Microbiology for the Health Sciences

Second Edition Spring 2018

“A person who won’t read has no advantage over one who can’t.”

-Mark Twain

“Computers are useless. They can only give you answers.”

-Pablo Picasso

1 Table of Contents

Lab Title Page # Introduction to Microscopy and Microbes 12 Cultivation of Microbes; Microbes in the Environment and on the Skin 25 Isolation of Bacteria by Dilution Techniques 31 Gram Stain 44 Acid Fast and Endospore Staining 54 Morphological Unknown 66 Microbial Growth; Differential, Selective and Enriched Media 76 How does the atmosphere affect microbial growth? 85 Carbohydrate Metabolism 96 Bacterial Transformation 105 Ames Test and UV radiation induced mutation 112 Control of Microbial Growth; Physical Methods 120 Chemical Methods of Control; Disinfectants and Antiseptics 126 Antimicrobial Drugs 132 How Does Antibiotic Susceptibility Differ Between Fungi and Bacteria? 142 Epidemiology: how a cold spreads through the dorm or lecture hall 149 Contaminated Spray Bottle and Water Contamination 155 Genus Unknown 163 Immunology; Innate Immunity 173 Blood Typing: ABO and Rh Blood Group Systems 180 White Blood Cell Identification 189 Rapid Antigen Detection Test for Group A Streptococcus 195 Public Service Announcement: Communicating Science to the Public 201 Universal Precautions in Action, Specimen Transport 207 Dipstick Urinalysis for the Detection of Urinary Tract Infections 213 Bacteria of the Gastrointestinal (GI) Tract 219

2 Note from the authors We want to impart onto you that a solid understanding of microbiology is valuable in all aspects of life and is absolutely critical in many fields including nursing, dietetics, respiratory therapy, health science, and health education & promotion. You are ultimately responsible for your learning. Those of you who enter the field of biology in some capacity whether it be the health sciences, basic sciences, or industry will be entering professions that will rapidly change during the course of your professional career. As such, you will need to be a life-long learner to remain current in your understanding of science and up to date on best practices. Take a genuine interest in your field and try to learn all you can while you’re still in school but you will continue to learn for the rest of your careers. We attempted to make this lab manual as clear, concise, engaging, and clinically relevant as possible. Please email David Huston ([email protected]) and Doug Bernstein ([email protected]) if you have suggestions or complaints regarding the lab manual that can be corrected for future semesters. Our goal is make the labs relevant to the health sciences and also interesting. Some of the labs that we will be performing are experiments in the truest sense of the word. As such, we as instructors and you as students have no idea exactly what the results will be. The great physicist Enrico Fermi stated, “There are two possible outcomes: if the result confirms the hypothesis, then you've made a measurement. If the result is contrary to the hypothesis, then you've made a discovery.” We will not always know what the result will be when we perform an experiment but your goal as students will be to interpret the results that you get to the best of your ability and draw conclusions from these results. Furthermore many of the labs we will perform will take multiple days. We will do the experiment one day and look at the results during the next lab period. This is intentional and unavoidable as microbes require many hours to grow.

We want to continue to improve this lab manual in the future. If you spot anything that is problematic or could be improved, please email David Huston ([email protected]) and Doug Bernstein ([email protected]). Use the subject heading “Regarding BIO 113 Lab Manual” and indicate specific page numbers if possible. For questions about your grade, missed labs, or if you are struggling in the lab, please contact your professor or lab GA.

Good luck and have a great semester!

Doug and David

3 Laboratory Safety Guidelines

• Read the appropriate section in your lab manual before you come to lab and watch the appropriate videos posted on Blackboard. Make sure that you understand the science behind the lab that you will be performing. If something is unclear, I strongly encourage you to reread parts that you do not understand. If things are still unclear, read about the subject in your textbook and online. If things are still unclear, talk to your professor during office hours or your GA before lab begins. Contact your professor or GA if you will be unable to meet with them during these times. • Again, read the lab manual and watch the Blackboard videos and make sure you understand their contents before you come to lab. There are pre-lab questions you will need to answer prior to coming to lab. These may be collected at the beginning of lab for credit. • Show up to lab on time. The lab periods are short, and there is a lot to do. In lab, be efficient, but be safe. Never compromise on safety. • Wash your hands with soap before and after lab. Sanitize your lab station before and after lab. Wear gloves when handling bodily fluids. If you come into contact with bodily fluids of another person, notify your lab GA immediately. If bodily fluids are spilled on the laboratory bench, cover them with paper towels and then soak with bleach or other similar disinfectant for 15 minutes. Discard waste in the appropriate containers. Ask your lab GA if you have questions. • Be a good lab citizen, make sure you place everything back where you found it when you are done. Reagents are expensive. Use what you need for the lab, but try not to be wasteful. • Label all of your plates with your name, lab section, and date. Place plates upside down in the incubator to prevent condensation from accumulating on the surface of your agar. If you use oil immersion, please clean the oil off of your microscope using a KimWipe or microscope paper when you are done. (Don’t use paper towels!!!!) • It is important that you follow all of the instructions that your lab GA provides. Although it may be tempting, do not play with fire. Turn off the gas when you are not using it. Make sure alcohol containers are not near open flames. Do not drink from the alcohol containers, you will not get drunk but you will get diarrhea and expelled from lab. • Whenever heating a tube point away from yourself and others. • Keep long hair pulled back during lab even when rock music is playing. Do not eat, drink or smoke in the lab. Avoid touching your face. Avoid touching the faces of others. • Do not wear open toed shoes or sandals in lab. If you do your lab instructor will ask you to leave the lab. • Make sure you know where the safety shower, eye wash station, fire blanket, fire extinguisher, fire alarms are and how to use them.

4 • In this lab, you will be working with several antibiotics. If you have known allergies to these antibiotics, please let your professor and/or lab GA know as soon as possible. • The microbial strains that you will be working with in this lab should not cause disease under normal circumstances. If you are immunocompromised, pregnant, or have open wounds, please let your professor and/or lab GA know and special precautions can be taken. • Despite best efforts to prevent accidents, they do happen. Notify your lab GA immediately, and take the appropriate steps to minimize damage. We understand that accidents happen. Just don’t attempt to hide them.

If you agree to abide by these rules of lab and the rule of common sense, please sign below

______Date______

5 Microscope Care and Use Contract

1. When you are finished using the oil immersion lens, move stage to lowest position. 2. Remove slide from stage clips. 3. Wipe oil off oil objective (100X) with lens paper. Clean/dry lens and stage with lens paper. 4. Put scanning power (4X) objective in center position. 5. Turn off light. 6. Unplug and wrap up power cord neatly. 7. Replace dust cover, if available. 8. Store microscope in correctly numbered cabinet with stage down and 4X objective in center position. Carry microscope with both hands at all times.

I have read and I agree to abide by the above rules for microscope maintenance at all times. If I am unsure of a rule I will ask the lab instructor for assistance. I understand that these microscopes are used by many students in many classes and as such must be maintained with the upmost care. Failure to abide by these rules will have a negative impact on my grade. Students may be held responsible for the cost of repairs or replacement if they are found to be handling the equipment improperly.

X______

6 Laboratory Safety Guidelines (student copy, to keep for your reference)

7 • Make sure you know where the safety shower, eye wash station, fire blanket, fire extinguisher, fire alarms are and how to use them. • In this lab, you will be working with several antibiotics. If you have known allergies to these antibiotics, please let your professor and/or lab GA know as soon as possible. • The microbial strains that you will be working with in this lab should not cause disease under normal circumstances. If you are immunocompromised, pregnant, or have open wounds, please let your professor and/or lab GA know and special precautions can be taken. • Despite best efforts to prevent accidents, they do happen. Notify your lab GA immediately, and take the appropriate steps to minimize damage. We understand that accidents happen. Just don’t attempt to hide them.

Keep this copy so you can refer back to it throughout the semester. If you ever have any questions ask your instructor or professor!

8 Microscope Care and Use Contract (student copy, to keep for your reference) 1. When you are finished using the oil immersion lens, move stage to lowest position. 2. Remove slide from stage clips. 3. Wipe oil off oil objective (100X) with lens paper. Clean/dry lens and stage with lens paper. 4. Put scanning power (4X) objective in center position. 5. Turn off light. 6. Unplug and wrap up power cord neatly. 7. Replace dust cover, if available. 8. Store microscope in correctly numbered cabinet with stage down and 4X objective in center position. Carry microscope with both hands at all times.

Keep this copy so you can refer back to it throughout the semester. If you ever have any questions regarding microscope care or use ask your instructor or professor!

9 Biosafety Levels

http://cnx.org/contents/5XZItubD@3/Controlling-Microbial-Growth

Note: we will be working with BSL-1/2 microbes in this lab.

10 Ways to Control Microbes on Surfaces

http://cnx.org/contents/5XZItubD@3/Controlling-Microbial-Growth

Fomite—a non-living material that can carry infectious agents and transfer them onto a new host (e.g. uniforms, blood-pressure cuffs, stethoscopes, forks, doorknobs)

Introduction to Microscopy and Microbes

“In the year of 1657 I discovered very small living creatures in rain water.” ―Antonie van Leeuwenhoek

Examining this water next day, I found floating therein divers earthy particles, and some green streaks, spirally wound serpent-wise.... I judge that some of these little creatures were above a thousand times smaller than the smallest ones I have ever yet seen, upon the rind of cheese, in wheaten flour, mould, and the like. ―Antonie van Leeuwenhoek

C. albicans 1000x

Name ______Section______Date______

Pre-Lab Questions: (You should read through the lab thoroughly before answering these)

1. What should I do immediately after using the 100X objective lens?

2. You view an image using the 4X objective lens. What is the total magnification of the image you are viewing?

3. Using your microscope, you observe round-looking bacteria. What would you call them?

4. Only one objective lens should ever have oil on it. Which objective lens is this? What is the total magnification of this lens?

5. Which objective lens should you always use first?

13 Name ______Section______Date______

Background:

Observation is key to understanding. The invention of light microscopes revolutionized science because it allowed humans to visualize cells (the fundamental unit of life) for the first time. This discovery led to our modern understanding of cell theory and later the acceptance of germ theory. These developments facilitated preventative techniques that have saved millions of lives (e.g. not dumping feces into drinking water, hand washing, disinfecting surgical instruments, pasteurization).

Only a very, very small fraction of microbes cause disease in humans. Many microbes live on us. Some are commensals—they benefit from us but we are not harmed. Others are mutualists—they benefit and we benefit (e.g. bacteria that metabolize the foods we eat and provide us with vitamins like vitamin K). And that small fraction are pathogens—the microbes benefit and we are harmed.

Some areas in the human body should not have microbes. These include the brain, spinal cord, interior of the eye, blood, and amniotic sac. Microbes in these areas indicate disease. Some areas that normally contain microbes in healthy individuals are the intestines, skin, and upper respiratory tract. Microbiota (mutualists and commensal bacteria) physically take up space on the human body and take up nutrients which causes competition with pathogenic microbes and thereby prevents overgrowth and infection of pathogens. Furthermore, many mutualists and commensals secrete chemicals which inhibit the growth of pathogens. Our understanding of the human microbiota is still limited but many researchers are in the process of working on understanding the roles these bacteria play in human health.

It is important to be able to identify what microbes are present on the human body because microbes cause specific diseases. For instance, the bacteria Borrelia burgdorferi causes Lymes Disease but not gonorrhea which is caused by Neisseria gonorrhoeae. Identification is also important because specific microbes are sensitive to specific certain classes of antibiotics. We will learn more about this as the semester progresses.

Bacteria come in a variety of morphologies (shapes and configurations). Bacteria that are spherical in shape are called coccus (plural is cocci). Bacteria that are rod-like are called bacillus (bacilli). Bacteria that are comma shaped are called vibrio (vibrios). Spiral shaped bacteria are usually a spirochete (spirochetes). Bacteria that grow in pairs are called diplo--. For example, bacteria grow in pairs like Neisseria meningitidis (causative agent of bacterial meningitis) would is classified as a diplococci. Bacteria that grow in chains are called strepto--. For example, bacteria that are rods and grow in chains like Streptobacillus moniliformis (causative agent of rat-bite fever) would be classified as streptobacilli. Bacteria that grow in clusters are called staphylo--. For example, bacteria that are spherical and grow in clusters like Staphylococus aureus (the causative agent of a number of infections) would be classified as staphylococci. Shown on the next page is table of these different morphologies and growth patterns.

14 Nomenclature Shape

Coccus Spherical

Bacillus Rod

Vibrio Comma

Spirochete Corkscrew

Nomenclature Growth Pattern

Diplo-- Pairs

Strepto-- Chains

Staphylo-- Clusters

Significance: Why should I care to know how to use a microscope?

Microscopes are essential for the study and diagnosis of many diseases including microbial infections and cancers. Although not every healthcare professional uses a microscopes on a daily basis, a basic understanding of microscopy and appreciate of the use and limitations of microscopes is key to being a well-informed member of a healthcare team.

What are microscopes used for? There are many different types of microscopes each of which have specific uses. We will focus on compound light microscopes which are useful for the identification of the morphology (structure) of bacteria, fungi, parasites, and human cells.

Notes for Lab:

Microscopes are expensive. To avoid damaging them, please be careful. When carrying your microscope, keep one hand on the base and one on the arm. Reread the microscope care and use contract at the beginning of this manual or ask your instructor if you have any questions regarding the microscope care.

Objective lens (There are four on a rotating turret—4X, 10X, 40X, and 100X) Ocular lens Arm (provides 10X magnification)

Stage clip (Holds slide in place)

Stage

Light source

Base

Coarse focus (for Fine focus (for refined rapid resolution resolution adjustments) adjustments)

Above is the compound light microscope that you will be using in lab. It is good for visualizing bacteria and parasites. Its resolution, however, is limited by the wavelength of visible light. Our compound light microscopes have two lenses. The ocular lens is in the eyepiece and magnifies objects 10X. One of four objective lenses can be used at a time. They magnify the image a further 4x, 10X, 40X, or 100X. The total magnification is the product of the magnification of the ocular and objective lens. For example, if you select the 40X objective lens, the total magnification is (10)(40)=400X. The image is magnified 400 times.

Below are several other types of microscopes. You will only need to be familiar with the use and anatomy of your compound light microscope but you should know why one might need to use one of these other microscopes.

Above is a confocal laser scanning microscope used for research in Cooper Science. This is used if you would like to create a 3d image of a cell. How this is done is beyond the scope of this laboratory.

Above is an inverted light and epiflourescent microscope use for research in the basement of Cooper Science. This is very similar to the microscopes that you be using in lab but as opposed to relying on stains for visualization it can also use fluorescence to visualize cellular structures. Also you can note that the objectives are underneath the sample which is different from the microscope you are using in lab.

Above is a transmission electron microscope (TEM) used for research in Cooper. It can create an image on the computer of internal structures of cells and viruses. The epifluorescent scope cost ~$25,000 while this one cost $1.2 million. This limits their utility as teaching tools .

Remember, not all microscopes are limited to the microbiological laboratory setting. Colposcopes, for example, are modified microscopes that magnify the structures of the female reproductive system and are useful in the diagnosis of many gynecological diseases including cervical cancer.

Materials:

• Compound light microscope • Immersion oil • Assorted pre-set microbial slides • Lens Paper

19 Name ______Section______Date______Laboratory Exercise:

1. Obtain specimen slides from you lab GA. Pull back the stage clip and place the slide on the mechanical stage. The slide must be level on the stage and should be securely held by the clip. (If it is not you will not be able to focus the image) Make sure that the stage is lowered all the way down possible prior to putting the slide on (use the coarse focusing knob to move the stage down) and the 4X objective lens is in the active position.

2. Plug the microscope in and turn it on. Adjust the intensity of the light as needed.

3. Look through eyepiece. You will see blurry light. Use the course focusing knob to bring the image into focus. To improve the clarity of the image, rotate the fine focusing knob as needed. Be mindful to go slowly. Slight changes in focus can lead to you losing your image. If you cannot find your specimen ask you lab instructor.

