Download bacteria: the good, the bad and the ugly

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INFECTIOUS DISEASES
by Dr. Jovanka Voyich-Kane
Department of Immunology and Infectious Disease, Montana State University
,
BioScience Montana is an immersive health sciences project for high school-aged Montana
4-H’ers. BioScience Montana is funded by the National Institutes of Health to help Montana
teens prepare for careers and studies in the health sciences and biomedical research.
4-H students from throughout Montana, along with adult team leaders, are chosen to
participate each year. Students are introduced to hands-on science and research projects
about how the brain makes choices, how scientists deal with infectious diseases, the
connections between nutrition and health, careers and studies in health science-related
fields, and digital media and social networking technologies.
The year-long program begins in August when students spend an immersive week on
the MSU-Bozeman campus, studying alongside faculty and students. Upon returning to
their home communities, students spend the remainder of the school year fully engaged
in experiments and science challenges. Participants use interactive technologies to
communicate with one another, to connect with MSU student mentors, and to present
what they have learned to family, schools and the statewide 4-H community.
BioScience Montana is made possible by Science Edcuation Partnership Award (SEPA)
funding from the National Institues of Health (NIH) awarded to Montana State University’s
Extended University, Montana 4-H Center for Youth Development, and the MSU Departments
of Cell Biology and Neuroscience, Chemistry and Biochemistry, and Immunology and
Infectious Diseases.
This curriculum booklet was developed by
Montana State University Extended University
P.O. Box 173860, Bozeman, MT 59717-3860
Production of these Infectious Diseases module materials:
Scientist and Author for this module...................................................Dr. Jovanka Voyich-Kane
Technology Coordinator.........................................................................................MJ Nehasil
Graphic Design............................................................................................. Marla Goodman
Other essential contributors to the project:
Project Investigators............................................................Jill Martz, Kim Obbink, John Miller
Project Director..............................................................................................Heather Rauser
Participant Coordinator.............................................................................. Stephanie Davison
Project Assistant...............................................................................................Sarah Rieger
Mentor Coordinator........................................................................... Dr. Shelia Nielsen-Preiss
Evaluator...........................................................................................................Becky Carroll
For more information visit out web site:
http://eu.montana.edu/bioscience/
TABLE OF CONTENTS
INTRODUCTION...................................................................................................................... 1
Vocabulary........................................................................................................................... 3
Laboratory Safety: Laboratory Standard Operating Procedures......................................... 6
Activity 1: Identify microbiota in your nose and throat............................................................... 7
Microbiota observations........................................................................................................ 9
Activity 2: Solve a microbiology case study............................................................................ 10
Case study observations..................................................................................................... 11
Agar info.............................................................................................................................. 12
mystery suspects..................................................................................................... 14-18
Escherichia coli (E. coli)........................................................................................................ 14
Salmonella enterica (Salmonellosis)...................................................................................... 15
Streptococcus pyogenes....................................................................................................... 16
Staphylococcus epidermidis................................................................................................. 17
Staphylococcus aureus........................................................................................................ 18
tips for your project................................................................................................ 19
Design your experiment............................................................................................ 20
glossary............................................................................................................................... 21
INTRODUCTION:
The following reading will give you a
basic introduction to bacteria and their
role in illness. It will explore some of the
details about their structure, the way they
reproduce, how they cause infection, and
the role of antibiotics in fighting them.
This information will help prepare you to
maximize your time in BioScience Montana
Infectious Disease Module, understand
the science behind the techniques you are
applying in the lab, and better understand
concepts you are exploring as part of your
time here on campus.
There are about 10 times as many bacterial cells living
inside your body as there are cells that make up your
body!
BACTERIA: THE GOOD
You are probably aware that you are host to a diverse
community of microorganisms that are happy to call you
home. The vast majority of the “non-you” inhabitants
living on and inside your body are bacteria. In fact, there
are about ten times as many bacterial cells living inside
your body as there are cells that make up your body.
For the most part, you are happy to have these tiny
helpers on board. They help fulfill such vital roles as
aiding in digestion and nutrient absorption in your
digestive tract, maintaining a healthy immune system,
reducing inflammatory response, and keeping your
In an average human being, 20 billion
E. coli are replicated each day.
skin healthy. You and your bacteria usually coexist
peacefully, largely unaware of one another. So maybe
you should know a little more about these microbes
that are such an important part of you.
Just what are bacteria? Bacteria are single celled
organisms that exist in a wide variety of environments
on our planet and on us too. Bacteria are classified as
prokaryotes, meaning they lack a “true” nucleus that is
enclosed in a membrane. Instead, their genetic material is
packed tightly into a ball-like structure called a nucleoid.
They have a single chromosome that contains about
3,000 genes, depending on the type of bacteria. Bac-
teria can be classified in a variety of different ways and
are most frequently divided into groups based on their
shape. The three main shapes are
• rod-like (bacillus),
• spherical (coccus), and
• spiral (spirillum).
Bacteria reproduce through a form of asexual reproduction called binary fission. Binary fission allows them to
clone themselves by replicating their DNA and then dividing. Bacteria are very good at replicating this way, and
in proper conditions can do so rapidly; it’s one of the
reasons why they are so successful on our planet and
in our bodies. Take for example Escherichia coli (E. coli),
a bacteria common in your intestinal tract. E. coli can
reproduce extremely rapidly, dividing every 20 minutes
under optimal growth conditions. That means one E. coli
in a petri dish can become two in 20 minutes, those two
become four in another 20, those four become eight in
another 20 minutes, and so on and so on. Perhaps not
so impressive while talking about numbers in the single
digits but if you keep the clock running, the numbers get
a lot more interesting!
