drying (removal of H2O): Most microorganisms cannot grow at reduced water activity. Often
used to preserve foods (e.g. fruits, grains, etc.). Methods involve removal of water from product
by heat, evaporation, freeze-drying, addition of salt or sugar.
4. Irradiation (microwave, UV, x-ray): destroys microorganisms as described under
"sterilization". Many spoilage organisms are easily killed by irradiation. In some parts of Europe,
fruits and vegetables are irradiated to increase their shelf life up to 500 percent. The practice
has not been accepted in the U.S.
B. Chemical means
Antimicrobial agents are chemicals that kill or inhibit the growth microorganisms. Antimicrobial
agents include chemical preservatives and antiseptics, as well as drugs used in the treatment of
infectious diseases of plants and animals. Antimicrobial agents may be of natural or synthetic
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origin, and they may have a static or cidal effect on microorganisms.
Some types of antimicrobial agents
1. Antiseptics: Agent that kill microorganisms (microbicidal) harmless enough to be applied to
the skin and mucous membrane; should not be taken internally (Table 2).
2. Disinfectants: Agents that kill microorganisms, but not necessarily their spores, not safe for
application to living tissues; they are used on inanimate objects such as tables, floors, utensils,
etc. (Table 2)
Note: disinfectants and antiseptics are distinguished on the basis of whether they are safe for
application to mucous membranes. Often, safety depends on the concentration of the
compound. For example, sodium hypochlorite (chlorine), as added to water is safe for drinking,
but "chlorox" (5% hypochlorite), an excellent disinfectant, is hardly safe to drink.
Table 21. Common antiseptics and disinfectants
Chemical
Action
Uses
Alcohols:
ethanol & isopropanol (50-70%)
Detergents
Phenolic compounds (e.g. carbolic
acid, lysol, hexylresorcinol,
hexachlorophene)
Denatures proteins and
solubilizes lipids
Disrupts cell membranes
Denatures protein and
disrupts cell membrane
Antiseptics at low concentrations;
disinfectants at high concentrations
Silver nitrate (AgNO3)
Precipitates proteins
General antiseptic and used in the
eyes of newborns
Tincture of Iodine (2% I2 in 70%
alcohol)
Inactivates proteins
Antiseptic used on skin
Chlorine (Cl2) gas
Forms hypochlorous acid
(HClO), a strong oxidizing
agent
Disinfect drinking water; general
disinfectant
Ethylene oxide gas
Alkylating agent
Disinfectant used to sterilize heatsensitive objects such as rubber and
plastics
Antiseptic used on skin
Skin antiseptics and disinfectants
3. Antibiotics: antimicrobial agents produced by microorganisms that kill or inhibit other
microorganisms. Antibiotics are low molecular-weight (non-protein) molecules produced mainly
by microorganisms that live in the soil. Among the moulds (eukaryotes in the Fungi Kingdom),
the notable antibiotic producers are Penicillium and Cephalosporium , which are the main
source of penicillin and its relatives. In Bacteria, the Actinomycetes, notably Streptomyces
species, produce a variety of types of antibiotics including streptomycin and neomycin (Table 3).
BIO152 2005
Real time evolution 1- 15
Table 32. The classes and properties of antibiotics used in this laboratory
Chemical class
Examples
Biological
source
Mode of action
Beta-lactams (penicillins and
cephalosporins)
Penicillin G
Penicillium
notatum
Inhibits steps in cell wall
(peptidoglycan) synthesis
Semisynthetic penicillin
Ampicillin
Inhibits steps in cell wall
(peptidoglycan) synthesis
Aminoglycosides
Streptomycin Streptomyces
Neomycin
griseus
Inhibits translation (protein
synthesis)
1&2
Kenneth Todar University of Wisconsin Department of Bacteriology, 2002
http://textbookofbacteriology.net/resantimicrobial.html accessed July 27, 2004
BIO152 2005