4. To magnify your image further, rotate the objective turret so that the 10X objective lens is in the active position. You shouldn’t need to adjust the coarse focus, adjust the fine focus as needed.

5. To magnify your image further, rotate the objective turret so that the 40X objective lens is in the active position. You shouldn’t need to adjust the coarse focus, adjust the fine focus as needed.

6. To magnify your image further, move the 40X out of the active position and keep another lens from entering the active position. This intermediate position should will allow you to freely access the slide without an objective lens interfering from above. In this position, drop a single drop of immersion oil onto the slide where the light is coming through. You will need very little oil. Then rotate the objective turret so that the 100X objective lens is in the active position. You shouldn’t need to adjust the coarse focus adjust the fine focus as needed. Be very careful doing this. The glass on the objective lens is very delicate—take care not to press the glass on the objective lens too firmly onto the slide. After viewing immediately, clean the slide and objective lens with Lens Paper. Do not move back to the 40X objective after viewing under the 100x oil lens. This will get oil on the 40x lens and ruin it.

You will be given many different slides with various microbes on them. Take note of the color the microbes is as a result of the stain, the size and shape of the microbe, and how the microbe is grouped in relation to other microbes. Make sure to share slides with your lab mates. Sketch what you see in the circles below. Be sure to record what sample you are viewing and the magnification.

20 Name ______Section______Date______

Sample:______Magnification:______Sample:______Magnification:_____

Sample:______Magnification:_____ Sample:______Magnification:_____

21 Name ______Section______Date______

Sample:______Magnification:_____ Sample:______Magnification:_____

When you are done, make sure you put everything away in its proper place. Don’t hesitate to ask questions.

22 Name ______Section______Date______

Post Lab Questions:

1. You see you a cluster of circular bacteria growing in chains. How can you describe these bacteria?

2. Dexter Morgan, a Floridian forensic scientist, finds a drop of the suspect’s blood at a crime scene. Morgan looks at the blood under a microscope and sees a spiral-shaped bacteria in the suspect’s blood. With a strong knowledge of microbiology, Morgan concludes that the suspect is infected with this sexually transmitted infection (STI). What STI/STD is the suspect infected with? You may need to reference your book or the internet to assist you with this.

3. Generally speaking, which are larger, Eukaryotes or Prokaryotes?

4. Why didn’t we look at viruses in this lab under the microscope?

23 Name ______Section______Date______

5. Lactobacillus species (sp.) are commensal bacteria in the human vagina that compete with Candida albicans (C. albicans), a yeast (single celled fungi). When antibiotics like doxycycline are used to treat bacterial infections they can kill Lactobacillus sp that are part of the healthy microbiota. A) What shape are the lactobacillus? B) What do you anticipate happening as a result of the Lactobacillus being killed?

6. What is the maximum magnification of the microscopes you used in lab?

7. Why is diagnosis of the causative agent of an infection not always possible by microscopy alone?

Cultivation of Microbes; Microbes in the Environment and on the Skin

“If you don't like bacteria, you're on the wrong planet.” Stewart Brand

25 Name ______Section______Date______

Pre-Lab Questions:

1. What does sterile mean in microbiology?

2. How should you place your plates in the incubator, why?

3. What kinds of surfaces do you anticipate will have more bacteria on than others?

26 Name ______Section______Date______Background:

Bacteria can be found almost everywhere on earth from the deepest sea vents to the tops of mountains. One of the first things any microbiologist must learn however is how to grow bacteria in a lab. We will be culturing bacteria from our skin and the local environment in this lab on Petri dishes. To do this we must use sterile agar (contains no live cells). You will learn more about procedures to sterilize media later in the semester.

Although we probably don’t think about it on a daily basis bacteria are all over us and our environment. However if we want to study these microbes we must first figure out how to grow them in the lab. In this exercise we will culture (grow) these microbes and compare the number and types of microbes found on our skin and our local environment.

Materials:

• Petri dishes with agar • Cotton-tipped applicators • Sterile water

27 Name ______Section______Date______

Laboratory Exercise: In groups of 4 In this lab, we will be sampling what microbes are growing in our environment or from our skin. To do this, we will need to culture them. What you swab and how you do it is largely up to you. Be sure to document what you did. A recommended procedure:

1. Wet a cotton tipped applicator with a drop of sterile water. 2. Swab a surface with the cotton tipped applicator. 3. Swab the cotton tipped applicator over your agar.

If you would like to place objects (e.g. coins or your fingers before and after washing your hands) directly on the media, this is okay as well.

Do the above procedure for two environmental or skin samples on separate plates. Predict which sample will produce the most colonies.

Be sure to label your petri dish with your name, your section, the date, and what you swabbed. When you are done, place your petri dishes in stacks and upside down in the incubator. Condensation will accumulate in the incubator on the inside surface of the bottom of your petri dish. You would rather have this occur on the lid than on your agar.

After 2 days your bacterial will have grown enough so that you can see them. At this point, you or your instructor will remove the plates from the incubator. You will observe the growth on your plates in the following lab period. Take note of the source, kind of growth, and number of colonies and answer the post lab questions.

28 Sample 1 Sample 2

Environmental Source

Number of Colonies

Color of Colonies

Size/Shape of Colonies

Other notes

Sample 1 Sample 2

Post-Lab Questions:

29 Name ______Section______Date______Post Lab Questions:

1. Was your prediction from question 3 in the prelab correct? If not why do you think your data came out the way they did?

2. Why was sterile water used to wet the cotton tipped applicator and not tap water?

3. Do you anticipate a pure culture will grow on your agar from your environmental swab and why?

4. If you were doing microbial sampling for a hospital, why would you need to keep your plate covered with a lid when you are not streaking?

5. Do you anticipate that all of the bacteria that are on the surface of whatever you swabbed will grow on the agar we used and why? Do all bacteria like the same environment?

Isolation of Bacteria by Dilution Techniques

31 Name ______Section______Date______

Pre-Lab Questions:

1. Why is it important to isolate a pure culture of bacteria?

2. Will you be able to see bacterial growth by the end of the first lab period?

3. You performed a dilution of bacteria in liquid media. You pipette 1mL of your diluted sample and count 287 bacterial colonies on your agar after a 24 hour incubation. If you were to pipette 0.1mL from this same diluted sample, how many bacterial colonies would you expect to count on your agar?

32 Name ______Section______Date______Background:

In this lab we will focus on isolating single species or pure culture of bacteria from a population of many bacteria. There are two primary reasons dilution techniques can used in labs.

1) Bacteria rarely live alone as a single species. In the environment and on you, many species of bacteria grow together resulting in complex microbial communities. For scientists studying bacteria, such biodiversity within a small area is challenging. To understand the physiology of a single species, scientists must isolate and grow a single species on growth media in the lab—this is called a pure culture (a culture with only one species and strain growing on it). When isolated and grown as a pure culture in lab, it is much easier to perform experiments to characterize and study bacteria.

2) In clinical samples of microbes, there would be far too many bacteria to count if we were to simply culture them. To determine the number of bacteria in a sample we will need to dilute the sample to make counting more manageable. Generally, scientists want to count between 30 and 300 microbes on a plate. If we dilute out sample too much and get very few colonies, variability will prevent us from getting an accurate count. If we don’t dilute our samples enough we will end up with more than 300 colonies and counting becomes burdensome and unreliable.

We will dilute our colonies two different ways. 1) The streak plate The streak plate technique is designed to isolate single colonies on agar. 2) The Spread Plate The spread plate technique quantitatively determines how many bacteria are in an original sample.

Materials:

Bacteria overnight liquid culture

E. coli on a plate

Sterile Media

Pipettes and pipette tips

Sterile Test tubes

Dilution media

Sharpie or wax marker

33 Name ______Section______Date______Laboratory Exercise: Streak plate: Groups of 4

To perform the streak plate technique, divide your plate into four quadrants. You may use a sharpie or wax marker to do this on the bottom of your plate. You will use a sterile loop to collect your sample and then streak it diagonally back and forth on the first quadrant. You will then sterilize your loop and collect a sample from the first quadrant and spread your loop diagonally for the second streak. You will then sterilize your loop to collect a sample from the second streak and then the third streak. You will then sterilize your loop to collect a sample from the third streak and then streak the fourth streak. After 24 hours of incubation, you should be able to observe single colonies growing, particularly in the fourth quadrant (which is the most diluted).

To sterilize your loop run it through a flame for a couple of seconds. Cool the loop by touching it to somewhere on the agar that does not contain bacteria. If you touch the bacteria with the hot loop it will kill the bacteria.

Each person in the group should try this. Below is a diagram illustrating how to streak plate four separate samples on one petri dish.

label First Streak

Second Streak

label

Third Streak

label Fourth Streak Divide Plate into ¼s and label each quadrant

Spread Plating Procedure: One set of plates for each section

We will dilute the sample in successive amounts of sterile media and then inoculate a specific amount of media onto the plate. After incubation, we will count the number of colonies and use math to determine how many bacteria are in our original sample.

1. Instructor perform the 1/100 dilutions shown below. Remember 100µL = 0.1mL, so adding 100µL to a tube of 9.9mL will bring the final volume to 10mL.

2. Using a micropipette, pipette 100µL (0.1mL) of each diluted sample to the center of labeled petri dishes. Make sure to label all of the dishes so you know which dilution was places on which dish. You should have 6 dishes total.

3. Place ~10 glass beads in your petri dishes.

4. Place the lid over the petri dish and gently swirl your glass beads all over your petri dish. Ensure that liquid has been spread evenly over the entire surface of your agar.

5. Double check that the bottom of your dish has been labeled with BIO113, the section, the date and time, the dilution, and the type of agar.

6. Incubate for 24 hours and then count your during the next class period. Count dishes with between 30-300. Samples with over 300 microbes are too numerous to count. Samples with under 30 are subject to variability that will interfere with an accurate estimation of the original sample.

7. Multiply the count by the inverse of your dilution to find the concentration of bacteria in your original sample. For instance if the plate from tube 2 had 200 colonies on it. You would multiply the dilution factor which is 100 because you did a single 1/100 dilution by 200 and you would get 20,000. This is the number of bacteria in 100µl of your original culture. To find the total number of bacteria in the original culture you have to again multiply by 100 because you had 10ml of original culture or 100* 100µl.

36 Name ______Section______Date______

Post-Lab Questions:

1. How many bacteria were in our starting culture, show your work?

2. Why might the above answer not be correct, hint do all bacteria from a culture necessarily grow when put on a plate?

3. What is the purpose of performing a streak plate?

4. What is the purpose of performing the dilution plate?

5. Were you surprised at the number of bacteria on either your skin or the object that you swabbed? Why and how so?

37 Name ______Section______Date______

6. You obtain a soil sample from your garden and perform a dilution series and plate the sample at 37⁰C to see how many bacteria are in your soil. Why might this not be an accurate way of determining how many bacteria are in your soil?

7. You perform a streak plate to isolate single colonies. You won’t be able to come back to the lab for the next five days. So if you were to streak plate today, your samples would have to wait in the incubator for five days before you can remove them to isolate single colonies. Why might this be problematic?

8. You swabbed the drinking fountain and perform a steak plate on TSA. After a 24 hour incubation, you observe single colonies that look grossly different from one another. What can you immediately conclude about the purity of your original sample?

9. You count 132 colonies on a plate that was spread plated with 1mL of 1/1000 dilution of the original sample. Estimate the concentration in your original sample.

38 Supplemental Guide to Serial Dilutions and Calculating CFUs/mL

Serial Dilution can be very challenging to understand and master. We have put together a small tutorial that to help students get a better handle on these topics. If you have any questions regarding these topics please ask your professor or instructor!!!!!!!!

Serial dilutions are widely used in biology and medicine. They refer to the stepwise dilution of a substance within a solution. Often, the dilution factor for each step remains constant, resulting in a logarithmic change in solute concentration. This provides the advantage of being able to generate a standard curve, a concentration curve, or for our purposes, to reduce the microorganisms present in a culture such that they may be accurately counted.

For many biological experiments, interpretation of results is determined by counting number of viable cells or colonies on special plates. The number of cells/colonies that can grow in a given time is concentration-dependent; the more microorganisms you start with, the more that can grow during the time of your experiment. The problem is that if you plate from a saturated or highly concentrated culture, your individual colonies will merge into one big field called a bacterial lawn. While lawns have their place in biology (ex: antibiotic resistance screening), they are not helpful if you need to have a specific cell count. By serially diluting your original culture, you can obtain countable plates and determine the cell density of the original culture. Every colony on the plate is derived from a single viable bacterial cell, known as a colony- forming unit (cfu). Determining the cfus/mL will give you an accurate estimate of the concentration or cell density of your starting culture.

To determine how many colony-forming units are in an undiluted culture of bacteria, we will use the Serial Dilution method, count # of colonies per dilution plate, and apply the following formula:

cfu/mL = (# colonies) x (Total Dilution Factor) x (inverse of fraction of 1 mL plated)

To determine the cfus (# of bacteria) of your original culture, you need to account for how dilute the culture was that you plated. You must also know how much of the diluted culture you plated, in mLs.

39 A. Determining Dilution Factors

To determine the Total Dilution Factor (TDF) for a given step in the series, you must multiply the dilution factors for each proceeding step together. The TDF will tell you how dilute the culture you are plating is, relative to the original bacterial culture. In the diagram below, if you want to know the TDF of Tube #4, you must multiply the dilution factors of Tubes 1, 2, 3, and 4 together. If you want the TDF of Tube #2, then you need only multiply the dilution factors of Tube 1 and 2.

T1 T2 T3 T4

This therefore means that you must first know the Individual Tube Dilution Factor (ITDF). IDFs represent how much of the culture was diluted in each individual tube.

Individual Dilution Factor = amount transferred (amount transferred + amount already in tube)

For tubes 1-4 above, the IDFs would be as follows:

T1 ITDF = 10 µL/ (10µL + 990 µL) = 10/1000 = 1/100 = 0.01 = 10-2 T2 ITDF = 10 µL/ (10µL + 990 µL) = 10/1000 = 1/100 = 0.01 = 10-2 T3 ITDF = 10 µL/ (10µL + 990 µL) = 10/1000 = 1/100 = 0.01 = 10-2 T4 ITDF = 10 µL/ (10µL + 990 µL) = 10/1000 = 1/100 = 0.01 = 10-2

For serial dilutions, the dilution factor for each step often remains constant. Notice how the dilution factor for each individual tube above is 10-2. This means that each tube was diluted

40 exactly the same. Since we represent dilution factors as the inverse of this number, it means each of the tubes above represent a 100-fold dilution.

However, since we want to know the cfu/mL of our original culture, we need to know the Total Dilution Factor (TDF) for each tube. This represents how much the culture was diluted overall in the tube, relative to the original culture. To calculate this you multiply the ITDFs of each tube up to the step you are interested in. So, using our example above: T1 TDF = T1 ITDF = 1/100 = 10-2

T2 TDF = (T1 ITDF) x (T2 ITDF) = (1/100) x (1/100) = 1/10,000 = 10-4

T3 TDF = (T1 ITDF) x (T2 ITDF) x (T3 ITDF) = (1/100) x (1/100) x (1/100) = 1/1,000,000 = 10-6

T4 TDF = (T1 ITDF) x (T2 ITDF) x (T3 ITDF) x (T4 ITDF) = (1/100) x (1/100) x (1/100) x (1/100) = 1/100,000,000 = 10-8

Notice that as you proceed through the step-wise dilutions, the TDF does not remain constant. While the first dilution is 100-fold, the dilution for tube 4 is 100 million-fold!

B. Determining the fraction of 1mL plated

Often for these types of serial dilutions, we plate our diluted culture in µL volumes, not mL. Remember though, that we are solving for cfus/mL. That means we also need to account for the fact that we plated our diluted culture in µL, and we multiply our colony # by the inverse of the fraction of 1 mL plated. To determine our multiplier, we must first convert the µL plated into mL.