After two hours the same petri dish will contain 64
bacteria. After three that number will be 512 and at hour
four it will be 4,096 bacteria. (How many of you could
you clone in four hours?) Now, keep in mind that there
are millions of bacteria throughout your digestive tract
and the following won’t surprise you. In an average human being 20 billion E. coli are replicated each day. This
is a staggering number but it also happens to be pretty
close to the amount lost every day. So on average your
1
on in bacteriological research, scientists noticed that the
two different groups of bacteria responded differently to
the stains they used in microscope work. One group of
bacteria absorbed the stain and were thus called Gram
positive.
The other type of bacteria didn’t, and were called Gram
negative. Structurally, it is the Gram positive bacteria
that construct a thick cell wall as the outside of their cell
and it was this peptidoglycan layer that absorbed the
stain. Gram negative bacteria are different: they use the
peptidoglycan as a cell wall layer between an inner and
outer membrane. Because it is sandwiched between two
membranes, it doesn’t interact with the stain and, therefore, the stain is not absorbed. In Figure 1 you can see
the basic structural differences between Gram positive
and Gram negative bacterial cell walls.
The structural differences between these Gram positive
and Gram negative bacteria have important implications
in that way that they make us sick and also how they
respond to antibiotics. Unfortunately, it is the harmful
aspects of bacteria’s interactions with us that we most
often associate with them. Bacteria can and do harm the
human body. Sometimes we get sick from outside invaders that are new to our system, but even our own helpful
bacteria can grow out of control and harm us under the
right circumstances and that’s when things get bad.
BACTERIA: THE BAD
Figure 1: A diagram showing the structural differences between
a Gram positive and Gram negative bacterium.
natural digestive flora (the microbes that live in your digestive tract) should stay pretty constant. Which is good,
because they do a lot of good things to help you digest
food and regulate your digestive tract.
Another interesting characteristic about bacteria is
that they have a cell wall made out of a material called
peptidoglycan. Although not structurally the same as a
plant cell wall (because plant cells make their cell walls
out something totally different, cellulose) it serves a similar function, helping to provide structure for the cell and
resist osmotic pressure. There is another important way
that bacterial cell walls are different from plant cell walls
and that is in their location. You may recall that plant
cells have their cell wall located on the outside of the
cell membrane. Some bacteria are like that, but not all.
As it turns out, there are two different structural layouts
for the cell wall of the bacteria, and this difference actually leads to another type of bacteria classification. Early
2
Scientists have a specific word for the things that make
us sick: pathogens. A pathogen is defined as a disease-causing particle or microorganism. Our bodies are
constantly exposed to foreign particles and microbes
and usually we don’t get sick. That’s because we have an
immune system and other defenses that do a fantastic
job of defending us the majority of the time. Therefore, in
order for us to get sick, a pathogen must invade us and
resist our defenses well enough that we become ill.
There are four major classes of pathogens:
•viruses,
•bacteria,
• parasites, and
•fungi.
Each of them has their own unique way to attack the
body and cause disease. Viruses and bacteria are very
different in many ways; particularly in what they are, but
also in how they replicate. Modern science takes these
differences into account in developing ways to fight
pathogens when they get out of control in the body and
cause illness.
Viruses are actually not classified as organisms. They
are not considered living things because they are not
made of cells and can’t reproduce on their own without
a host. They also don’t need to eat and don’t grow or
develop. Structurally, viruses are simple and yet surprisingly elegant.
Viruses have an outer coat, called a capsid, that is
made of proteins. Their capsids are constructed of geometric patterns, often in elaborate arrangements. Inside
the capsid lies the heart of the virus, its DNA or RNA. This
is what holds the genetic code for the virus and, more importantly, the directions for how to make more of the virus.
Viruses can’t replicate on their own so they must find a
suitable host cell. They do this entirely by chance as they
float through the environment. If it happens to encounter a
cell that is a good host, the virus particle attaches to the
outside of the cell and injects its DNA or RNA into the cell
through the cell membrane. The virus DNA or RNA then
takes over the cell and forces it to make copies of the virus.
The cell fills with new virus particles until it bursts,
releasing more viruses to infect more cells in the host
organism. A virus infects your cells and attacks them
causing them burst. This makes you feel icky. Your body
reacts to this attack in a variety of ways, depending on
the type of virus, and the result is that you feel sick.
Bacteria make you feel sick too, but mostly for a
different reason. There are two main ways bacteria cause
illness: by destroying tissue in the host organism and by
making toxins. Bacteria that cause the diseases of tuberculosis, gonorrhea, and leprosy actually invade the tissues they infect and destroy them. Obviously this causes
damage to the individual and the living tissues the bacteria have invaded. These are not the most common forms
of pathogenic bacteria, however. More common are the
toxin producing varieties. There are two types of toxins
made by bacteria, exotoxins and endotoxins.
Exotoxins are types of toxins released by bacteria; they
are extremely potent and even small amounts can kill.
Botulinum toxin, a neurotoxin produced by the bacterium
Clostridium botulinum is one of the world’s most potent
toxins. One gram of the toxin is potent enough to kill a
million people.
Endotoxins are toxins that are actually present on the
bacteria themselves. They are the lipopolysaccharides on
the outside of a bacteria’s outer membrane (illustration
b on Figure 1). Because the endotoxin is the lipopolysaccharide chain found on the outer membrane, endotoxins
are found only in Gram negative bacteria.
