Bacteriathe good,

the bad,

and the ugly

InfectIous DIseasesby 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 UniversityP.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 .................................................................................................Carrie Benke

Participant Coordinator .......................................................................................Todd Kesner

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

1

there are about 10 times as many bacterial cells living inside your body as there are cells that make up your body!

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.

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

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 reproduc-tion called binary fission. Binary fission allows them to clone themselves by replicating their DNA and then di-viding. 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 hu-man 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

In an average human being, 20 billion E. coli are replicated each day.

2

natural digestive flora (the microbes that live in your di-gestive 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 simi-lar 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 actu-ally leads to another type of bacteria classification. Early

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, there-fore, 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 invad-ers 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 Scientists have a specific word for the things that make us sick: pathogens. A pathogen is defined as a dis-ease-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

figure 1: A diagram showing the structural differences between a Gram positive and Gram negative bacterium.

3

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 surpris-ingly elegant.

Viruses have an outer coat, called a capsid, that is made of proteins. Their capsids are constructed of geo-metric 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 im-portantly, 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 tu-berculosis, gonorrhea, and leprosy actually invade the tis-sues they infect and destroy them. Obviously this causes damage to the individual and the living tissues the bacte-ria 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 lipopolysac-charide 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

vocaBUlaryTerm 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)

PeptidoglycanA 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 negativeBacteria 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

4

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 bacteri-al 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 differ-ence 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 bacteri-um 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 anti-biotics 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 per-fect 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 resis-tant bacteria? This is a term used to describe bacteria that have developed a way to cope with antibiotics. Antibiotic resistance can have some disastrous impli-cations 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 charac-teristics 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!

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.

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.

5

Liz and emma carlson examine their horse microbi-ome project plates. the sisters from Lewis and clark county investigated the number and type of bacteria in horses of different ages.

Bacteria are extremely adaptable through natural selection because of their very rapid rates of reproduc-tion, 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 antibi-otic, 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 liv-ing in the petri dish had some mutation that made it re-sistant 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 muta-tion. 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 Staph-ylococcus 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

overwhelms your immune system and you get a “Staph” infection, but these are commonly treated with antibiot-ics and rarely cause for concern. MRSA is different. This type of bacteria is resistant to a large class of antibiot-ics, 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.

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 bio-sciences 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).

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!

6

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.

laBoratory safety

7

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 1throat 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.

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.

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.

8

streaking for isolation1. After dipping your nasal or throat swab

into sterile PBS (in the 15 mL conical tube labeled PBS), move it rapidly over the full-width of the plate as indicated below in Circle 1. Place your swab in the 15 mL conical.

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.

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).

4. Invert and incubate your plates overnight (science assistants will take your plates to the incubator).

9

Day 2use 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

NASAL TSAAre the colonies all the same size?

MSADid the colonies ferment mannitol?

BAWhat kind of hemolytic reaction did you observe?

MACDid the colonies ferment lactose?

EMBDid the colonies create a green sheen?

CNAAre the colonies all the same size?

Characteristics of growth on plate

Amount of Growth (plus system)

Throat TSAAre the colonies all the same size?

MSADid the colonies ferment mannitol?

BAWhat kind of hemolytic reaction did you observe?

MACDid the colonies ferment lactose?

EMBDid the colonies create a green sheen?

CNAAre the colonies all the same size?

Characteristics of growth on plate

Amount of Growth (plus system)

For the recording of the characteristics, refer to the sheets in your notebook demonstrating growth charac-teristics of the different agars (pages 12–13). Record color changes and types of hemolysis, plus any other observations you note (different types of bacterial colo-nies shapes and sizes).

microBiota oBservations

10

activity 2 solve a microBiology case stUDy

Day 11. 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.

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 21. 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

catalase testSome 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.

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).

11

case stUDy oBservationsuse 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 (differ-ent types of bacterial colonies shapes and sizes).

Unknown # TSAAre the colonies all the same size?

MSADid the colonies ferment mannitol?

BAWhat kind of hemolytic reaction did you observe?

MACDid the colonies ferment lactose?

EMBDid the colonies create a green sheen?

CNAAre 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:

12

Mannitol salt agar (Msa)A selective and differential agar which con-tains 7.5% salt to select for certain Gram-pos-itive 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 man-nitol as its energy source, the agar will turn yellow due to a drop in the pH which causes a color change. For example, both Staphylococ-cus aureus (section A) and Staphylococcus epidermidis (section B) will grow on MSA but only Staphylococcus aureus can ferment man-nitol. Section C demonstrates no growth.

Blood agar (Ba)A nutrient rich medium that contains 5% sheep blood. This agar will grow most organ-isms and will help you identify what bacteria is growing based on the ability of the bacteria to lyse blood cells. Different bacteria have differ-ent 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 differen-tiate bacteria based on their ability to ferment lactose. If bacteria can use lactose, they will turn a pink color. If bacteria cannot use lac-tose, they will remain colorless or yellow. E. coli will ferment lactose but Salmonella sp. cannot.

agar info

a B

c

13

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 antibiot-ics 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 micro-organisms, 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

agar info

14

Mystery Suspects

✔ 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 examplePatients 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

Escherichia coli (E. coli)

15

Mystery Suspects

✔ Gram-negative rod

✔ 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 exampleA veterinary student complains to the doctor of diarrhea and abdominal tender-ness. He also had nausea and vomited the day before. He notes that he recently handled with his pet turtle.

S. enterica on MacConkey Agar

Salmonella enterica (Salmonellosis)

16

✔ 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 exampleA 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 admin-isters penicillin G.

S. pyogenes on blood agar

Streptococcus pyogenesa.k.a. Group A Streptococcus

Mystery Suspects

17

✔ Gram-positive grows in clusters

✔ 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 exampleTen days after undergoing chemother-apy for cancer, a middle-aged man develops a fever. On exam, he has ery-thema 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.

S. epidermidis on Mannitol Salt Agar (MSA)

Staphylococcus epidermidis (S. epi)

Mystery Suspects

18

✔ 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 exampleA college basketball player presents to the clinic with several red painful purulent boils on his upper arms. Gram stain of the purulent materi-al reveals Gram-positive clusters. Culture is resistant to penicillin and methicillin.

S. aureus on Mannitol Salt Agar (MSA)

Staphylococcus aureus (S. aureus)

Mystery Suspects

19

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.

20

Design yoUr experimenthypothesis:

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 EMB CNA

additional observations:

Implications of your findings:

What would you do differently if you had the chance to re-do the experiment?

21

Abscess: a collection of pus on the body that causes pain, swelling, and inflammation around it; typically due to an infection

Aliquot: a portion of a solution

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

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”

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

Colonization: establishment of a microbial population in the animal/human host

Colony: a visible group or cluster of bacteria derived from one bacterium, you count colonies on an agar plate to quantify bacterial growth

Crystal Violet: a dye used in Gram staining; it remains inside bacterial walls containing higher amounts of peptidoglycan

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

Flora: a population of microbes that inhabit the internal and external body surfaces of healthy humans and animals (see: microbiota)

Furuncles: a boil; results in a painful swollen area on the skin filled with pus and dead tissue

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

glossary

22

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

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

23

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