So since in our experiment we will plate 50 µL, the inverse fraction would be as follows:

41 50 µL x (1ml/1000µL) = 0.05 mL = 5/100 = 1/20 ….. the inverse of this is 20

If we had plated 25 µL, then the inverse fraction would be:

25 µL x (1ml/1000µL) = 0.025 mL = 25/1000 = 1/40 = 40

C. Practice Calculation You are given the following information. Determine the cfu/mL for the undiluted culture and tubes 1-4 given the following data.

T1 T2 T3 T4

Volume plated: 100 L 10 L 200 L 100 L

μ μ μ μ

# colonies: lawn 437 134 5

ANSWER KEY:

Tube 1- Since the plate is confluent, there are too many colonies to count. DISREGARD PLATE.

Tube 2- The TDF for this tube is: (T1 ITDF x T2 ITDF) = (1/100) x (1/100) = 1/10,000 = 10-4

The inverse fraction of 1mL is: 10 µL x (1ml/1000µL) = 0.01 mL = 1/100 = 100

cfu/mL = (437 colonies) x (10,000) x (100) = 437,000,000 = 4.37 x 108 cfus/mL

Tube 3- The TDF for this tube is: (T1 ITDF x T2 ITDF x T3 ITDF) = (1/100) x (1/100) x (1/100) = 1/1,000,000 = 10-6

The inverse fraction of 1mL is: 200 µL x (1ml/1000 µL) = 0.2 mL = 2/10 = 1/5 = 5

cfu/mL = (134 colonies) x (1,000,000) x (5) = 6.7 x 108 cfus/mL

Tube 4- Too few colonies to count. DISREGARD PLATE.

To get the most accurate cfu/mL for the original culture, you would average together the cfus/mL of the two plates we were able to count:

[(4.37 x 108 cfus/mL) + (6.7 x 108 cfus/mL)]/2 = ~5.535 x 108

Gram Stain

44 Name ______Section______Date______Pre-Lab Questions:

1. What color will Gram positive bacteria be? What color will Gram negative bacteria be?

2. What structure is more prominent in Gram positive bacteria than Gram negative bacteria?

3. Why should I not spray water aggressively or directly on my bacteria on my slide?

Background:

Bacteria are small but despite their size one of the more straight forward ways to identify and categorize them is by looking at them. Unfortunately, unless you have a very powerful/expensive microscope they are nearly impossible to see. Scientists have come up with many unique solutions to this problem including a wide variety of stains that color bacteria just like you were staining a deck or tie dyeing a tee shirt. As with any other object that we would like to stain, bacteria absorb different stains to different degrees and the. Furthermore, since bacteria vary so extraordinarily between species some species will absorb some stains while others will absorb different stains.

When a microbiologist given a sample of an unknown bacterium to identify, often times the first test that they perform is a Gram stain. The rationale being that, most bacteria are either Gram positive (G+) or Gram negative (G-), so often times the Gram stain which is a quick cheap test can eliminate many bacteria as potential candidates. In addition, the antibacterial drug susceptibility of Gram+ vs Gram- bacteria differ significantly, so often one can gain medically relevant information from this test.

Gram positive bacteria have a thick outer peptidoglycan (PG) layer while Gram negative have a thinner peptidoglycan layer that is covered by an outer phospholipid membrane. In both Gram positive and Gram negative bacteria, peptidoglycan is a primary constituent of bacterial cell walls that help bacterial cells maintain their shape and size. Peptidoglycan is a polymer (structure composed of repeating subunits) of modified sugars and amino acids called N-acetylglucosamine (NAG) and N- acetylmuramic acid (NAM) and we will learn about this more in lecture.

After a Gram staining procedure, Gram positive bacteria’s thick peptidoglycan layer will retain the primary stain called crystal violet and will consequently appear purple under the microscope. Gram negative bacteria’s thin peptidoglycan layer will be unable to retain crystal violet, but will take up the counterstain called safranin and will appear pink.

Gram Positive Bacteria Gram Negative Bacteria Peptidoglycan (PG) layer Thick Thin Number of Membranes 1; inside of PG layer 2; 1 inside and 1 outside of PG layer Stain Retained after Gram Staining Crystal violet Safranin Color after Gram Staining Purple Pink

46 Materials:

• Escherichia coli • Bacillus cereus • Mixed culture of Escherichia coli and Bacillus cereus • Crystal violet • Safranin • Ethanol • Iodine • Distilled water • Compound light microscope • Microscope slides • Bunsen burner and striker • Blot book or paper towels

****To heat fix your bacteria hold the slide by one edge with a clothespin, pass it slowly through a Bunsen burner flame. You can hold the slide with a clothes pin, but you do not want to move so slowly that the edge of the slide you're holding heats to uncomfortable levels. This heat fixation step denatures bacterial proteins causing the cells to stick to the slide while also killing the bacteria.

Note: the table above is for your reference and should not be used as a substitution for the steps listed in the procedure.

47 Procedure: Working in Groups of 4 you will gram stain all three of the cultures provided.

1. Flame your loop to sterilize it and wait for it to cool.

2. Scoop a very small amount of bacteria off the surface of your petri dish using a loop.

3. Scratch off the bacteria to your slide. Try to spread the bacteria out as thin as you can. The less clumped the bacteria are, the easier they will be to visualize under your microscope. You may want to make room so that you can place two different samples on opposite ends of your slide. (You don’t need a lot of bacteria, billions or trillions of bacteria can fit on a loop.)

4. Briefly guide the bottom (side that does not have bacteria on it) over the Bunsen burner’s open flame 3 times. You can hold your slide with a clothespin to keep your hands away from the flame. Do not keep the slide over the flame for more than half a second. This is called heat fixing and prevents the bacteria from coming off your slide during later steps.

5. Add a few drops of purple crystal violet (the primary stain) to your samples and wait 30 seconds to one minute. Take care not to get the crystal violet on your hands or clothes because it will stain. The crystal violet will initially stain all cells purple.

6. After 30 seconds to one minute, gently wash the excess crystal violet from the slide using a distilled water squirt bottle. Aim the water above the area with bacteria and point the slide down at a 45˚ angle. Take care not to aim the water stream directly at the bacteria because this will wash away your sample.

7. Add a few drops of iodine directly onto your samples and wait 10 seconds. Iodine will react with crystal violet and form a larger complex. Gram positive bacteria have thick peptidoglycan layers, and so the crystal violet-iodine complex will become entrapped. Gram negative bacteria, on the other hand, have thin peptidoglycan layers and the crystal violet-iodine complexes will be washed away in Step 9. Iodine is called a mordant because it helps affix crystal violet to peptidoglycan.

8. After 10 seconds, gently wash away the excess iodine using a distilled water squirt bottle. Again, aim the water above the area with bacteria and point the slide down at a 45˚ angle. Take care not to aim the water stream directly at the bacteria because this will wash away your sample.

9. Add ethanol (ethyl alcohol) to your samples. Ethanol acts as a decolorizing agent because it breaks apart the outer phospholipid bilayer of Gram negative cells which allows the Gram violet- iodine complex to leave. After this step, Gram positive cells should be purple (because they will retain the crystal violet-iodine complex) and Gram negative cells will be clear (because the crystal violet-iodine complex is being washed away).

10. Add pink safranin (the counterstain) to your samples and wait 30 seconds to one minute. Safranin will not have an appreciable effect on purple Gram positive cells, but safranin will stain Gram negative cells pink. Imagine dumping some pink dye on a dark purple coat, it may stain a little but it won’t be as noticeable as if it was a white coat.

11. After 30 seconds to one minute, wash away the excess safranin using a distilled water squirt bottle. Again, aim the water above the area with bacteria and point the slide down at a 45˚ angle. Take care not to aim the water stream directly at the bacteria because this will wash away your sample.

12. Blot your samples dry by placing your slide in a blotbook or laying on paper towels and gently closing it and applying gentle pressure. Avoid smearing your samples.

Above is an example of what your slides might look like.

13. View your slides under the microscope. Remember to use the 4X objective lens first.

14. Draw your what you see below. Are they Gram positive or negative?

49 Name ______Section______Date______

Sample:______Magnification:_____ Sample:______Magnification:_____

50 Name ______Section______Date______

Sample:______Magnification:_____ Sample:______Magnification:_____

51 Name ______Section______Date______

Post-Lab Questions:

1. Looking at your slides which bacteria are gram positive and which are gram negative?

2. How you do you know which are gram + and which are gram -?

3. Are your results expected given what is known about these species of bacteria? If not, explain why you may not have gotten the expected results.

4. β-lactam (beta-lactam) antibiotics inhibit the synthesis of peptidoglycan. What group of bacteria do you suspect are generally more susceptible to treatment with β-lactams? Why?

5. Lysozyme is an enzyme that hydrolyzes β-1,4-cross-linkages between N-acetylmuramic acid and N- acetylglucosamine. It is found in tears, saliva, milk and other secretions in the human body. Why

52 would the body produce this enzyme? What group of bacteria would be more susceptible to its effects?

6. Why is it necessary to use the counter stain during the gram staining procedure, what would happen if we did not add this stain at the end of the procedure?

7. You run out of ethanol in the lab. Your lab partner wants to go ahead and perform a Gram stain without it. Do you suspect that you will get accurate results? Why?

Acid Fast and Endospore Staining

Colorized scanning electron microscope (SCM) image of Mycobacterium tuberculosis.

54 Name ______Section______Date______Pre-Lab Questions:

1. What color will endospore negative bacteria be after endospore staining? What color will mycobacteria bacteria after mycolic acid staining? (see the endospore and acid fast tables)

2. What species of bacteria are we going to use to look at endospores and what species will we use for mycolic acid staining?

3. Why do we briefly heat our bacteria on the slide before staining?

55 Background:

As we discussed in the Gram stain lab a wide variety of stains exist to distinguish bacteria from one another. In this lab we will explore two other popular staining procedures the Acid Fast stain and the Endospore stain. Many of the techniques that you will be applying in this lab will be analogous (similar) to those you used in the Gram stain lab and thus this should give everyone a little extra practice manipulating slides and microscopes.

Acid Fast bacteria have a distinct cell wall that is made up of mycolic acid. Because of this they are resistant to many common antibiotics. Perhaps the most well-known of these bacteria are Mycobacterium tuberculosis, the causative agent of tuberculosis (TB) the greatest killer of man the world has ever known. It is estimated in the last 200 years, 1 billion people have been killed by TB. This is an astounding source of mortality considering the advent of antibiotics. As you can imagine, being able to visualize this bacteria or its close relatives is extremely important for health care workers.

Endospores are a structure that some species of bacteria can secrete which acts almost like armor for the bacteria. Endospores make bacteria resistant to a wide variety of stresses including wet and dry heat, UV and gamma radiation, oxidizing agents, chemicals, and extremes of both vacuum and ultrahigh hydrostatic pressure. Since many bacteria that make these spores can contaminate food being able to identify these spores so proper sterilization techniques can be applied to ensure food safety is very important.

Materials:

• Escherichia coli • Mycobacterium phlei • Bacillus subtilis (72 hours growth) • Bacillus subtilis (24 hours growth) • Carbolfuchsin • Acid-alcohol • Methylene Blue • Malachite green • Tin Can for steaming • Distilled water • Compound light microscope • Microscope slides • Bunsen burner and striker • Blot book or paper towels

56 ****To heat fix your bacteria hold the slide by one edge with a clothespin, pass it slowly through a Bunsen burner flame. You can hold the slide with a clothes pins but you do not want to move so slowly that the edge of the slide you're holding heats to uncomfortable levels. This heat fixation step denatures bacterial proteins causing the cells to stick to the slide while also killing the bacteria.

Note: The table below are here for your reference and should not be used as a substitution for steps in the procedure.

57 Procedure: Working in Groups of 4 (Acid Fast Stain)

Flame your loop to sterilize it and wait for it to cool.

1. Scoop a very small amount of E. coli off the surface of your petri dish using a loop.

2. Scratch off the bacteria to your slide. Try to spread the bacteria out as thin as you can. The less clumped the bacteria are, the easier they will be to visualize under your microscope. You may want to make room so that you can place two different samples on opposite ends of your slide. (You don’t need a lot of bacteria, billions or trillions of bacteria can fit on a loop.)

• Repeat step 1 and 2 with Mycobacterium phlei and put it on a separate slide.

3. Briefly guide the bottom of the slide (side that does not have bacteria on it) over the Bunsen burner’s open flame 3 times. You can hold your slide with a clothespin to keep your hands away from the flame. Do not keep the slide over the flame for more than half a second. This is called heat fixing and prevents the bacteria from coming off your slide during later steps.

4. Add a few drops of carbolfuchsin (the primary stain) to your samples and wait 5 minutes. The carbolfuchsin binds tightly to mycolic acid if it is present in the cell wall of the bacteria.

5. After 5 minutes, wash the excess carbolfuchsin from the slide using a distilled water squirt bottle. Aim the water above the area with bacteria and point the slide down at a 45˚ angle. Take care not to aim the water stream directly at the bacteria because this will wash away your sample.

6. Wash the smear with acid alcohol. This is the decolorizer. Continue to do this until the acid alcohol runs off the slide clear.

7. Wash the slide again with distilled water.

8. We now need to add the counterstain, in this case methylene blue. Place a few drops of methylene blue on the bacteria and let sit for 1 minute.

9. Wash the slide again with distilled water and blot dry.

10. View your slides under the microscope. Remember to use the 4X objective lens first.

11. Draw your what you see below. Which bacteria are acid fast + and which are -.

58 Name ______Section______Date______

Sample:______Magnification:_____ Sample:______Magnification:_____

59 Name ______Section______Date______

Sample:______Magnification:_____ Sample:______Magnification:_____

60 Procedure: Working in Groups of 4 (Endospore Stain)

Flame your loop to sterilize it and wait for it to cool.

1. Scoop a very small amount of Bacillus subtilis (72 hours growth) off the surface of your petri dish using a loop.

• Repeat step 1 and 2 with Bacillus subtilis (24 hours growth) and put it on a separate slide.

4. Put a small piece of paper towel over each smear, this will keep the dye from evaporating too fast during the steam step.

5. Cover the smears and paper towel with malachite green and steam the slide by placing face up on a tin can with boiling waters. As the stain evaporates add more stain so that is it constantly wet.

6. Remove the paper towel and put it in the garbage (not the sink).

7. Wash the slide with distilled water.

8. We now need to add the counterstain, in this case safranin. Place a few drops of safranin on the bacteria and let sit for 1 minute.

9. Wash the slide again with distilled water and blot dry.

10. View your slides under the microscope. Remember to use the 4X objective lens first.

11. Draw your what you see below. Which bacteria have endospores and which do not?

61 Name ______Section______Date______

Sample:______Magnification:_____ Sample:______Magnification:_____

62 Name ______Section______Date______

Sample:______Magnification:_____ Sample:______Magnification:_____

63 Name ______Section______Date______

Post-Lab Questions:

1. Looking at your slides which bacteria are acid fast + and which are acid fast -?

2. How you do you know which bacteria are acid fast + and which are acid fast -?

3. Are your results expected given what is known about mycolic acid and this species of bacteria? If not, explain why you may not have gotten the expected results.

4. Looking at your slides which bacteria are endospore + and which are endospore -?

64 5. How you do you know which bacteria are endospore + and which are endospore -?

6. Are your results expected given what is known about endospores? If not, explain why you may not have gotten the expected results.

7. β-lactam (beta-lactam) antibiotics inhibit the synthesis of peptidoglycan. In your acid fast experiment so you suspect either of the bacteria you worked with will be susceptible to these antibiotics. (Think back to the gram stain lab.)

8. Why is it necessary to counter stain the bacteria for each procedure? What would happened if we did not counterstain?

9. You are in a clinic and a patient comes in coughing up phlegm, weight loss, and night sweats—all symptoms of TB. Unfortunately you don’t have access to a TB test (we’ll learn about them more later in the semester). How can you test this patient for TB and what course of treatment should be started? Remember many antibiotics are not effective against TB so be specific.