Salmonella and Escherichia coli are both examples of
Gram negative bacteria that have endotoxins. When they
VOCABULARY
Term
Definition
Bacteria
(Eubacteria)
A domain of microorganisms, only micrometers in length, with a primitive nucleus
Flora
A population of microbes that inhabit the internal and external body surfaces of healthy humans
and animals (see: microbiota)
Peptidoglycan
A major component of the bacterial cell wall that contributes to the shape of the bacteria; amount
of peptidoglycan is used to differentiate bacteria in the Gram stain (see Gram-positive and
Gram-negative)
Gram positive
Bacteria that retain the crystal violet stain due to a high amount of peptidoglycan in the cell wall;
these bacteria typically stain purple during Gram staining
Gram negative
Bacteria that do not retain the crystal violet stain due to a low amount of peptidoglycan in the cell
wall; these bacteria typically stain red during Gram staining (they retain the color of safranin, the
counter-stain)
Microbiota
A population of microbes that inhabit the internal and external body surfaces of healthy humans
and animals (see: flora)
MRSA
Methicillin-resistant Staphylococcus aureus are a form of Staphylococcus that has developed
resistance to a large class of antibiotics
Pathogen
A microorganism capable of producing disease in a healthy animal or human host
3
Often, your doctor will tell you that a
virus has run its course. Generally,
there’s not a drug you can take to help
your body fight the infection. This is not
the case for bacterial infection.
grow unchecked in your digestive system you feel sick
largely because of endotoxins present in your intestinal
tract. The net result of the toxins, whether they are an
endotoxin or an exotoxin, is that they make you sick and
produce the symptoms of illness.
When a virus infects a host’s cells it is actually hiding
inside the host’s cells. Therefore it’s hard to find ways to
fight a virus without damaging the host cell. That’s why
your doctor will tell you that you have to let a virus run
it’s course: Generally there’s not a drug you can take to
help your body fight the infection.
This is not the case for bacterial infections. For bacterial pathogens we can take an antibiotic. Antibiotic comes
from Greek terms that literally mean “against life.” There
are different types of antibiotics that attack bacteria in
different ways, but all basically either attack the bacteria
or disrupt the way it functions. To fight bacteria we need
a selective poison, one that works against bacterial cells
but does no harm to your cells.
Luckily, bacteria have a very distinct structural difference from your cells. Can you recall what it is? If you are
thinking about a cell wall, you’re right! Many antibiotics
work by keeping the bacteria from properly assembling a
truly functional cell wall, or by causing the peptidoglycan
that makes up the cell wall to disintegrate. The bacterium then succumbs to osmotic pressure, bursts, and is
destroyed. Antibiotics that work this way are extremely
effective against Gram positive bacteria, but tend to be
less effective against Gram negative bacteria because
Gram negative bacteria have their peptidoglycan layer
sandwiched between two membranes. For Gram negative
bacteria other classes of antibiotics are more effective.
The other classes of antibiotics work against bacteria
in a variety of ways. Some reduce the bacteria’s ability
to properly make proteins so the structure of their cell
suffers. Others interfere with their ability to copy their
DNA so they can’t reproduce. Still others make it difficult
for them to manufacture energy from glucose. The end
result is that the reproductive rate of the bacteria slows
down so your immune system can catch up and defeat
the bacteria. In the end, your immune system wins and
you feel better. The unfortunate side effect is that antibiotics target all the bacteria in your body, both the good
and the bad, but it is a small price to pay when you are
really overrun and ill from a bacterial infection. In a perfect world you get sick, you take a pill, and you get better.
But bacteria are tenacious and they have some distinct
advantages when it comes to adapting to the drugs we
throw at them. Without really trying, they sometimes find
a way to get by when we try to wipe them out. That’s
when things get ugly.
BACTERIA: THE UGLY
Have you ever heard the term antibiotic or drug resistant bacteria? This is a term used to describe bacteria
that have developed a way to cope with antibiotics.
Antibiotic resistance can have some disastrous implications for those who have to deal with one of these
so-called “Superbugs.” The whole idea of antibiotic
resistance actually stems from a pretty cool aspect
of bacteria: their ability to rapidly evolve to adapt to
their environment. It happens to be one of the characteristics that has helped them to be so successful on
our planet. It would be really impressive if we weren’t
talking about them infecting us!
Figure 2: For their Bioscience Montana project, the 4-H team in Cascade County compared the cleanliness of a dog’s mouth to a
human’s mouth.
4
Bacteria are extremely adaptable through natural
selection because of their very rapid rates of reproduction, but also because they mutate. You may recall that
bacteria don’t reproduce sexually, they clone themselves.
So their genetic variation comes from random mutations
that occur when the DNA is copied in preparation for cell
division. If one of those random mutations happens to
help a bacterium to be resistant to effects of an antibiotic, it will have an rapid impact on the genetics of the
bacterial population. For example, let’s say we had a
group of bacteria living and replicating in a petri dish.
Now say we introduced an antibiotic to that petri dish.
Ideally, the antibiotic would kill all the bacteria in the
dish, but what if it didn’t? What if just one bacterium living in the petri dish had some mutation that made it resistant to the antibiotic? That single survivor would go on
to replicate and after four hours it would have more than
4,000 antibiotic resistant buddies to keep it company.
In fact the entire new population of bacteria in the petri
dish would have this antibiotic resistant genetic mutation. This petri dish scenario is similar to what happens
in your body, except you are talking about much larger
populations of bacteria with a much broader range of
mutations and a lot more genetic diversity to start with.