Morphological Unknown

Each new discovery furnishes a step which leads on to the complete truth. ?Sherlock Holmes

66 Name ______Section______Date______

Pre-Lab Questions:

1. Fill out the following chart. This should help you deduce which tube is which.

Gram status Endospore Acid Fast S. epidermidis E. coli M. phlei B. subtilis

2. What is the primary and secondary stains for the Gram stain? Briefly discuss what they will stain.

3. What is the primary and secondary stains for the endospore stain? Briefly discuss what they will stain.

4. What are the primary and secondary stains for the acid fast stain? Briefly discuss what they will stain.

67 Name ______Section______Date______Background:

Alright, I admit it, we screwed up. Someone sprayed the tubes of our bacterial freezer stocks with ethanol and the names have gotten wiped off! Now we don’t know which tube has which bacteria in them. We know that the tubes in question contained either S. epidermidis, E. coli, Mycobacterium phlei, or Bacillus. Luckily for us these bacteria can be distinguished using the staining procedures you have learned so for this semester; Gram, endospore, and acid fast. Your mission is to determine which unknown is which so that we can relabel the tubes.

Materials:

Unknown Samples Crystal violet Safranin Ethanol Iodine Carbolfuchsin Acid-alcohol Methylene Blue Malachite green Tin Can for steaming Distilled water Compound light microscope Microscope slides Bunsen burner and striker Blot book or paper towels

Laboratory Exercise: In Groups of 4

Do the following procedures in whatever order you deem appropriate. You will be assigned your unknown by your lab instructor. If you figure out your unknown and you would like to work on others you are more than welcome to.

Gram Stain:

1. Flame your loop to sterilize it, and wait for it to cool.

2. Scoop a very small amount of bacteria off the surface of your petri dish using a loop.

68 3. Scratch off the bacteria to your slide. Try to spread the bacteria out as thin as you can. The less clumped the bacteria are, the easier they will be to visualize under your microscope. You may want to make room so that you can place two different samples on opposite ends of your slide. (You don’t need a lot of bacteria, billions or trillions of bacteria can fit on a loop.)

9. Add ethanol (ethyl alcohol) to your samples. Ethanol acts as a decolorizing agent because it breaks apart the outer phospholipid bilayer of Gram negative cells which allows the Gram violet-iodine complex to leave. After this step, Gram positive cells should be purple (because they will retain the crystal violet-iodine complex) and Gram negative cells will be clear (because the crystal violet-iodine complex is being washed away).

69 Name ______Section______Date______

12. Blot your samples dry by placing your slide in a blotbook or laying on paper towels and gently closing it and applying gentle pressure. Avoid smearing your samples.

13. View your slides under the microscope. Remember to use the 4X objective lens first.

14. Draw your what you see below. Are they Gram positive or negative?

Sample:______Magnification:_____ Sample:______Magnification:_____

70 Name ______Section______Date______Endospore Stain:

1. Flame your loop to sterilize it and wait for it to cool.

2. Scoop a very small amount of your sample off the surface of your petri dish using a loop.

4. Briefly guide the bottom of the slide (side that does not have bacteria on it) over the Bunsen burner’s open flame 3 times. You can hold your slide with a clothespin to keep your hands away from the flame. Do not keep the slide over the flame for more than half a second. This is called heat fixing and prevents the bacteria from coming off your slide during later steps.

5. Put a small piece of paper towel over each smear, this will keep the dye from evaporating too fast during the steam step.

6. Cover the smears and paper towel with malachite green and steam the slide by placing face up on a tin can with boiling waters. As the stain evaporates add more stain so that is it constantly wet.

7. Remove the paper towel and put it in the garbage (not the sink).

8. Wash the slide with distilled water.

9. We now need to add the counterstain, in this case safranin. Place a few drops of safranin on the bacteria and let sit for 1 minute.

10. Wash the slide again with distilled water and blot dry.

11. View your slides under the microscope. Remember to use the 4X objective lens first.

12. Draw what you see below. Which bacteria have endospores and which do not?

71 Name ______Section______Date______

Sample:______Magnification:_____ Sample:______Magnification:_____

72 Acid Fast Stain:

1. Flame your loop to sterilize it and wait for it to cool.

2. Scoop a very small amount of the sample off the surface of your petri dish using a loop.

5. Add a few drops of carbolfuchsin (the primary stain) to your samples and wait 5 minutes. The carbolfuchsin binds tightly to mycolic acid if it is present in the cell wall of the bacteria.

6. After 5 minutes, wash the excess carbolfuchsin from the slide using a distilled water squirt bottle. Aim the water above the area with bacteria and point the slide down at a 45˚ angle. Take care not to aim the water stream directly at the bacteria because this will wash away your sample.

7. Wash the smear with acid alcohol. This is the decolorizer. Continue to do this until the acid alcohol runs off the slide clear.

8. Wash the slide again with distilled water.

9. We now need to add the counterstain, in this case methylene blue. Place a few drops of methylene blue on the bacteria and let sit for 1 minute.

73 Name ______Section______Date______

10. Wash the slide again with distilled water and blot dry.

11. View your slides under the microscope. Remember to use the 4X objective lens first.

12. Draw what you see below. Which bacteria are acid fast + and which are -?

Sample:______Magnification:_____ Sample:______Magnification:_____

74 Post-Lab Questions:

1. Fill out the following chart with the results from your tests and the results from the other groups.

Gram status Endospores Acid Fast Sample 1 Sample 2 Sample 3 Sample 4

2. What is the identity of each sample?

3. How do you know the identity of each sample?

4. Would more testing be needed on these samples if we did not know there were only four possibilities? Explain.

Microbial Growth; Differential, Selective and Enriched Media

77 Name ______Section______Date______

Pre Lab Questions:

1. Briefly describe selective and differential media.

2. Staphylococcus aureus is a Gram positive cocci. Do you anticipate it growing well on MacConkey agar?

3. Bacteria grow within a variety of certain temperature ranges and grow optimally at an even narrower ranges. You can control the temperature in your incubator. Is temperature differential or selective?

78 Name ______Section______Date______

Background:

Microbiologists know that certain microbes can grow and others cannot in certain media. Furthermore, species that do grow in these distinct media may grow differently. Scientists leverage such differences to the identity microbes. These types of media are called differential and or selective media. Selective media have chemicals within them which will permit the growth of some microbes but not others. For example, agar containing the antibiotic penicillin will allow the growth of penicillin-resistant- Streptococcus pyogenes but will kill any strains of Streptococcus pyogenes which are not resistant to penicillin. Differential media distinguish between two microbes which both grow on that media but differ biochemically. For example, blood agar is used to differentiate between species which degrade red blood cells differently. There are three different groups: gamma (γ) hemolytic, beta (β) hemolytic, and alpha (α) hemolytic bacteria. γ-hemolytic bacteria do not degrade red blood cells. The media surrounding these bacteria will remain red. α-hemolytic bacteria produce hydrogen peroxide which oxidizes red hemoglobin to green methemoglobin. The media surrounding these bacteria will appear dark green. β-hemolytic bacteria lyse red blood cells and completely degrade hemoglobin. You will observe swaths of yellow surrounding β-hemolytic bacteria.

Some media can be either differential or selective depending upon the bacteria being grown. For instance, mannitol salt agar (MSA) is both a selective and differential media. MSA contains a high salt concentration which kills most microbes except for Staphylococcus species (selective). MSA also contains the carbohydrate mannitol and the pH indicator phenol red. pH indicators change color depending on the pH. Phenol red, for example, is yellow under acidic conditions but is red under alkaline (basic) conditions. Some species of Staphylococcus such as Staphylococcus aureus can ferment mannitol (use it for energy). The byproduct of fermentation is acid. So, when Staphylococcus aureus grows on MSA, it ferments mannitol, produces acid, and consequently appears yellow. Staphylococcus epidermidis, on the other hand, lacks the enzyme required to begin mannitol fermentation and so acid is not produced. So, Staphylococcus epidermidis will appear red when growing on MSA. Although both can grow on MSA, Staphylococcus aureus will appear yellow and Staphylococcus epidermidis will appear red when growing on MSA (differential). However, if you were to culture E. coli and Staphylococcus on MSA it would be selective as E. coli will not grow. Thus one media can be both selective and differential depending upon the bacteria being cultured.

MacConkey agar (MA) is also both a selective and differential media. MA contains crystal violet which inhibits Gram positive bacteria, but allows for the growth of Gram negative bacteria (selective). MA contains the pH indicator neutral red which is red under acidic conditions and yellow under alkaline conditions. MA also contains lactose. Coliform bacteria (lactose fermenting, Gram negative bacilli), such as E. coli, Citrobacter and Enterobacter, produce acid as a byproduct of lactose fermentation. Non- coliform enteric bacteria, such as Salmonella typhimurium, can grow on MA, but do not ferment lactose. As a result, coliform bacteria will change the media red and non-coliform enteric bacteria will not change the color of the media (differential). Remember, Gram positive bacteria will not grow on MA (selective).

Eosin-methylene blue agar (EMB) is also both a selective and differential media. EMB contains eosin Y and methylene blue pH indicator dyes which inhibit the growth of Gram positive bacteria, but permit the

79 growth of Gram negative bacteria (selective). Eosin Y and methylene blue pH indicator dyes are dark purple under acidic conditions and are red under neutral and alkaline conditions. Non-lactose fermenters will appear red. Lactose fermenters will appear purple. E. coli will have dark metallic-green growth.

The goal of this lab is to observe how different bacteria grow when on different types of media. We will plate four species of bacteria on differential and selective media and observe how they grow.

Materials:

7.5% sodium chloride agar

Mannitol salt agar (MSA)

Blood agar

MacConkey agar

Eosin-methylene blue agar

Staphylococcus epidermidis

Staphylococcus aureus

Escherichia coli

Salmonella typhimurium

80 Name ______Section______Date______Laboratory Exercise: In Groups of 4 Day 1:

1. Divide a petri dishes of 7.5% sodium chloride agar, Mannitol salt agar, MacConkey agar, and Eosin- methylene blue agar into quarters as shown below and label the bottom of the plate. Label the bottom of the plates.

2. Streak each quadrant with the bacteria that is labeled on the quadrant with your loop. Make sure to flame the loop after each inoculation to keep from have contamination.

3. Your lab instructor will inoculate the blood agar plate.

4. Place all plates in the 37°C incubator overnight or until next class period.

81 Name ______Section______Date______Day2:

1. Examine the plates you inoculated on Day1 draw what you see below. Make sure to label the plates below so that we know which drawing corresponds to which plate. Make sure to note how much growth you see as well as any color changes that have occurred in media surrounding the growth.

82 Name ______Section______Date______Post Lab Questions:

1. Which media were selective and for which organisms were they selective?

2. How could you tell the media were selective?

3. Which media were differential and for which organisms were they differential?

4. How could you tell the media were differential?

83 Name ______Section______Date______

5. Adding phenylethyl alcohol to agar inhibits the growth of gram negative bacteria and fungi while permitting the growth of gram positive bacteria. Is phenylethyl alcohol agar a differential or selective media?

6. A strain of Moraxella catarrhalis is able to grow on agar containing the antibiotic amoxicillin. You suspect this strain may contain the enzyme β-lactamase which inactivates amoxicillin, making it ineffective as an antibiotic. You grow the same strain in media containing amoxicillin and clavulanic acid (a β-lactamase inhibitor). What do expect the results to be; growth or no growth? Explain.

7. A patient comes into a hospital and has what appears to be an infection. Your microscope is broken so staining is not an option. Design an experiment to determine if the infection is caused by a gram positive or gram negative organism.

8. As you can see on the blood agar plates, some microbes can lyse red blood cells releasing nutrients into the surrounding area. What might the advantage be for a bacteria to be able to do this? Do you think this could have an effect on the bacteria’s virulence? Explain.

How does the atmosphere affect microbial growth?

It is better to have your head in the clouds, and know where you are... than to breathe the clearer atmosphere below them, and think that you are in paradise. Henry David Thoreau

There is good evidence that Venus once had liquid water and a much thinner atmosphere, similar to Earth billions of years ago. But today the surface of Venus is dry as a bone, hot enough to melt lead, there are clouds of sulfuric acid that reach a hundred miles high and the air is so thick it's like being 900 meters deep in the ocean. Bill Nye

85 Pre Lab Questions:

1) Can all bacteria grow without oxygen?

2) Would you expect it is more likely to find Anaerobic or Aerobic bacteria in your colon?

3) Microaerophiles would likely grow best in______?

a) Your stomach

b) Your skin

c) Your Colon

d) Not on your body

86 Caution Liquid Nitrogen and Dry Ice can burn exposed skin make sure follow all instructions when using them! Introduction:

Without food humans can live a couple of weeks, without water a couple of days, without oxygen (O2) humans die in minutes. If you slow your metabolism by falling in a frozen lake or something of that nature you might be able to survive for a bit longer, maybe even dozens of minutes but the fact remains in a relatively short period of time you will die due to lack of oxygen. We use oxygen as the terminal electron acceptor in the electron transport chain. Without a functioning electron transport chain we cannot make ATP in quantities necessary to allow our brain to function. As such, we are considered aerobic organisms, we require oxygen. However as we have learned not all bacteria require oxygen to survive and in fact oxygen is toxic to many bacteria. Thinking about oxygen toxicity is counterintuitive to us, but oxygen is very reactive and can thus can cause large amounts of damage to cells under the wrong circumstances.

One of the important take home messages from biology 113 is that bacteria can survive anywhere on earth from the bottom of deep sea vents to inside or on our bodies. This variety of habitats means that different species of bacteria have evolved different mechanisms to leverage their environments. As oxygen levels vary in these different environments it is unsurprising that different species of bacteria have distinct requirements for oxygen. ~Gas content of earth’s atmosphere:

78.09% nitrogen, 20.95% oxygen, 0.93% argon, 0.04% carbon dioxide ~Gas in your stomach

15% oxygen, 5% CO2, 80% Nitrogen ~Gas in your large intestine

Depends upon you diet and bacteria that are present but typically

Little oxygen, More CO2, Hydrogen gas, Sulfur Compounds, as well as others

How we classify bacteria according to oxygen requirements?

Obligate aerobic bacteria are like humans in that they cannot grow unless oxygen is present. They use oxygen as their terminal electron acceptor.

Obligate anaerobes are the opposite, if oxygen is present they die they require an environment that lacks oxygen. These microbes (e.g. Clostridium) lack catalase, an enzyme critical to the breakdown of

H2O2 (hydrogen peroxide). H2O2 is toxic to cells. Other genera of bacteria such as Streptococcus,

87 Enterococcus, and Lactobacillus lack catalase but have developed other ways to limit H2O2 toxicity by limiting the production of H2O2.

Facultative anaerobes (majority of bacteria) can use oxygen or they can use other molecules as terminal electron acceptors. An example of a facultative is E. coli, we grow E. coli in the lab where oxygen is present is relatively high levels but E. coli is also found in our guts where oxygen is not present in high quantities.

Aerotolerant organisms cannot utilize oxygen but at the same time are not damaged by oxygen to the point that it inhibits growth.

Microaerophiles grow best in an atmosphere with higher levels of CO2 (typically 5-10%) and lower levels of oxygen.

Studying anaerobic organisms in the lab requires that we remove most of the oxygen form the environment where we grow the bacteria. This can be done in a number of different ways depending upon how much oxygen you would like to remove and how much bacteria you need to grow. In this lab we are going to grow organisms without oxygen.

1) Anaerobic incubators -- In a perfect world where money and space was not a limitation we could use Anaerobic incubators to do our experiments. These are incubators that are sealed and have the atmosphere pumped out

88 of them and simultaneously flooded with a mixture of Nitrogen and CO2. These are expensive large pieces of equipment that are impractical for an undergraduate lab class. Shown below.

http://www.pro-lab-direct.com/product-p/plt1147.htm

We are going to make a homemade anaerobic incubator in class.

2) Growth on reducing media – This media contains chemicals that combine with oxygen and remove some of the oxygen from the environment. These are very convenient and can be bought or made.

One classic example of this is sodium thioglycolate media (HSCH2COONa). Usually a dye is used show

where oxygen is present in the media.