MRSA is a superbug that has gotten quite a bit a
press lately. MRSA stands for Methicillin-resistant Staphylococcus aureus. Methicillin is a class of antibiotics
related to Penicillin. Staphylococcus aureus is a type of
bacterium that is very common on your skin and normally
lives there without bothering you. Sometimes the bug
Bacteria can rapidly evolve to adapt to
their environment. This characteristic
has helped them to be successful on
our planet and it’s really impressive...
if we weren’t talking about them
infecting us!
overwhelms your immune system and you get a “Staph”
infection, but these are commonly treated with antibiotics and rarely cause for concern. MRSA is different. This
type of bacteria is resistant to a large class of antibiotics, thus limiting treatment options. This has researchers
working hard to develop new lines of antibiotics, and
doctors mindful to be conservative and appropriate in
their use of antibiotics.
Liz and Emma Carlson examine their horse microbiome project plates. The sisters from Lewis and Clark
County investigated the number and type of bacteria in
horses of different ages.
ENJOY, EXPLORE, LEARN,
AND DISCOVER!
As you delve more deeply into
concepts of microbiology and
apply the techniques used in the
research lab during this BioScience
Montana learning module, think
about how your newly acquired
knowledge can impact your life.
How will you use this knowledge
and experience to build a better
community, build leadership, and
develop life skills?
Dr. Jovanka Voyich-Kane is a molecular biosciences professor in the Montana State
University Department of Immunology and
Infectious Disease. She is one of three
MSU professors who lead modules for 4-H
teens as part of the Bioscience Montana
project, which is funded by the National
Institutes of Health’s Science Education
Partnership Awards (SEPA).
5
LABORATORY SAFETY
LABORATORY STANDARD
OPERATING PROCEDURES
1. Personal protective equipment including gloves, eye protection, and a
lab coat must be worn when working with human cells or bacteria.
Do not wear personal protective equipment outside of the lab. Open-toed
shoes may not be worn in the lab.
2. No food or beverages in lab. Food is stored outside the work area in
cabinets or refrigerators designated for this purpose only.
3. Disposable labware is to be discarded in biohazard bags. Samples
containing ≤ 1 mL volume can be discarded directly into biohazard bags.
Petri dishes containing cultures are discarded directly into biohazard
bags.
4. Sink area must be kept reasonably clean throughout the day. Eyewash
station must be readily accessible. Sink must be free of all labware prior
to leaving for the night.
5. Lab benches where work has been conducted must be decontaminated
at the completion of work or at the end of the day and after any spill or
splash of viable material with 70% EtOH, 10% Bleach, or Lysol.
6. If a spill occurs spray Lysol, or 70% EtOH, or a 10% Bleach solution
on spill and place a paper towel on top of the spill to prevent further
spreading. After ≥ 30 min remove paper towel and thoroughly
decontaminate area using more of the disinfectant. If you are unsure
about how to handle a spill contact Dr. Voyich or a science assistant.
7. Personnel must wash their hands after they handle viable materials,
after removing gloves, and before leaving the laboratory. Bacteria can
be easily passed on by human contact, and is commonly spread by the
hands.
6
Activity 1
IDENTIFY
MICROBIOTA
IN YOUR NOSE
AND THROAT
Goal: Identify normal flora
microorganisms from both nose and
throat and look for Staphylococcus
aureus and Streptococcus species.
DAY 1
Throat swab:
1. Put on your lab coat, goggles, and gloves.
2. Obtain an aliquot of sterile phosphate buffered
saline (PBS), and sterile swabs. Obtain 2 plates
each of Mannitol Salt Agar (MSA), Blood Agar (BA),
MacConkey Agar (MAC), Eosin Methylene Blue
Agar (EMB), Colistin-Nalidixic Acid Agar (CNA), and
Tryptic Soy Agar (TSA).
3. Wet the swab with PBS. Say “Ahhhhhh.” Swab both
of your tonsils. Place swab in sterile 15 mL tube
containing a small amount of PBS. Discard swabs
in biohazard.
Nasal swab:
1. Put on your lab coat, goggles, and gloves
but you don’t want a partner for this one!
2. Obtain an aliquot of sterile PBS. Swab
one of your nostrils as demonstrated by
the science team and streak for isolation
on MSA, followed by BA, MAC, EMB, CNA,
and TSA.
3. Let the science team know when you are
done and one of them will put your plates
in the incubator.
4. Take the swab containing your partner’s normal
microbiota and streak for isolation (page 8) on
BA, put the swab back in the 15 mL tube with
the PBS, repeat the procedure and streak for
isolation on MSA, MAC, EMB, CNA, and TSA. Follow
the directions for streaking for isolation on the
board and ask for help if you need it. Discard all
materials used for streaking for isolation in the
biohazard.
5. Let the science team know when you are done and
one of them will put your plates in the incubator.
7
Streaking for Isolation
8
1. After dipping your nasal or throat swab
into sterile PBS (in the 15 mL conical tube
labeled PBS), move it rapidly over the fullwidth of the plate as indicated below in Circle
1. Place your swab in the 15 mL conical.
3. Get a new inoculating loop, turn the plate
again 90° and repeat, this time make sure
you have a portion of the streak that does
not re-enter the previous streaks (see
Circle 3).
2. Turn the plate 90°, use an inoculating loop
and start your streak at the end of the
primary inoculation zone. Make 3- 4 passes
over the primary inoculation zone as shown
in Circle 2. Dispose of your inoculating loop
in the biohazard bag.
4. Invert and incubate your plates overnight
(science assistants will take your plates to
the incubator).
DAY 2
Use the charts below to record your observations from
the nose and throat experiments.