3) Brewer anaerobic jar – You can place growth media, usually in a petri dish in a jar and then place a gas pack and catalyst that will remove the oxygen from the environment and replace it with CO2. To look at your plates you must open the jar at this point your experiment has been exposed to oxygen so you cannot take multiple data points. Shown below.

The point of all of these techniques is to limit/control the amount of oxygen that is present during the experiment. We are going to use two techniques to limit the amount of O2 present. 1) Grow cells on thioglycolate media. 2) Make homemade anaerobic chambers using liquid Nitrogen or Dry Ice or a vacuum sealer. Materials:

2 Petri Dishes containing nutrient agar or LB agar per group of 4 students

89 2 Petri Dish containing YPD per group of 4 students

Tubes containing thioglycolate media 2 tubes per section

Food saver and bags

Ziplock Bags

Liquid Nitrogen

Dry Ice (Solid CO2)

Inoculating loop Microbes In Petri Dishes: Fungi C. albicans

S. cerevisiae

S. pombe

C. glabrata

Bacteria Alcaligenes faecalis

Clostridium sporogenes

Enterococcus faecalis

Escherichia coli Procedure: Day1 Instructor

1) Instructor -- label 2 Thioglycolate tubes with a bacterial species we have streaked on a plate. Inoculate each tube with a loop of bacteria.

2) Incubate these tubes upside down on a counter designated by your instructor at room temperature until the next class period. Between all of the sections we should be able to have all four of the bacteria grown in this broth. Students

90 1) With a sharpie divide each of your four petri dishes into four sections.

2) Label each section of the YPD plates with the yeast you will streak into that section. Label all plates with your group and section number. Label one plate as aerobic and one as anaerobic. As shown above.

3) Label each section of the LB agar plates with the bacteria you will streak into that section. Label one plate as aerobic and one as anaerobic. Label all plates with your group and section number. Always label the bottom of the plates not the lids. This is not shown but is the similar to the figure above.

4) From the stock plate take your sterile loop and streak a single line of bacteria or yeast onto the labeled sections of the agar. If you want to be creative you can make something other than an straight line, this is microbiology it is supposed to be . After each streak make sure to sterilize your loop in the flame.

5) Place the 2 aerobic plates upside down on a counter designated by your instructor at room temperature overnight.

6) Groups 1 and 4 vacuum seal your anaerobic plates using the vacuum sealer.

7) Groups 2 and 5 place your anaerobic plates in a 1 gallon zip lock bag and place one or two small pieces of dry ice in the bag and seal the bag. As the bag fills with gas let the gas out of the bag until the dry ice has dissolved and then seal the inflated bag.

8) Groups 3 and 6 place your anaerobic plates in a 1 gallon zip lock bag and pipette 2-3 mL’s of Liquid Nitrogen into the bag and seal the bag. As the bag fills with gas let the gas out of the bag until the Liquid Nitrogen has dissolved and then seal the inflated bag. (If you want help with the liquid nitrogen ask your instructor.)

9) Place the anaerobic plates upside down on a counter designated by your instructor at room temperature overnight.

Day2

1) Draw what you see in the thioglycolate cultures including where the indicators turned blue or pink.

1) Retrieve your plates and record the results for your experiments as well as the other two anaerobic plates your group did not perform in the chart below. (Instructors can make a chart on the board where everyone can put their data to make sure everyone gets all data)

Aerobic Vacuum Liquid Nitrogen Dry Ice Catalase

Candida albicans Candida glabrata Saccharomyces cerevisiae Schizosaccharomyces pombe Alcaligenes faecalis Clostridium sporogenes Enterococcus faecalis Escherichia coli

92 2) Perform the catalase test on the cultures that grew by taking a small amount of culture and transferring it to a microscope slide. Then drop a couple of drops of 3% H2O2 on the slide. If the microbes contain catalase you will see bubbles.

93 Name ______Section______Date______Post-Lab Questions: (Use your data from class to answer 1-4)

1) Which cultures grew best in aerobic conditions? Is this what you expected why or why not?

2) Which cultures grew best in nitrogen? Is this what you expected why or why not?

3) Which cultures grew best in CO2? Is this what you expected why or why not?

4) Which cultures grew best in the vacuum? Is this what you expected why or why not?

5) Which cultures were catalase positive and which were negative? Is this what you expected why or why not?

94 6) Did any cultures grow more slowly without oxygen? How could you tell? What does this tell about their metabolism?

7) Based on our results is it possible classify each of the organisms tested as either an obligate aerobic, obligate anaerobe, aerotolerant, microaerophile, or facultative anaerobe. If you cannot definitely say what they are provide the list of possible classifications each organism could have.

8) You are a space explorer and discover an organism on another planet where the atmosphere does not contain any oxygen. Describe an experiment you could do to distinguish it between an obligate aerobic, obligate anaerobe, aerotolerant, microaerophile, or facultative anaerobe.

Carbohydrate Metabolism

96 Name ______Section______Date______Pre-Lab Questions:

1. What does amylase degrade? How will we know if amylase is present?

2. What type of catabolism discussed in this lab does not require the presence of molecular oxygen (O2)?

3. How many OF-glucose tubes will be inoculating with for each bacterial strain?

97 Name ______Section______Date______Background:

All organisms need energy to survive and replicate. This energy can be obtained in one of two ways, they can either gather it from sunlight through photosynthesis or they can get it by breaking chemical bonds and harnessing the energy in those bonds. In this lab we will examine organisms that break the chemical bonds of carbohydrates and harness this energy. I am sure many of you have heard of carbohydrates (carbs). Just like bacteria we eat carbohydrates to get energy. When we eat a candy bar, drink a soda, or eat a piece of bread we are taking in carbohydrates. We have a much more limited diet than bacteria. For instance, if you were to try to eat grass or the bark of a tree you would not be able to gather any nutrients from it even though these are carbohydrates, but some bacteria can digest these types of carbohydrates.

To harness the energy of carbohydrates, bacteria utilize two general strategies: oxidative catabolism and fermentative catabolism. Oxidative catabolism, as the name implies, requires oxygen (O2). Fermentative catabolism does not require oxygen, but can still occur even if oxygen is present. In this lab we will test if bacteria are using oxidative catabolism or fermentative catabolism by growing them on oxidative- fermentative (OF) medium.

OF medium contains a high concentration of glucose (or another carbohydrate) and low concentration of peptone (partially digested proteins). For one bacterial sample, we will use two tubes. One tube will be open to the air (this will permit oxidative catabolism). We will put mineral oil on the top of the agar in the other tube to prevent air from permeating the agar creating a mostly anaerobic environment (if growth occurs in this tube, then the bacterium is capable of fermentative catabolism). The agar contains the pH indicator bromthymol blue is yellow in an acidic environment but is green in neutral conditions and blue in alkaline environments. Remember acid is a byproduct of fermentation, so if fermentation occurs, we will turn the media yellow. If protein metabolism predominates, alkaline ammonia will be produced which will turn the media blue.

Let us go through some possibilities. Tubes 1 and 2 (below) were inoculated with the same bacterium. Tube 1 does not have mineral oil at the top allowing for an oxidative environment. Tube 2 does have mineral oil at the top creating an anaerobic environment. As you can see, the top of column 1 is yellow, but the bottom is green and all of the agar in tube 2 is green. Since only the top of the agar is yellow (where most of the oxygen is), we can conclude that the bacterium is utilizing oxidative catabolism for glucose. Since the bottom of 1 and all of 2 are green, we know that metabolism has not occurred and therefore the bacterium is not capable of fermentative catabolism in these anaerobic environments.

Tubes 3 and 4 (above) were inoculated with the same bacterium. Tube 3 does not have mineral oil at the top allowing for an oxidative environment. Tube 4 does have mineral oil at the top creating an anaerobic environment. As you can see, both tubes are completely yellow. Because they are yellow, we know it is an acidic environment due to the production of acid from fermentation. We can therefore conclude that this bacterium is capable of oxidative fermentation.

Tubes 5 and 6 (above) were inoculated with the same bacterium. Tube 5 does not have mineral oil at the top allowing for an oxidative environment. Tube 6 does have mineral oil at the top creating an anaerobic environment. As you can see tube 5 is blue at the top but green at the bottom. This indicates that this bacterium is capable of oxidative catabolism of proteins producing alkaline ammonia turning the agar blue. This bacterium is not capable of fermentation because we don’t observe any metabolic activity in anaerobic environments. (The flow chart on the following page will provide more clarification on how to interpret this type of data.)

The second assay that we will perform will be took look at starch metabolism. Starch is a huge carbohydrate composed of ~10,000 carbohydrate molecules. It is the sugar that plants use to store energy. Not surprisingly it is found in abundance in food staples such as corn, wheat, rice, and potatoes. We will test which bacteria are able to metabolize starch by growing bacteria on plates that only have starch as a sugar source.

To determine if starch hydrolysis is occurring outside of the bacterial cells, only within the cells, or not at all; we will use the starch hydrolysis test. To do this, we will use agar containing starch which have a bacterium streaked onto them. We will then add enough Gram’s iodine to cover the bottom of the plate. Iodine binds to starch creating a blue/black compound. Areas where starch has been hydrolyzed will appear tan. If there are tan areas only where bacteria are, then starch is hydrolyzed only within cells. If there are tan areas that extend beyond the perimeters of bacterial growth, then we can conclude that the bacteria are secreting amylase which degrades the starch outside of the cells.

99 100 Name ______Section______Date______Materials:

Starch agar

Oxidative/fermentation glucose medium

Inoculating needles

Gram’s iodine

Bacillus subtillis

Escherichia coli

Pseudomonas aeruginosa Laboratory Exercise: In Groups of 4

Oxidative-Fermentative Glucose Medium: Day1

1. Collect bacteria on an inoculating needle from the stock plate.

2. Stab the needle into a tube. Repeat with the second tube. Be sure to label your tubes

3. Add mineral oil to the top of one of your tubes.

4. Repeat steps 1-3 for all of the species of bacteria. Be sure to flame your inoculating needles in between samples to prevent contamination.

5. Incubate your samples for 24 hours.

Day2

Look at your tubes and complete the post lab questions using your results.

Starch Hydrolysis Test: Day1

1. Divide your plate into three sections and label each section with the bacteria that will be streaked in that section.

101 2. Using your sterile inoculating loop streak Bacillus subtillis, Escherichia coli, and Pseudomonas aeruginosa onto the plate.

Day2

1. Flood your plates with Gram’s iodine.

2. Observe and record borders of bacterial growth and discoloration due to Gram’s iodine.

102 Name ______Section______Date______Post-Lab Questions:

1. Which bacteria grew on the starch plate?

2. Which bacteria were able to metabolize starch?

3. Which bacteria were able to grow on the OF-glucose media?

4. What if any color changes did you observe in the OF-glucose media? Were these color changes dependent upon the presence of oxygen?

5. What do these changes in color tell you about how the bacteria are metabolizing the glucose?

6. You spit on a starch plate and then incubate it for several hours. You then add Gram’s iodine. What do you predict will be the result?

103 Name ______Section______Date______7. You are unsure if the bacterium you are studying hydrolyzes starch in an anaerobic environment. Design an experiment to determine this.

8. You’ve read that the particular bacterium you’re studying is capable of fermenting using proteins as a carbon source. What do you anticipate your tubes will look like?

9. You’ve read that the particular bacterium you’re studying is capable of both fermenting glucose and oxidizing glucose. What do you anticipate your tubes will look like?

10. You’ve read that the particular bacterium you’re studying is capable of fermenting glucose only in anaerobic environments because it dies in the presence of high concentrations of oxygen. What do you anticipate your tubes will look like?

104 Bacterial Transformation

Because all of biology is connected, one can often make a breakthrough with an organism that exaggerates a particular phenomenon, and later explore the generality. Thomas R. Cech

105 Name ______Section______Date______Prelab Questions:

1) In this lab we will transform what species/strain of bacteria?

2) What phenotype will the bacteria gain by the transformation?

3) How will we know if our transformation worked?

106 Name ______Section______Date______Introduction:

The quote by Nobel Laureate Thomas Cech on the cover page of this lab underscores the importance of the material we will discuss this week. Transformation was discovered in bacteria by Griffith in 1928 and this has been directly responsible for some of the greatest advancements in medicine and science. As significant as this sounds this actually downplays the importance of transformation in bacteria. The realization that we could change the genetic makeup of an organism and in turn its phenotype has opened the door to genetic engineering in dozens of other species including mammalian cells. It is not an understatement to say that Griffith’s experiment will influence science for hundreds of years.

Bacterial transformation occurs when a bacteria takes up a small piece of DNA. This DNA generally comes from another bacteria that has died. The new piece of DNA can give the bacteria new phenotypic characteristics (it can change how well it will grow.) In this lab we will be transforming Escherichia coli (E. coli) bacteria with a gene that allows it to grow in the presence of the antibiotic Ampicillin. We are in essence make antibiotic resistant bacteria. We are using a special strain of E. coli called DH5 alpha. This strain has been specially engineered to be good for lab experiments. This also makes it unlikely to be unable to survive in the human gut. Thus it is safe to work with and the antibiotic resistance that we give it in our experiment is not dangerous.

This strain of E. coli bacteria is not naturally competent at a frequency we could easily observe in the lab. To make them competent we have grown them in media that will make them more receptive to foreign DNA. This media alters the charge of the outside of the cells and makes the cells slightly permeable. Exposure to heat makes the cells even more permeable. This will give us the best chance to successfully transform our bacteria with plasmid DNA. As you may remember from lecture, plasmids are small circular pieces of DNA that are easily exchanged between bacteria. We are going to feed our competent E. coli plasmid in hopes that the bacteria takes in the plasmid DNA. On the plasmid is a gene that makes the bacteria resistant to Ampicillin a common antibiotic used in the lab. If our experiment is successful our transformed bacteria will be able to grow in the presence of the antibiotic Ampicillin.

Materials:

1 tube competent cells (CC)

1 tube plasmid (P)

Liquid LB media

Glass Beads

4 petri dishes two with Amp Agar (green stripe) and two with plain agar (red stripe)

107 Name ______Section______Date______Procedure Day 1: Working in Groups of 4

1) Mix 50ul competent cells and 1ul plasmid in a labeled 1.5 ml Eppendorf tube (P).

Leave the other 50ul of competent cells 1.5 Eppendorf tube (CC)

2) Close both tubes and set both tubes on ice for 10 minutes.

3) Heat shock tubes of cells by putting them in the 42 C water bath for 45 seconds.

4) Remove the tube from the bath and place on ice for 3 minutes.

5) Add 200ul LB liquid media

6) Place at 37 degrees C for 30 minutes

7) Pipette 125ul of plasmid mixture onto the Amp agar plate (label plate)

8) Pipette 125ul of plasmid mixture onto the agar plate (label plate)

9) Pipette 125ul of cells that did not get plasmid onto the Amp agar plate (label plate)

10) Pipette 125ul of cells that did not get plasmid onto the agar plate (label plate)

11) Pour 10-20 glass beads onto each plate and put the lib back on each plate. Shake the plates for 20 seconds. Pour the glass beads into the dirty bead container.

11) Place all four plates upside down in 37 C incubator.

108 Name ______Section______Date______Procedure Day 2:

12) Remove plates from incubator and record findings.

Plate Name______Plate Name______

Number of colonies______Number of colonies______

Plate Name______Plate Name______

Number of colonies______Number of colonies______

109 Name ______Section______Date______Questions: 1) Which plates did you expect to have the most colonies? Why?

2) Which plates did you expect to have the least colonies? Why?

3) Did your results match what you expected?

4) If your results did not match your expectations why do you think this is?

5) If you plated your transformation on plates that contained a different type of antibiotic would you expect the bacteria to grow?

110 Name ______Section______Date______

6) If you took your bacterial cells that have been transformed and let them replicate without the presence of antibiotics and then plated the cells on antibiotic media do you expect all of the bacteria could still grow in the presence of antibiotic? Why?