For the recording growth use the plus system. This can
be estimated by determining the zone where you ended
up with single colonies (indicated on the Circle diagram
page 8).
1–2+ = growth in the initial 1/3 to 1/2 of the plate.
3+ = growth in the middle 1/3 of the plate
4+ = growth in all zones
For the recording of the characteristics, refer to the
sheets in your notebook demonstrating growth characteristics of the different agars (pages 12–13). Record
color changes and types of hemolysis, plus any other
observations you note (different types of bacterial colonies shapes and sizes).
MICROBIOTA OBSERVATIONS
NASAL
TSA
Are the
colonies all
the same
size?
MSA
Did the
colonies
ferment
mannitol?
BA
What kind
of hemolytic
reaction did
you observe?
MAC
Did the
colonies
ferment
lactose?
EMB
Did the
colonies
create a
green sheen?
CNA
Are the
colonies all
the same
size?
TSA
Are the
colonies all
the same
size?
MSA
Did the
colonies
ferment
mannitol?
BA
What kind
of hemolytic
reaction did
you observe?
MAC
Did the
colonies
ferment
lactose?
EMB
Did the
colonies
create a
green sheen?
CNA
Are the
colonies all
the same
size?
Characteristics of
growth on plate
Amount of Growth
(plus system)
Throat
Characteristics of
growth on plate
Amount of Growth
(plus system)
9
Activity 2
SOLVE A
MICROBIOLOGY
CASE STUDY
Goal: To use knowledge you gained
in the previous activities to identify
the microorganism from a mixed
culture and present your diagnosis
to the other students.
DAY 1
1. Obtain a mystery sample and a case study
from the science team. The mystery may
contain Gram positive or Gram negative
microorganisms (or both).
2. Using a swab, streak your unknown on each
agar and using an inoculating loop streak for
isolation on BA, MSA, TSA, MAC, EMB, and
CNA. Also streak on the agar that contains an
antibiotic (science team will explain).
3. Give your plates to the science team to
incubate overnight.
Catalase Test
Some bacteria such as Staphylococcus aureus
and Staphylococcus epidermidis, contain the
enzyme catalase which reacts with hydrogen
peroxide and forms water and bubbles of oxygen.
Other types of bacteria, such as Streptococci
species do not contain catalase. Thus, a drop
of hydrogen peroxide on a sample of bacteria
can help to differentiate what type of bacteria is
present.
1 Obtain a single culture and pick up the
culture with an inoculating loop. Spread the
colony on a glass slide.
2 Ask for a science mentor to come and assist
you with the catalase test. You will pipette a
small amount of hydrogen peroxide onto your
sample. Record the results.
10
DAY 2
1. Prepare a Gram stain of your mystery sample.
A. Draw 3 circles on a slide using a wax pen.
B. In circle 1, take a single colony from the
Staphylococcus plate. This will be your control,
demonstrating a Gram-positive result (ask a
science team member for help).
C. In circle 3, take a single colony from the
Escherichia coli plate. This will be your
control, demonstrating a Gram-negative result
(ask a science team member for help).
D. In circle 2, take a single colony from your
unknown sample.
E. Follow the steps on the Gram Stain Procedure
Handout.
?
Staphylococcus
Unknown
Escherichia coli
2. Perform catalase test (instructions shown at left).
3. Write down your results and discuss your findings
with your team.
4. Report the most likely culprit based on laboratory
results and based on the clues given in the case
study (i.e. how did the person get infected, what
area of the body is experiencing the illness).
CASE STUDY OBSERVATIONS
Use the chart below to record your observations from your case study.
For the recording growth, use the plus system. This can be estimated by determining the zone where you ended up
with single colonies (indicated on the Circle diagram page 8).
For the recording of the characteristics, refer to the info sheets demonstrating growth characteristics of the different
agars on pages 12 and 13. Record color changes and types of hemolysis, plus any other observations you note (different types of bacterial colonies shapes and sizes).
Unknown #
TSA
Are the
colonies all
the same
size?
MSA
Did the
colonies
ferment
mannitol?
BA
What kind
of hemolytic
reaction did
you observe?
MAC
Did the
colonies
ferment
lactose?
EMB
Did the
colonies
create a
green sheen?
CNA
Are the
colonies all
the same
size?
Characteristics of
growth on plate
Amount of Growth
(plus system)
Additional observations:
Gram stain results:
Catalase Results:
Clues from the case study:
Diagnosis:
11
AGAR INFO
Mannitol Salt Agar (MSA)
B
A
C
A selective and differential agar which contains 7.5% salt to select for certain Gram-positive bacteria such as Staphylococci. The
high salt concentration selects for halophilic
organisms (organisms that like salt). It also
contains mannitol sugar and phenol red (a pH
indicator), which will select for organisms that
ferment mannitol. If an organism can use mannitol as its energy source, the agar will turn
yellow due to a drop in the pH which causes a
color change. For example, both Staphylococcus aureus (section A) and Staphylococcus
epidermidis (section B) will grow on MSA but
only Staphylococcus aureus can ferment mannitol. Section C demonstrates no growth.
Blood Agar (BA)
A nutrient rich medium that contains 5%
sheep blood. This agar will grow most organisms and will help you identify what bacteria is
growing based on the ability of the bacteria to
lyse blood cells. Different bacteria have different types of hemolysins: α, β, and δ hemolysin.
The blood agar will look different depending on
what type of hemolysins the bacteria have.
MacConkey Agar (MAC)
A selective and differential medium used to
select Gram-negative bacteria. It can differentiate bacteria based on their ability to ferment
lactose. If bacteria can use lactose, they will
turn a pink color. If bacteria cannot use lactose, they will remain colorless or yellow. E. coli
will ferment lactose but Salmonella sp. cannot.