111

Ames Test and UV radiation induced mutation

Almost all the world is natural chemicals, so it really makes you re-think everything. A cup of coffee is filled with chemicals. They've identified a thousand chemicals in a cup of coffee. But we only found 22 that have been tested in animal cancer tests out of this thousand. And of those, 17 are carcinogens. There are ten milligrams of known carcinogens in a cup of coffee and that’s more carcinogens than you’re likely to get from pesticide residues for a year! Bruce Ames

In microbiology the roles of mutation and selection in evolution are coming to be better understood through the use of bacterial cultures of mutant strains. Edward Laurie Tatum

This second quote may seem obvious but at the time it was made we were just beginning to understand what a gene was and how it caused a phenotype. The first quote is just there to scare coffee drinkers.

112 Name ______Section______Date______Prelab Questions: 1) What does the Ames test test for?

2) Usually the Ames test uses ______but for our test we will use ______.

3) What mutation will allow our microbe to grow on the plates lacking Tryptophan?

113 Introduction: Mutation is interesting. This may seem like an odd statement to start a lab but let’s face it Hollywood has made billions of dollars from movies that use mutation as a premise. Countless superheroes and super villains supposedly have become the way they are due to mutation. If it was not interesting people would not want to see these stories. These stories are obviously fantastical, but mutation and natural selection are responsible for all of the diversity that we see on our planet. Even though we can’t see it, all around us mutation is occurring on a daily basis. Most mutations have very little effect on organisms, but some mutations can have dramatic and deadly effects. Mutations in microbes can lead them to become dangerous pathogens or resistant to antimicrobial drugs. Mutations in our genomes can lead to 100’s of different diseases including cancer. In this lab we are going to intentionally mutate the genomes of microbes using chemicals and UV radiation to observe how mutations effect phenotype.

The Ames test as described or as will be described in lecture is a way to determine if a chemical compound is mutagenic. This has numerous real world applications including determining if a compound is more likely to be a carcinogen. Although historically this test has been performed with bacteria we are going to use the yeast S. cerevisiae. S. cerevisiae is used to leaven bread, brew beer, and ferment wine. S. cerevisiae is also a very useful as a research tool as it is very amenable to genetic analysis. We are going to use a strain of S. cerevisiae that cannot grow without the amino acid Tryptophan. The reason our strain cannot grow without Tryptophan is that a mutation has been made in a gene important for synthesizing Tryptophan. As such, if we give the yeast Tryptophan it grows very well. However if we do not give the yeast Tryptophan they will not grow because they cannot make Tryptophan and in turn cannot make proteins. However if we make another mutation that reverses the first mutation the cells can then make Tryptophan and can grow on media without Tryptophan! We will test if chemicals (Ames Test) or UV radiation will make mutations that allow our strain to grow on media that does not contain Tryptophan. Thus we are testing to see if we can reverse the first mutation.

Materials per group of 4 4 paper disks

2 petri dishes

3 chemicals to test

EthBr

S. cerevisiae culture

114 Name ______Section______Date______Day1 Ames Test:

1) Pipette 200ul of S. cerevisiae culture onto a SD-trp plate.

2) Pour 10-20 glass beads onto each plate and put the lid back on each plate. Shake the plates for 30 seconds. Pour the glass beads into the dirty bead container. Let the plates dry.

3) With tweezers place four circular disks spaced evenly on the plate.

4) Pipette 15ul of test chemicals you brought from home or provided by the lab onto a disk.

5) Place the plate in the 30 C incubator overnight.

UV Radiation Mutation:

1) Pipette 200ul of S. cerevisiae culture on 2 SD-trp plates. Label one plate UV and the other control.

2) Pour 10-20 glass beads onto each plate and put the lib back on each plate. Shake the plates for 30 seconds. Pour the glass beads into the dirty bead container. Let the plates dry.

3) Each group will radiate their plate for a different length of time. 5 seconds, 30 seconds, 60 seconds, 2 minutes. Power level 1200. Each lab section will leave one of their plates not irradiated.

4) Put all plates in the 30 C incubator for 2 -5 days.

115 Name ______Section______Date______Day2 Ames Test

1) Remove plate from incubator and record results.

Draw what you see on the plate

Number of colonies around each disk______

Ames Test Questions 1. Were their colonies around any of the test substances?

2. If there were colonies so what does this mean?

3. If there were no colonies why do you think that this is?

116 Name ______Section______Date______

4. If the substance that you placed on paper was both mutagenic and toxic to yeast what result would you expect?

5. Could a substance that was perfectly safe for yeast and bacteria (never caused mutations) still be mutagenic to humans? Explain your answer.

117 Name ______Section______Date______Day 2 UV Radiation Mutation: 1) Count the number of colonies on the irradiated plates vs non-irradiated plate and record these numbers below.

______Number of colonies non-irradiated

______Number of colonies 5 seconds irradiation

______Number of colonies 10 seconds irradiation

______Number of colonies 20 seconds irradiation

______Number of colonies 30 seconds irradiation Questions: 1. Did irradiation mutate the yeast so that they were able to grow?

2. If you did not see growth does this mean that the yeast were not mutated by the radiation? Why or why not?

3. Let us say you wanted to fix a mutation in the Tryptophan gene. What are some of the drawbacks to fixing the Tryptophan gene in the way that we did it in our exercise?

118 Name ______Section______Date______

4. If a human had a defect in a gene, could we fix it by irradiating the cells with the defective gene in the person, why or why not?

119

Control of Microbial Growth; Physical Methods

120 Name ______Section______Date______Pre-Lab Questions:

1. Predict results for this experiment for each time and temperature. High, medium, minimal, no growth?

63⁰C Prediction Time Point Amount of Growth 0 seconds 30 seconds 2 minutes 5 minutes 15 minutes

100⁰C Prediction Time Point Amount of Growth 0 seconds 30 seconds 2 minutes 5 minutes 15 minutes

2. How do you predict the above results might be different from a spore forming bacterium?

3. Which would transfer heat more quickly, boiling or baking in an oven at 100°C for the same length of time?

121 Name ______Section______Date______Background:

Humans have been using heat to kill microbes for thousands of years. Of course in the beginning they did not realized this was why they cooking food or sealing wounds (cauterization) but they realized food stayed longer and wounds were less likely to get infected when they treated them with heat. Although there are many bacteria which thrive under what we consider “extreme conditions” (they are called extremophiles), unsurprisingly bacteria that infect humans typically like living at or close to the temperature of the human body. Perhaps it is also not surprising that many of these bacteria cannot survive at high temperatures and experience inhibited growth at low temperatures. There are additional ways that bacterial growth can be inhibited such as gamma-rays which are high energy photons. In practice, gamma-rays can be used to sterilize foods and medical equipment. In lecture we will talk about how limiting water through desiccation or salting prevents bacterial growth. In this lab we will only be using heat to inhibit bacterial growth.

There are two kinds of heat: dry and moist. Dry heat doesn’t transfer thermal energy quite as efficiently as moist heat (think of putting your hand in boiling water as opposed to an oven turned to 212⁰F). Methods that use moist heat to control microbial growth include pasteurization, boiling, and autoclaving. One of the inherent limits of moist heat however is that liquid water can only get to 212⁰F degrees when boiling and many microbes can withstand that amount of heat for a short time. So how can we increase the heat of water beyond boiling? Autoclaving sterilizes (kills all bacteria and inactivates bacterial spores) surgical equipment and lab ware. Autoclaving uses higher pressure (~20- 30psi, while atmospheric pressure is only ~15psi) to increase the maximum temperature of water (~250⁰F, 121⁰C) for 15 minutes. In other words, the high pressure increases the boiling point of water which allows the steam to carry more thermal energy and transfer this energy to microbes leading to death.

In this lab, we will be determining the effects that moist heat has on the survival of E. coli. We will not be autoclaving anything because that would just kill all of the microbes. Instead, we will compare the number of colonies that grow from a sample exposed to moist heat at varying lengths of time using two different temperatures and microbes that remain at room temperature. Materials:

Sharpie

3 TSA plates E. coli

Inoculating loop

Test tube

Beaker

Hot plate

122 Name ______Section______Date______Laboratory Exercise: Working in Groups of 4

1. A water bath should be heated to 63°C. In a metal can or glass beaker boil water.

2. Using a Sharpie, divide the bottom of a TSA plate into five equal sections and label them “0 seconds”, “30 seconds”, “2 minutes”, “5 minutes”, “15 minutes”.

3. Flick the bottom of your test tube with liquid LB media and E. coli to suspend the bacteria.

4. Stick your inoculating loop in the LB media and streak it onto the “0 seconds” section of your TSA plate. Try to be consistent with subsequent streaking of the other sections. (Be sure to flame your loop in between inoculations to prevent misleading results.)

5. Place your test tube in either water bath for 30 seconds. Remove it and then flick the bottom to suspend the bacteria. Stick your inoculating loop in and streak it onto the “30 seconds” section. (You can split the work and have one person from the group perform one temp and another person perform a different temp.) (Be careful with the boiling water as it is hot)

6. Repeat this for 2 minutes, 5 minutes, and 15 minutes.

7. Be sure that all of your plates are labeled with your BIO 113, your section, your group number, the date and time.

8. Incubate your plate until the next lab period in the 37 incubator and record the amount of growth in each section.

25⁰C ~ room temp Time Point Amount of Growth 0 seconds 30 seconds 2 minutes 5 minutes 15 minutes

123 Name ______Section______Date______

63⁰C Time Point Amount of Growth 0 seconds 30 seconds 2 minutes 5 minutes 15 minutes

100⁰C Time Point Amount of Growth 0 seconds 30 seconds 2 minutes 5 minutes 15 minutes

124 Name ______Section______Date______

Post-Lab Questions:

1. Which method resulted in the fewest living bacteria? Did your results match up with your predictions from the prelab?

2. Using a similar procedure, how could you kill more bacteria in a shorter period of time?

3. Would you predict the same results if we used the same temperatures but used a dry heating method, an over for instance?

4. Would you predict different results if we used a bacterium which could form spores? Explain.

125

Chemical Methods of Control; Disinfectants and Antiseptics

126 Name ______Section______Date______Pre-Lab Questions:

1. Am I allowed to bring in and test my own antimicrobial for this lab?

2. How will we suspend our bacteria in this lab?

3. What should I remember to do in between samples to prevent contamination that might lead to misleading results?

127 Name ______Section______Date______Background:

We have all used chemical methods to control microbial. Whether it is the use of hand sanitizer, detergent, soap, or bleach—at some point we have needed to kill microbes. This is extremely important especially in hospitals where human pathogens can spread among a community very quickly.

In this lab, we will be testing the effectiveness of various antimicrobial agents on E. coli. For this lab, you are welcome and in fact encouraged to bring your own antimicrobial agent (e.g. hand sanitizer, bleach, kitchen spray, contact lens fluid) to test. Find out if your mouthwash actually kills bacteria, is pine sol better than pine glow, etc. Although these antimicrobial agents have a broad spectrum of activity (they kill or inactivate many different kinds of microbes), we will only be testing their effectiveness against E. coli.

Read through the chart below for a disambiguation of relevant related terms.

http://cnx.org/contents/5XZItubD@3/Controlling-Microbial-Growth

128 Materials:

Sharpie

TSA plate

Test tubes

E. coli

Antimicrobial agents

Inoculating loop

Procedures: In groups of 4

1. Using a Sharpie, divide the bottom of your TSA into five equal sections. Label these sections “0 seconds”, “30 seconds”, “2 minutes”, “5 minutes”, and “15 minutes”.

2. Flick to suspend your E. coli in your test tube.

3. Use an inoculating loop to get a sample of E. coli and streak it onto the “0 seconds” section of your TSA plate. Be sure to flame your loop in between sample to prevent misleading results.

4. Add 2.5mL of your antimicrobial agent into a clean test tube.

5. Add 2.5mL of your E. coli sample into this test tube. Flick the test tube to mix.

6. 30 seconds after mixing your samples, use your inoculating loop to get a sample and then streak onto the corresponding section of your TSA plate.

7. Repeat this for each time point. Be sure to flame your loop in between sample to prevent misleading results.

8. Be sure that your plate is labeled with your BIO 113, your section, your group number, the date and time.

9. Incubate your plate for 24 hours and then record the amount of growth in each section.

129 Name ______Section______Date______

Agent Used: Time Point Amount of Growth 0 seconds 30 seconds 2 minutes 5 minutes 15 minutes

Post-Lab Questions:

1. There are stronger antimicrobials available than the ones we use on a day-to-day basis. Why don’t we use the stronger antimicrobials?

2. How do you predict this results might be different from a spore forming bacterium?

3. Resistance is a problem not only antibiotics but also for disinfectants and antiseptics. Design an experiment to demonstrate that bacteria can develop resistance to a particular antimicrobial agent.

130 Name ______Section______Date______

4. Why can’t sterility be guaranteed if a paper package of sterile surgical gloves gets wet?

5. Although the number of living microbes can be reduced dramatically to almost non-detectable levels, why can’t you sterilize human skin prior to surgery?

131

Antimicrobial Drugs

When antibiotics first came out, nobody could have imagined we'd have the resistance problem we face today. We didn't give bacteria credit for being able to change and adapt so fast.

Bonnie Bassler

132 Name ______Section______Date______Prelab Questions:

1 2

Paper disk 1, 2, and 3 are each saturated with a different antibiotic.

1) In the above figure how many species of microbes do you see on the plate?

2) Which antibiotic had the broadest effect (effective against the most bacteria on the plate?

3) Did antibiotic 1 kill any microbes, can you be sure?

133 Background Microbes have been waging microscopic war with each other for hundreds of millions of years. One of the major ways that microbes fight/compete in these battles is through the secretion of small molecules into the surrounding environment making it more challenging for other microbes to grow. Some microbes secrete compounds that change the pH of the surrounding environment thus inhibiting the growth of organisms that are unable to survive the altered pH. Others such as S. cerevisiae secrete small molecules such as ethanol which broadly inhibit the growth of other organisms. Still others manufacture and secrete compounds that are explicitly active against specific biological components of other microbes. Any substance that is secreted by a microbe and inhibits another microbe is considered an antibiotic. For the purposes of this lab we are going to be investigating antimicrobial drugs, compounds that can be taken by a patient to cure or stem unwanted microbial growth in the body.

For instance beta lactam antibiotics such as penicillin inhibit the synthesis of peptidoglycan, a key component of many bacterial cell walls. Beta lactam antibiotics are secreted by fungi whose cell walls do not contain peptidoglycan and are thus not affected by this class of compounds. Another example of an antimicrobial drug is streptomycin. This class of antibiotics is actually secreted by Streptomyces bacteria and targets protein synthesis in bacteria. Antibiotic drug resistance has been around long before humans and was reported in some of the very first papers characterizing antibiotic activity. Antibiotic resistance is prevalent in nature and is becoming more prevalent as the world uses more and more antibiotics. In this lab we will perform experiments to examine if bacteria that live on our body or in our environment are susceptible to antibiotics.

Disk-Diffusion Method The first procedure that we will perform is a disk diffusion experiment. We will inoculate a Petri dish with a lawn of bacteria. A lawn of bacteria means that as the bacteria grow they will cover the entire Petri dish. After the initial spread of the bacteria you will not be able to see the bacteria but over time as the bacteria grow the lawn will become visible. We will place paper disks on the plate that contain different classes of antibiotics. The antibiotics will slowly diffuse away from the disks and enter the media. As the antibiotic gets farther away from the disk it will become more and more dilute. Close to the disk the antibiotic will be at relatively high concentration and may inhibit bacterial growth. As we move farther away the decreased amount of antibiotic will become less likely to inhibit growth until finally a concentration is reached that is no longer inhibitory to the bacteria. From this experiment we will identify relative susceptibility of the bacterial lawn to antibiotics. A larger diameter of inhibition means the bacteria are more susceptible to the antibiotic. Similar tests are used in companies to determine antibiotic susceptibility and potency. The tests we perform in lab are not going to be as well controlled as they would be at a company. In industry, the diffusion rate of the compounds and plate depth are precisely controlled, we will not control for these aspects.

134 Name ______Section______Date______Minimum Inhibitory Concentration The second procedure we will perform in this lab will test the minimum inhibitory concentration of an antibiotic (MIC). We will do this by growing bacteria in media with different concentrations of antibiotic. The MIC is the lowest dose of antibiotic that still inhibits bacterial growth.