12
Eosin Methylene Blue Agar (EMB)
A selective and differential medium that
allows growth of Gram-negative bacteria.
The agar contains lactose and two dyes:
eosin and methylene blue. When a bacteria
is able to ferment lactose, it produces acid
and turns the bacterial colony a dark purple
to black. Colonies that do not use lactose will
be colorless.
Colistin-Nalidixic Acid Agar (CNA)
A selective medium containing the antibiotics colistin and nalidixic acid which selects
for Gram-positive bacteria. S. aureus, S.
epidermidis, and Group A Streptococcus will
grow on this medium.
Tryptic Soy Agar (TSA)
A supportive medium which contains glucose
and amino acids for the growth of most microorganisms, it will not select for Gram-positive
or Gram-negative (both groups will grow on
this agar).
Bacteriological Media, http://faculty.
mc3.edu/jearl/ML/ml-8.htm
13
Mystery Suspects
Escherichia
coli (E. coli)
✔ Gram-negative rod
✔ Ferments lactose
✔ Catalase positive
Clinical
Presentation
✔ Watery or bloody diarrhea
✔ Urinary Tract Infection (UTI)
✔ Septic Shock
Pathobiology
✔ In normal GI flora
✔ Animal sources of infection
✔ Transmits via fecal-­oral
Clinical Case
Example
Patients in a small town visit
the hospital complaining about
bloody diarrhea, fatigue, and
confusion. After interviewing the
patients, the doctors discover
that each patient frequents the
same fast-­food burger joint. The
doctors identify the causative
agent using serological testing
and stool cultures.
E. coli on MacConkey agar
14
Salmonella
enterica (Salmonellosis)
✔ Motile by flagella
✔ Produces hydrogen-sulfide
✔ Does not ferment lactose
✔ Catalase positive
Clinical Presentation
✔Gastroenteritis
Pathobiology
✔ Carried in animals and humans
✔ Transmits fecal-oral
Clinical Case Example
A veterinary student complains to the
doctor of diarrhea and abdominal tenderness. He also had nausea and vomited
the day before. He notes that he recently
handled with his pet turtle.
Mystery Suspects
✔ Gram-negative rod
S. enterica on MacConkey Agar
15
Streptococcus
pyogenes
Mystery Suspects
a.k.a. Group A Streptococcus
✔ Gram positive cocci in chains
✔ Typically infects throat or skin
✔ B-hemolytic on blood agar
✔ Catalase negative
Clinical
Presentation
✔ Pharyngitis (Strep throat)
✔Impetigo
✔Cellulitis
Pathobiology
✔ Transmits human to human via
respiratory droplets, saliva
✔ Trauma introduces bacteria into
skin
Clinical Case
Example
A young child presents with a fever and
skin rash localized around the lips and
on his arms. The rash appears to have
pustules with yellow crusts. Cultures
from the skin show Gram pos. cocci
and are β-hemolytic. The doctor administers penicillin G.
S. pyogenes on blood agar
16
Staphylococcus
epidermidis (S. epi)
✔ Catalase positive
✔ Coagulase negative
Clinical Presentation
✔ Infection on indwelling medical
devices such as catheters or
prosthetic joints
Pathobiology
✔ Normal flora on skin
✔ Forms biofilms and adheres to
medical device
Clinical Case Example
Ten days after undergoing chemotherapy for cancer, a middle-aged man
develops a fever. On exam, he has erythema and tenderness at the insertion
site of the IV catheter. Blood cultures
are positive for Gram-positive bacteria.
The original catheter is removed and
the patient is started on antibiotics.
Mystery Suspects
✔ Gram-positive grows in clusters
S. epidermidis on Mannitol Salt Agar (MSA)
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Mystery Suspects
Staphylococcus
aureus (S. aureus)
✔ Gram positive clusters
✔ Catalase positive
✔ Coagulase positive
✔ Antibiotic resistance is a problem
(MRSA = methicillin-resistant S.
aureus)
Clinical
Presentation
✔ Skin/subcutaneous: impetigo,
cellulitis, boils
✔ Sepsis, Endocarditis
Pathobiology
✔ Colonize skin or pharynx and
immune system responds
and causes inflammation and
abscess development
✔ Entry into blood via ruptures in
skin
Clinical Case
Example
A college basketball player presents
to the clinic with several red painful
purulent boils on his upper arms.
Gram stain of the purulent material reveals Gram-positive clusters.
Culture is resistant to penicillin and
methicillin.
S. aureus on Mannitol Salt Agar (MSA)
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TIPS FOR YOUR PROJECT
Read this over before the Infectious
Disease virtual lab meeting. During the
virtual meetings we will answer questions
about testing a hypothesis and on your
experimental design.
1. You can work in groups or work by yourself.
2. Develop a hypothesis to be tested. Think of a
topic you are interested in learning more about.
For example, have you ever wondered if hand
sanitizers actually work? Or how fast they actually
work? Do they work better than plain old soap and
water? Figure out what is known about the topic:
Perform an internet search, read an article about
it, interview an expert. Develop an educated guess
(i.e. a hypothesis) based on your knowledge.
I hypothesize that 60 seconds of rubbing
my hands with waterless hand sanitizer will
reduce bacterial numbers and diversity on
my hands compared to washing my hands
with soap and water for 60 seconds.
3. Write down your experimental procedure. How will
you test your hypothesis? What will the variables
be? How many plates do you need? Since we are
shipping supplies in October you can tell us if
you need more plates and or swabs to test your
particular hypothesis. Of course, there is a limit
on how large you can make your experiment, but
we can accommodate a few extra materials.