In lecture you have or will learn about the different mechanisms by which antibiotic resistance can occur. For this lab you will test how susceptible the microbes of your skin or from a lab surface are to antibiotics. The outcome of these assays are not a diagnosis or cause for alarm. As stated above we all has microbes on our skin that are resistant to antibiotics to some degree.

Materials

Each Group of 4

1 TSA or LB plate per group of 2 students

1 test tube 2 ml of LB liquid media

4 small paper disks

Forceps

2 Cotton Swabs per group

Ruler

Antibiotics

Each Lab Section Set of 10 test tubes each with 2 ml of media in them.

1 2 ml liquid LB overnight culture of S. aureus

135 Procedure Working in Groups of 4

Day 0 Inoculate 2ml of liquid media. This will not require an entire lab period. Can be performed after a quiz or at the end of another lab.

1. Get a test tube that contains two ml of media from the rack.

2. Label the tube with your names/group name using sharpie or wax pencil

3. Wet a sterile swab and swab wherever you would like to collect microbes. You can collect from you skin, the door handle, your mouth, the floor etc

4. Place the swab in the media and mix 20 times

5. Place the media at 37 C°.

Day 1 Spread bacteria and put antibiotic disks on Petri Dish At this point the cultures you have inoculated will have grown and they will look cloudy, the microbes may have settled and you may need to mix or vortex the culture to suspend the culture and make it look cloudy.

1. Take a clean swab and mix your culture. Then streak the swab over the plate to inoculate the culture. (Figure 1) (Be careful to not damage the agar if you feel the swab is drying out you can get more bacteria at any time. You will not over inoculate)

2. Take the same swab remix in your culture and perform the second swab

3. Take the same swab remix in your culture and perform the third swab

4. Repeat steps 1-3 at least once. Your entire plate needs to be covered with bacteria. You will not be able to see these bacteria at this time but as they grow they will form the lawn of bacteria. The bacteria will not however be able to fill in large gaps (greater than 1mM) that you have failed to inoculate.

If you plate seems a little wet let it dry for 5-10 minutes either on your bench or in the incubator with the lid off.

136

5) Using forceps place 4 sterile paper disks on the Petri Dish.

6) Pipette 10 ul antibiotic onto three of the disks. Each disk only gets one antibiotic and each disk should get a different antibiotic. Pipette water onto the 4th disk.

137 Name ______Section______Date______

Label bottom of the Petri dish with a sharpie or wax pencil with your name and which disk has which antibiotic. (Labeling the lid will not be useful as there is no guarantee it will be put back on the dish in the same orientation every time.)

7. Place the Petri dish in the 37 degree incubator upside down. This prevents condensation from dripping off of your lid onto the plate. ℃ 8. Incubate the plates until next lab period.

Day 1 (continued) Inoculate Minimum Inhibitory Concentration Assay Instructor label tubes 1-10 with sharpie or wax pencil each will have 2 ml of media already in it.

1. Instructor will add 2 ul of Ampicillin to tube number one and mix. The final concentration of antibiotic in tube 1 will be 100 micromolar.

2. Take .5 ml of media from tube 1 and place it in tube 2. This is a 1/5 dilution and will give a final concentration of 20 micromolar.

3. Take .5 ml of media from tube 2 and inoculate tube 3.

4. Continue this procedure until all tubes have been inoculated.

5. Inoculate each tube with 3 microliters of overnight liquid S. aureus culture.

6. Incubate the 10 test tubes at 37 C° overnight. Instructor will take out and put in the refrigerator or cold room to stop bacterial growth.

138 Name ______Section______Date______Day 2 Record Results and Questions -- Zone of Inhibition 1. Draw the growth that has occurred on your plate. Label all disks with which antibiotic was on them. If multiple colony morphologies indicate this in the drawing.

2. Measure the diameter of each zone of inhibition.

Antibiotic Tested Zone of Inhibition (mM)

3. Which antibiotic was most effective, why do you say this?

139 Name ______Section______Date______

4. Which antibiotic was least effective, why would you say this?

5. How many types of bacteria/microbes do you see?

6. Are all bacteria inhibited equally by all of the antibiotics?

7. What is the mechanism of action and the spectrum of activity for the antibiotics that you choose to use?

8. What species of bacteria did you expect to find on the surface that you swabbed and are the zones of inhibition what you would have predicted given the mechanisms of action of the antibiotics that you used?

9. Do you think that all of the bacteria from the surface that you swabbed grew in your overnight culture and were equally represented on your plate why?

140 Name ______Section______Date______

Day 2 Record Results and Questions – Minimum Inhibitory Concentration Assay 1. Record the concentration of antibiotic in each of the 10 tubes.

2. Record if bacterial growth occurred in each tube. How can you tell if bacteria grew in the tube?

3. What is the MIC for this antibiotic for S. aureus?

4. Would you expect the same MIC for another species of bacteria, why or why not?

5. If we were to test a non-pure culture such as bacteria from our hand or desk, would you expect a higher or lower MIC? Explain.

141

How Does Antibiotic Susceptibility Differ Between Bacteria and Fungi?

142 Name ______Section______Date______

Pre-Lab Questions:

1. Which antimicrobial compounds will we be working with in this lab?

2. Which bacteria and fungi will we be plating in this lab?

3. How will be inoculate our plates?

143 Name ______Section______Date______

Background

Microbes are microbes are microbes. Bacteria, Fungi, Viruses they are all just single cells they are all the same! If you have an infection take antibiotics you’ll feel better! This is the prevailing knowledge among much of the population, but as microbiologists we know that nothing could be further from the truth. Bacteria, Yeast, and Viruses differ in very significant ways including their size, complexity, method of replication, and susceptibility to drugs. In this lab we are going to compare and contrast Yeast vs Bacteria as both of these organisms are amenable to study in the lab. They are relatively easy and cheap to grow, are visible under a light microscope, and strains that we will use are susceptible to a wide variety of drugs.

Materials needed for each group of 4

Cultures of S. cerevisiae (yeast) and E. coli (bacteria) grown in liquid media overnight

1 Petri Dish of YPD agar (for yeast)

1 Petri Dish of LB agar (for bacteria)

Sterile Swabs sharpie

8 paper disks

P20 or P200 pipettman and tips

Antimicrobial drugs:

Nourseothricin (Nat)

Fluconazole (Flu)

Streptomycin (Strep)

Ampicillin (Amp) ruler

144 Name ______Section______Date______Day 1 Spread bacteria and put antibiotic disks on Petri Dish

The overnight cultures of S. cerevisiae and E. coli will have grown and they will look cloudy

1. Take a clean swab and mix the E. coli culture. Then streak the swab over the LB agar plate to inoculate the plate. (Figure 1) (Be careful to not damage the agar if you feel the swab is drying out you can get more E. coli at any time. You will not over inoculate)

2. Take the same swab remix in E. coli culture and perform the second swab of the LB agar plate.

3. Take the same swab remix in E. coli culture and perform the third swab of the LB agar plate.

5. Take a clean swab and mix the S. cerevisiae culture. Then streak the swab over the YPD agar plate to inoculate the plate. (Figure 1) (Be careful to not damage the agar if you feel the swab is drying out you can get more yeast at any time. You cannot over inoculate)

6. Take the same swab remix in S. cerevisiae culture and perform the second swab of the LB agar plate.

7. Take the same swab remix in S. cerevisiae culture and perform the third swab of the LB agar plate.

8. Repeat steps 1-3 at least once. Your entire plate needs to be covered with S. cerevisiae. You will not be able to see these S. cerevisiae at this time but as they grow they will form the lawn of S. cerevisiae. The S. cerevisiae will not however be able to fill in large gaps (greater than 1mM) that you have failed to inoculate. Throw out your swab in the biohazard waste.

If your plate seems a little wet let it dry for 5-10 minutes either on your bench or in the incubator with the lid off.

Figure 1

145

Name ______Section______Date______

9. Using tweezers (forceps) place 4 sterile paper disks onto each Petri Dish.

10. Pipette 10 ul of Nourseothricin onto disk 1, 10 ul of Fluconazole onto disk 2, 10 ul of Steptomycin onto disk 3, 10 ul of Ampicillin onto disk 4. Each disk only gets one antibiotic and each disk should get a different antibiotic.

Label bottom of both of your Petri dishes with a sharpie or wax pencil with your name and which disk has which antibiotic, the microbe, and your group and section number. (Labeling the lid will not be useful as there is no guarantee it will be put back on the dish in the same orientation every time.)

11. Place the Petri dishes in the 37 degree incubator upside down. This prevent condensation from dripping off of your lid onto the plate. ℃ 12. Incubate the plates overnight or until next lab period.

Day 2 Record Results and Questions -- Zone of Inhibition 1. Draw the growth that has occurred on your plates. Label all disks with which antibiotic was on them. If multiple colony morphologies indicate this in the drawing.

LB YPD

146

Name ______Section______Date______

2. Measure the diameter of each zone of inhibition.

Antibiotic Tested YPD Zone of Inhibition (mm)

Antibiotic Tested LB Zone of Inhibition (mm)

3. Which antibiotic was most effective on each plate?

4. Which antibiotic was least effective on each plate?

147

5. Are the bacteria and yeast equally inhibited by all of the antibiotics?

6. What is the mechanism of action and the spectrum of activity for the antibiotics that you used? (You will be able to find this information online)

7. If any of the antibiotics did not work provide a rational for why they did not work.

8. If any of the antibiotics inhibited both bacteria and yeast provide a rational considering they are form different kingdoms of life.

9. Do you think any of the drugs you we used in lab would be effective against viral infections, why or why not?

148

Epidemiology: how a cold spreads through the dorm or lecture hall

149 Name ______Section______Date______

Pre Lab Questions:

1. Which hand are you going to shake other people’s hand?

2. How many classmates are you going to shake hands with?

3. How will you determine if you have been infected with the florescentitis disease?

150 Name ______Section______Date______Introduction:

As many of you living in the dorms have probably noticed by now, when one person gets a cold in a dorm that cold spreads from person to person quickly. There is a variety of reasons for why this occurs and we will discuss some of them in other labs and in lecture. However, the study of how disease spreads throughout a population is Epidemiology. This study can take many forms. Scientists can study where a disease originated, through what vector the disease spreads, how fast it spreads, and whom it is spreading to or likely to spread to. The field of epidemiology like most fields in biology is constantly evolving, and now does not just look at infective disease but also the correlation between lifestyle and disease. One of the most famous and controversial (at the time) epidemiological studies was done by Richard Doll and Austin Bradford Hill which showed a very strong correlation between tobacco smoking and lung cancer. As you can imagine this is extremely important to the medical community and worldwide health because if we do not know how or where a disease is likely to spread it is almost impossible to contain a disease through policy.

Materials:

Dust Canisters

Paper

Pencil

Hand

Procedure:

For this lab we will be dealing with the mysterious and luckily harmless condition of florescentitis. This disease occurs mainly in 113 students that are exposed to fluorescent dust during lab. Don’t worry, this is a harmless condition that can cured by washing ones hands at the end of class.

1. Randomly select a dust canister and liberally spread the powder on your right hand. If you do not have a right hand or your right hand is in a cast use your left hand. Just make sure to shake with the hand that has the powder on it. Make sure to note which number canister you picked.

2. Shake hands (using your right hand) with 3 other students in random order. Shake firmly. BE SURE TO RECORD THE FOLLOWING FOR EACH PERSON you shake hands with: Name, canister number, and the order that you shook their hand.

151 3. Then we will screen everyone's right hands with the black light. Record on the spreadsheet the number of infected people as "Round 1" total.

4. Repeat steps 1-3 for round 2, and then again for round 3

Round 1: Round 2: Round 3: Container Infected: 1=Y/ Container Infected: Container Infected: Number: 0= N number: 1/0 number: 1/0 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7 7 7 8 8 8 9 9 9 10 10 10 11 11 11 12 12 12 13 13 13 14 14 14 15 15 15 16 16 16 17 17 17 18 18 18 19 19 19 20 20 20 21 21 21 22 22 22 23 23 23 24 24 24

Total Infected: 0 Total Infected: 0 Total Infected: 0

152 Name ______Section______Date______

Post Lab Questions:

1. Who is the source of the infection, if you can’t determine the source why can’t you and who are the likely candidates?

2. What number was the “infected” canister? How did you arrive at this conclusion?

3. How can an epidemic stop without medical intervention (i.e. no quarantine, vaccines, or treatment)?

4. Diagram how the disease spread around the classroom below, feel free to draw the benches and where people are sitting.

153

5. Why are dorms and lecture hall good places for disease to spread?

6. You are on a cruise and a passenger comes down with an intestinal virus. A fellow passenger is very worried about contracting this virus and says that he needs to find out who was the first person to have the virus so they can stay away from this person. List 2 reasons why this probably not going to be a very helpful in keeping this passenger from contracting the virus.

7. Name something the passenger could do to decrease the likelihood of contracting the virus.

154 Contaminated Spray Bottle and Water Contamination

If truth prevails, the contributions of a courageous physician and a brilliant engineer to the conquest of waterborne disease will still be remembered in another hundred years.” ― Michael J. McGuire, The Chlorine Revolution: Water Disinfection and the Fight to Save Lives

738 million people worldwide do not have access to clean drinking water this is roughly twice the population of the United States. -unfun fact-

155 Name ______Section______Date______Prelab Questions:

1) How much water will you run through your filter?

2) What do you use to pick up the filter after running the water through it? Why can’t you use your hands?

3) What type of bacteria do we think contaminated the spray bottle and who do we think contaminated it?

156 Name ______Section______Date______Background:

Microbes can be spread in a variety of ways. In lecture we have/will talk about microbes that are spread by variety of different mechanisms. Two of the most prevalent ways that microbes can be spread is through the air and water. If you ever lived in a dorm or have gone to a grammar school, you know that when one person gets a cold, it tends to spread to another person and then another and then another. This is often through one person contracting an illness transmitting this disease to additional members of the group. While all of us are familiar with the spread of disease from person to person the spread of disease via water is probably not something that we think about very often. In the developed world the spread of disease through water is not nearly as prevalent as is was 200-300 years ago because we have systems in place that make sure that we limit the number of bacteria that enter our food and water supply. The important part about the previous statement is that though we limit the number of bacteria, we do not eliminate all bacteria from all of the water we drink or come in contact with. For instance the water bottle that you are drinking out of and touching your mouth to has many bacteria from your mouth that now reside on the water bottle. The water fountain that is sitting out in the open all day long collects bacteria on it. Even the water that comes from the tap has bacteria in it. The key to all of these and the reason that we don’t get sick is the type of bacteria on these sources are generally bacteria that do not cause us harm or they are in such low numbers that it is unlikely that they will ever make us sick. However, throughout history and even now in areas where water quality standards are lower or nonexistent many become ill from water born illnesses. Examples of Waterborne illnesses:

Viruses: SARS and Polio

Eukaryotes: Giardiasis (Protozoa), Cryptosporidiosis (Protozoa)

Bacteria: Cholera, Dysentery, Typhoid, and Legionnaires Disease

Materials:

5 LB plates

Spray bottle

Buchner funnel

Filter Paper

1 liter flask

157 Name ______Section______Date______Procedures:

Somehow the spray water bottle in lab has become contaminated. I talked to Microbiology 313 they said that is possible that some of one of their cultures contaminated our spray bottle while it was sitting open in the sink. They said that any combination of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Micrococcus luteus, Bacillus cereus, and Salmonella typhimurium could have contaminated our bottle. They don’t give us 113ers any respect, always using our stuff and trashing it. Unfortunately there is nothing we can do about this now, but because every time we spray the bottle we get bacterial contamination on what we spray we can’t use the bottle. Since we are microbiologists we are interested what kind/s of bacteria are contaminating our water bottle, “at least you should be”. Your professor and lab instructor think that this is an excellent opportunity to demonstrate bacteria aerosolization and at the same time test if other water sources on campus are contaminated with bacteria. Day1: Groups of Four Students

Spray Bottle Test: To determine which type of bacteria are contaminating our water bottle we must isolate single colonies of bacteria. We will do this and at the same time demonstrate aerosolization.