Experimental Procedure:
A. Identify your control. How do you obtain a
baseline of bacteria from your hands? Should
you wash your hands first before beginning
the experiment? Should the test be done on
the same day or on a different day? What is
the more controlled experiment? The control
will be compared to the experimental groups
to assess how the treatment altered the
outcome.
B. Design a procedure where the hands will be
equally dirty. Make sure you are consistent
with how you dirty your hands! Some possible
methods include washing your hands with
soap and water (to keep the baseline bacteria
on your hands similar between tests) and
then:
1. touching raw chicken for 60 seconds.
2. brushing your horse without gloves for 5
minutes.
3. typing on a public computer for 5 minutes
(at a library or computer lab at school).
C. Decide how to sample the bacteria on your
hands. Will you swab each finger? Will you
swab 3 fingers on each hand – on one hand
only? Will you combine the swab material? If
so how – on the petri dish? Make sure you
are consistent with how you collect samples!!
D. Standardize how you “wash” your hands.
Your hypothesis says 60 seconds, but doesn’t
specify how vigorous you’ll be washing. You
will probably want to make sure the motion
is very similar between washing with the
waterless hand sanitizer and with soap and
water. Now, again, you need to sample the
bacteria on your hands. Do this exactly as you
did after touching the contaminated material.
4. Record exactly how you are doing your
experiment so you can determine if you have
introduced more variables that are complicating
your results.
5. Record results, take pictures, and fill out table
for growth on media. How much growth (use the
plus system from the module, 1-2 +, 3+, 4+), and
which plates supported growth (diversity of the
microorganisms)?
6. If possible, repeat!
7. Was your hypothesis correct? What are the
implications of your findings? How does this
support or add to the information that is already
available on soap and water versus waterless
hand sanitizers? If you were to perform your
experiment again what would you change?
8. Put together a presentation. You can either have
a poster or oral presentation for our meeting at
Montana State University in January.
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DESIGN YOUR EXPERIMENT
Hypothesis:
Background Facts:
Methods:
Variables:
Results:
Which media supported growth? How much growth did you find? Review your materials from the hands-on module at
Montana State University.
TSA
MSA
BA
MAC
Additional observations:
Implications of your findings:
What would you do differently if you had the chance to re-do the experiment?
20
EMB
CNA
GLOSSARY
Abscess: a collection of pus on the body that causes
pain, swelling, and inflammation around it; typically
due to an infection
Colony: a visible group or cluster of bacteria derived from
one bacterium, you count colonies on an agar plate
to quantify bacterial growth
Aliquot: a portion of a solution
Crystal Violet: a dye used in Gram staining; it remains
inside bacterial walls containing higher amounts of
peptidoglycan
Antibiotic Resistance: a microorganism that is able to
survive exposure to an antibiotic
Antibiotics: a substance or compound that slows down or
prevents the growth of bacteria
Bacteria (Eubacteria): a domain of microorganisms, only
micrometers in length, with a primitive nucleus
Buffer AE: a solution that binds to DNA in order to
remove purified DNA from a column
Buffer AL: a solution that disrupts protein structures
Buffer AW1: a solution that denatures (destroys) proteins
in order to purify DNA
Buffer AW2: a solution that contains ethanol, which
removes cellular salts from a sample in order to
purify DNA
Carrier: an individual who is colonized (infected) with
a pathogen, but free of disease, who is capable of
acting as a source of infection for others
Catalase: an enzyme that degrades hydrogen peroxide
into hydrogen and water
Catheter: a tube that can be inserted into a body
cavity, duct, or vessel that allows for drainage,
administration of fluids or gases, or access by
surgical instruments; an example is a urinary
catheter inserted in the bladder to drain urine
Cellulitis: a common skin infection caused by bacteria,
symptoms commonly include: redness, inflammation,
and soreness of the skin; also can cause fever-like
symptoms
Deoxyribonucleic acid (DNA): found in all cells and
contains the genetic instructions used for
development and function; consist of two sugar
backbones (containing deoxyribose sugar) with linked
nucleotides in between; the nucleotides can be one
of four bases and the sequence of these bases
determines genetic make-up of the organism
Disease: a condition that is accompanied by an impaired
body function
Echocardiogram: a type of ultrasound that images the
heart; can provide an assessment of the state of
heart tissue
Endocarditis: an inflammation of the inside lining of the
heart chambers and heart valves
Endotoxin: a type of toxin found on Gram-negative
bacteria that is made up of the lipopsaccharide chain
found on the outer membrane of the bacterium
Erythema: redness of the skin occurring with skin
infection, injury, or inflammation
Ethanol: an alcohol used for many biological purposes;
can be used to disrupt protein structure and remove
salts to help purify DNA; can be also used as a
disinfectant due to its ability to penetrate bacterial
cell walls and is used to decolorize bacterial cells in
the Gram staining procedure
Exotoxin: a potent toxin secreted by some forms of
bacteria
Centrifuge: a piece of equipment that puts an object
in circular motion, causing denser substances to
settle to the bottom of the tube- “the pellet”- while
the less dense constituents remain on top- “the
supernatants”
Flora: a population of microbes that inhabit the internal
and external body surfaces of healthy humans and
animals (see: microbiota)
Coagulase: a protein produced by some microorganisms
that converts fibrinogen to fibrin, resulting in
clumping of blood; used to distinguish between
different types of Staphylococcus species
Gastroenteritis: a medical condition characterized by
inflammation of the GI tract, including the stomach
and small intestine; results in a combination of
diarrhea, vomiting, abdominal pain, and cramping
Furuncles: a boil; results in a painful swollen area on the
skin filled with pus and dead tissue
Colonization: establishment of a microbial population in
the animal/human host
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Gel Electrophoresis: the separation of DNA, Ribonucleic
Acid (RNA), or protein, based on size and charge;
electric currents are applied to move molecules
through the matrix
Gram Stain: a method of differentiating bacterial species
into Gram-positive and Gram-negative bacteria based
on the amount of peptidoglycan in their cell walls
Gram-negative: bacteria that do not retain the crystal
violet stain due to a low amount of peptidoglycan in
the cell wall; these bacteria typically stain red during
Gram staining (they retain the color of safranin, the
counter-stain)
Gram-positive: bacteria that retain the crystal violet
stain due to a high amount of peptidoglycan in the
cell wall; these bacteria typically stain purple during
Gram staining
gyrB: a gene present in all Staphylococcus aureus; used
as a control during PCR to confirm DNA was isolated
from Staphylococcus aureus
Hemolysis: from the Greek word meaning “release of
blood”, is the rupturing of red blood cells followed by
the release of hemoglobin into surrounding medium,
there are three types of hemolysis: alpha (α) reduces
the iron in the blood (bacteria colonies form “a green
halo” on blood agar), beta (β) completely ruptures
the blood cells (bacteria colonies form a “clear halo”
on blood agar), and gamma (γ) is a lack of hemolysis
(colonies often appear grey with no halo on blood
agar)
Hypothesis: a proposed explanation for a phenomenon/
occurrence, often based on previous observations
that can be tested with a set of experiments
Impetigo: a common skin infection characterized by a
single, or many, blisters filled with pus and, when
broken, leaves a reddish, raw-looking base
Infection: results from a microbe that penetrates body
surfaces, gaining access to tissues and multiplying
which then induces a host response
Iodine: a trapping agent that binds to crystal violet
making it a larger molecule to trap the dye in the
peptidoglycan
Lactose: a sugar found in milk formed by galactose and
glucose
Lipopsaccharide chain: found on the outside of the outer
envelope of a Gram-negative bacteria, often called
endotoxin
22
Lyse: to burst or cause dissolution or destruction of cells
Lysostaphin: an antibacterial enzyme that can cleave
components of Staphylococcus aureus cell wall; this
is used in DNA extraction to release the genetic
makeup (DNA) out of bacteria
Mannitol: a sugar alcohol, typically of a lower (acidic) pH;
component of Mannitol Salt Agar (MSA); colonies of
bacteria that can ferment mannitol appear yellow on
the plate while those that cannot appear pink
mecA: a gene found in bacteria that confers resistance
to a large class of antibiotics including penicillin
and methicillin; Staphylococcus aureus strains
that have this gene are called methicillin-resistant
Staphylococcus aureus (or MRSA)
Media: a liquid or gel designed to support the growth of
microorganisms or cells
Meningitis: a bacterial infection of the membranes
covering the brain and spinal cord
Microbiota: a population of microbes that inhabit the
internal and external body surfaces of healthy
humans and animals (see: flora)
Microorganism: a microscopic organism that can consist
of a single cell, cell cluster, or a multicellular complex
organism; includes bacteria, viruses, algae, fungi, etc.
Morphology: the form and structure of organisms and
their specific features
MRSA: methicillin-resistant Staphylococcus aureus
is a form of Staphylococcus that has developed
resistance to a large class of antibiotics (see mecA)
Opportunistic Pathogen: microorganism that is free living
or a part of the host’s normal microbiota but may
become pathogenic under certain circumstances,
such as when the immune system is compromised
Pathogen: a microorganism capable of producing disease
in a healthy animal or human host
Pathogenicity: ability to cause disease
Peptidoglycan: a major component of the bacterial cell
wall that contributes to the shape of the bacteria;
amount of peptidoglycan is used to differentiate
bacteria in the Gram stain (see Gram-positive and
Gram-negative)
Pharyngitis: inflammation of the throat; also known as a
sore throat
Pharynx: the throat
Phosphate Buffered Saline (PBS): a buffer solution used
in biological research; a water-based salt solution
that is non-toxic to cells
PI Buffer: a buffer used in the isolation of DNA that
prevents cellular clumping and degrades the RNA
that is present
Polymerase Chain Reaction (PCR): a technique used
to amplify DNA in order to generate thousands
to millions of copies of a specific sequence; may
be used for DNA cloning, identifying evolutionary
ancestors, diagnosing genetic diseases, and
identifying genetic fingerprints used in forensic
science labs
Primer: a start point for DNA synthesis and replication;
primers are typically short strands of nucleic acid
(the building blocks of DNA) that are used to bind a
specific sequence of DNA in order to amplify it during
a PCR reaction
Proteinase K: destroys proteins that degrade DNA and
RNA
Reagents: a substance or compound added to a system
in order to cause a reaction
Ribonucleic acid (RNA): like DNA, found in all cells;
consists of one sugar backbone (containing ribose
sugar) with nucleotides bound to the backbone; RNA
is created based on a DNA template and is then
used to make proteins necessary for cellular function
Safranin: a biological stain used as a counterstain in
Gram staining
Sepsis: a medical condition characterized by a whole
body inflammatory response to an infection; also
known as blood poisoning
Serological Testing: a test used to determine the
presence of a microorganism in the blood
Supernatant: the liquid above a solid residue (pellet) after
centrifugation
Virulence: attributes of a microbe that enhance its
pathogenicity
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