1) Line up 4 four LB agar plates on the bench about 12 inches away from one another and remove the lids. Make sure the plates are labeled with the distance that they are from where you are spraying your group and section number.

2) Stand at the end of the bench with everyone behind you, you don’t want to spray anyone with bacteria.

3) Spray the spray bottle on the mist setting once from the end of the bench. To make sure that it is on the mist setting spray it once or twice into the sink.

4) Put the lids back on the plates and place them in the 37 incubator overnight. Your lab instructor will take them out the next day. Note where spray landed on the bench, how far from the spray site. ℃

Water Contamination Experiment: We are going to test how many bacteria are in the water of Cooper. This is an experiment, I have never done this before so we/I don’t know what the outcome will be. We should not just do drinking water, anything that has water is fair game including toilets, waterbaths, ice machines, drips from the ceiling if you can find one (although I hope you can’t get a liter water from a leak in the ceiling.)

1) Collect 1 liter of water from somewhere in the building in your sterile 1 liter bottle. This can be from anywhere but please don’t steal water from someone’s water bottle or experiment without asking. Note where you got the water from.

2) Filter the water through the Buchner funnel with a filter paper into a flask. To do this you will need to hook up a vacuum to your funnel. Your lab instructor will show you how to do this. If you have a flask that is less than 1 liter you will need to stop the filtration to empty the flask and then restart the filtration so the flask does not overflow.

158 Name ______Section______Date______3) Take filter paper using sterile tweezers or gloved hands and lay the filter on a petri dish.

4) Place the petri dish in the 37 incubator until the next lab period.

℃

Above is an example of what your plate may look like after incubation (if the water you test has a particularly large microbial load).

Day2: Spray Bottle Test: Determine how far a spray bottle sprays bacteria.

1) Look at your plates and record how many colonies are growing on each in the table below.

Plate 1 Plate 2 Plate 3 Plate 4

159 Name ______Section______Date______

Questions for Spray Bottle Test:

1) How many colonies do you see on each plate? If you don’t have distinct colonies and are therefore unable to perform a colony count with confidence, give a qualitative estimate of how dense the microbial growth is.

2) Based on the diversity of colony morphology, at least how many species of bacteria are contaminating our water bottle?

3) Can you determine which species of bacteria are contaminating the water bottle just by looking at the colonies, why or why not?

4) How far did the bacteria reach?

5) Did you expect the bacteria to go the distance that they went, why or why not?

6) Describe one benefit of letting the plates grow for more days in the incubator? (How might this give us more accurate data?)

7) Describe one drawback of letting the plates grow for more days in the incubator? How might this give us less accurate data?

160 Name ______Section______Date______

Day 2 Water Contamination Experiment: Determine which water source has the most bacteria.

Put your data on the board so everyone can use the data for the below questions.

1) Count how many colonies are on your plate and the number of different types of colonies?

Building location Number of colonies ~# Types of colonies

Questions Water Contamination Experiment:

1) Which location has the most bacteria in the water?

2) Did this surprise you, why or why not?

3) Which location has the least bacteria in the water?

4) Did this surprise you why or why not?

161 Name ______Section______Date______5) Does our experiment tell us which source has the most microbes why or why not?

6) Does this experiment tell you the exact number of different types of bacteria in a particular water source, why or why not.

7) Does the plate with the most bacterial growth mean that this water is the most dangerous why/why not?

8) If a plate does not have any growth does this mean that the water is definitely safe to drink why/why not?

162

Genus Unknown

163 Name ______Section______Date______

Pre-Lab Questions:

1. What will the first test be that we perform in this lab?

2. If our unknown bacterium expresses urease, what color will the urea broth be?

3. What metal will hydrogen sulfide react with in the SIM test? What color will the resulting compound be?

164 Name ______Section______Date______

Background:

If we were to take a microbial sample from a patient and then isolate single colonies and grow a pure culture, how would we identify what bacterium it is? Earlier in the semester we showed you that many bacteria can distinguished from one another based on morphology, but what if bacteria look the same under the microscope. In this lab, we will utilize the different biochemical abilities of microbes to try to identify them from a few potential options. As you will see, we have many biochemical tests in our arsenal—more than we will need for the purpose of this lab, in fact. We will use some familiar tests and some new tests. The new tests will be discussed below.

To determine if a microbe can ferment lactose, dextrose, and sucrose, we will use three different phenol red broths. Each broth will have lactose, dextrose, or sucrose. All of the broths will have the pH indicator phenol red. If the inoculated bacterium is capable of fermenting that sugar, acid will be produced and the tube will turn yellow. If fermentation does not occur, the tube will remain red. There are upside down Durham tubes (pretty much a small test tube). If the bacterium produces, gas a small bubble will collect in the Durham tube.

To determine if our bacterium is capable of hydrogen sulfide (H2S) production, we will use SIM media. SIM stands for Sulfur reduction, Indole production, and Motility. We will only be analyzing the reduction of sulfur to hydrogen sulfide. Similar to TSI, our SIM has iron in it which will combine with any hydrogen sulfide that is created to produce a black pigment. If no hydrogen sulfide is produced, the media will remain yellow.

The methyl red test is another test to determine if your bacterial unknown is secreting acid. This test determines if your bacteria is making acetic, lactic, or formic acids from pyruvic acid, the product of glycolysis. Bacteria that are doing this are considered methyl red positive. If your bacteria is taking pyruvic acid and turning it into a neutral product they are considered methyl red negative.

To determine if bacteria have urease and can metabolize urea (a compound that is the end product of amino acid metabolism in humans), we will utilize the urea test. We will use urea broth which contains phenol red which is light orange under neutral conditions but is pink at an alkaline pH (basic). If urease is present, it will convert urea to carbon dioxide and ammonia which will increase the pH of the media which can be visualized by a pink color change.

To determine if bacteria have catalase and can degrade hydrogen peroxide (H2O2, an oxidizing chemical produced as a byproduct of other metabolic reactions), we will utilize the catalase test. To do this, we will place hydrogen peroxide on a sample of our bacteria. Remember, catalase degrades H2O2 to H2O and O2. So we should quickly see oxygen bubbles form if the bacterium produces catalase.

In this lab you will be given a sample of bacteria that could be any of bacteria listed on the chart below. You will use the staining techniques and biochemical techniques to determine which bacteria you have been given.

165 Name ______Section______Date______

Escherichia Pseudomonas Staphylococcus Micrococcus Bacillus Salmonella coli aeruginosa aureus luteus cereus typhimurium Morphology Rod Rod Sphere Sphere Rod Rod Gram Status Negative Negative Positive Positive Positive Negative Lactose Acid and Negative Acid Negative Negative Negative Fermentation gas Dextrose Acid and Negative Acid Negative Acid Acid and Gas Fermentation gas Sucrose Acid Negative Acid Produced Negative Acid Acid Fermentation Produced Produced Produced H2S Negative Negative Negative Negative Negative Positive Production Methyl Red Positive Negative Positive Negative Negative Positive Test Urease Negative Negative Negative Positive Negative Negative Activity Catalase Positive Positive Positive Positive Positive Positive Activity

Materials: 4 Unknown samples

Inoculating loop

Crystal violet

Safranin

Ethanol

Iodine

Distilled water

Compound light microscope

Microscope slides

Bunsen burner and striker

Blot book or paper towels

Phenol red lactose broth

166 Name ______Section______Date______Phenol red dextrose broth

Phenol red sucrose broth

Methyl red broth

Hydrogen peroxide

Trypticase soy agar plate

Laboratory Exercise:

Begin this lab by performing a Gram stain. Then perform the biochemical tests you feel you need to complete to identify your bacterium.

Gram Staining:

1. Flame your loop to sterilize it, and wait for it to cool.

2. Scoop a very small amount of bacteria off the surface of your petri dish using a loop.

167 Name ______Section______Date______

9. Add ethanol (ethyl alcohol) to your samples. Ethanol acts as a decolorizing agent because it breaks apart the outer phospholipid bilayer of Gram negative cells which allows the Gram violet-iodine complex to leave. After this step, Gram positive cells should be purple (because they will retain the crystal violet-iodine complex) and Gram negative cells will be clear (because the crystal violet-iodine complex is being washed away).

10. Gently wash away the ethanol using a distilled water squirt bottle. Again, aim the water above the area with bacteria and point the slide down at a 45˚ angle. Take care not to aim the water stream directly at the bacteria because this will wash away your sample.

11. Add pink safranin (the counterstain) to your samples and wait 30 seconds to one minute. Safranin will not have an appreciable effect on purple Gram positive cells, but safranin will stain Gram negative cells pink. Imagine dumping some pink dye on a dark purple coat, it may stain a little but it won’t be as noticeable as if it was a white coat.

12. After 30 seconds to one minute, wash away the excess safranin using a distilled water squirt bottle. Again, aim the water above the area with bacteria and point the slide down at a 45˚ angle. Take care not to aim the water stream directly at the bacteria because this will wash away your sample.

13. Blot your samples dry by placing your slide in a blotbook or laying on paper towels and gently closing it and applying gentle pressure. Avoid smearing your samples.

14. View your slides under the microscope. Remember to use the 4X objective lens first.

15. Draw what you see below. Are they Gram positive or negative?

168 Name ______Section______Date______

Sample:______Magnification:_____

Lactose, Dextrose, and Sucrose Fermentation:

1. Flame your loop and wait for it to cool.

2. Use your loop to scoop a small amount of your unknown bacterium.

3. Insert your loop into the test tube with either lactose, dextrose, or sucrose.

4. Incubate your test tube for 24 hours.

SIM Test for H2S Production:

1. Flame your inoculating needle.

2. Use your needle to scoop a small amount of your unknown bacterium.

3. Insert your needle straight down into the SIM media.

4. Incubate your test tube for 24 hours.

169 Name ______Section______Date______

Methyl Red Test:

1. Flame your loop and wait for it to cool.

2. Use your loop to scoop a small amount of your unknown bacterium.

3. Insert your loop into the test tube with methyl red indicator.

4. Incubate your test tube for 24 hours.

Urease Test:

1. Flame your loop and wait for it to cool.

2. Use your loop to scoop a small amount of your unknown bacterium.

3. Insert your loop into the urea broth test tube.

4. Incubate your test tube for 24 hours.

Catalase Test:

1. Flame your loop and wait for it to cool.

2. Use your loop to scoop a small amount of your unknown bacterium.

3. Scrape this onto a microscope slide.

4. Add a drop of hydrogen peroxide. Observe for bubbles.

170 Name ______Section______Date______

Post-Lab Questions:

1. Fill out the table as much as you need to. What is your unknown microbe?

Your Unknown Sample Morphology Gram Status

Lactose Fermentation Dextrose Fermentation Sucrose Fermentation

H2S Production Methyl Red Test Urease Activity Catalase Activity

2. Did you need to use every biochemical test to identify your unknown bacteria from the list of possible options? Explain. When might you need to use more tests?

3. Some bacteria like Proteus mirabilis express urease and cause UTIs. How do you think they impact the pH of urine? Remember, urea is secreted in urine.

171 Name ______Section______Date______

4. E. coli and Proteus mirabilis are leading causes of UTI’s. Using the urease test, could you distinguish between E. coli and Proteus mirabilis?

5. Helicobacter pylori is a causative agent of stomach ulcers. Why might it be beneficial for this bacterium to express urease?

6. Why might it be beneficial for bacteria living in an oxygen-rich environment to express catalase?

172

Immunology; Innate Immunity

173 Name ______Section______Date______

Pre-Lab Questions:

1. Is Streptococcus pyogenes Gram positive or Gram negative?

2. What protein from the innate immune system will we be working with today?

3. What does the OD540 tell us?

174 Name ______Section______Date______Background:

Our immune systems defend us against pathogens. The adaptive immune system mounts a delayed, yet powerful and specific attack to invaders. Vaccines stimulate B and T cells of the adaptive immune system to “remember” invaders so that the immune system may promptly respond to potential invaders. The innate immune system, on the other hand, does not target specific pathogens and does not “remember” past invaders, but it does provide rapid responses that thwart infection. The innate immune system is comprised of tissues such as the skin, mucus, neutrophils, dendritic cells, macrophages, mast cells, natural killer cells, the complement system, stomach acid, and a wide range of chemicals. One of those chemicals, lysozyme, a protein present in saliva, tears, mucus, and breast milk (and egg whites). Lysozyme protects the tissues it is present on by degrading the linkages holding peptidoglycan together.

In this lab we will indirectly test the function of salivary lysozyme on Streptococcus pyogenes. To do this, we will be using a spectrophotometer. A spectrophotometer or “spec” emits photons (light waves/particles) through a sample and then measures how many photons pass through. The spectrophotometer then reports this measurement in terms of absorbance—how many photons are absorbed by the sample. The higher the absorbance, the more photons are absorbed by the sample. The absorbance is reported in terms of optical density (OD). The spectrophotometer is capable of emitting photons at a spectrum of wavelengths. Today, we will be using photons with 540nm wavelengths. So we will be measuring the OD540 of our sample. If you have a sample with lots of bacteria it will absorb lots of light. These cultures will look cloudy and this will result in a higher absorbance. If however you have a culture without a lot of bacteria the media will almost look clear. This will not absorb lots of light and will result in a lower absorbance.

In this lab exercise we are going to test the effects of lysozyme on the bacteria Streptococcus pyogenes by seeing how the absorbance of our culture changes over time.

Materials:

Working salivary glands

Tears (although not required)

Lysozyme buffer

Spectrophotometer

Spectrophotometer tubes

Assorted pipettes and tips

Centrifuge tubes

Lysozyme

175 Name ______Section______Date______

Laboratory Exercise: In Groups of 4

1. Get two test tubes and label them.

2. TUBE 1: Collect 0.5mL of saliva (or tears, this is our source of lysozyme) in one of the tubes. (This does not have to be exact but must be close.)

3. TUBE 2: In the second tube add 2.5ml lysozyme solution.

4. Add 4.5mL of lysozyme buffer to Tube 1.

5. Add 2.5ml of lysozyme buffer to Tube 2.

6. Transfer 2.5mL from Tube 1 into spectrophotometric tube.

7. Repeat step 6 for Tube 2

8. Set the spectrophotometer to OD540. Zero the spec with pure lysozyme buffer solution. Place the spectrophotometer tube in the spectrophotometer.

9. Add 2.5mL of the Streptococcus pyogenes culture to each of these spectrophotometric tubes. Each tube should now have 5 ml.

10. Pipette the mix of bacteria and buffer solution up and down gently three times. (Take care not to create bubbles as this will skew our data.)

11. Start timer

12. Record the absorption values at OD540 for both tubes after 30 seconds, 60 seconds, 120 seconds, 180 seconds, 240 seconds, 5 minutes, 8 minutes, and 12 minutes. Designate someone in your group to record the time on their phone or in the lab manual below.

13. Graph your data on the graph paper provided below with time on the X axis and A450 on the Y axis.

176

Time OD540 30sec 60sec 120sec 180sec 240sec 5 min 8 min 12 min

177

Post-Lab Questions:

1. What happened to the optical density of the bacterial solution for each tube?

2. Explain your above results from question 1.

3. If we were to boil the lysozyme solution prior to adding S. pyogenes, do you anticipate getting the same data? Why or why not.

178

4. Xerostomia, dry mouth, can be caused by a number of factors including mouth breathing, dehydration, some medical conditions, and some drugs. People with xerostomia experience a higher incidence higher incidence of dental caries (cavities). From a microbiological perspective, how do you explain this?

5. Knowing that lysozyme degrades the linkages holding peptidoglycan together, do you anticipate lysozyme being as effective against Gram negative bacteria, why or why not? Would lysozyme be effective against fungi why or why not?

179

Blood Typing: ABO and Rh Blood Group Systems

180

Name ______Section______Date______Pre-Lab Questions:

1. Why are people with type O blood called universal donors? Why are people with type AB blood called universal recipients?

2. If someone observes clumping (hemagglutination) in the A and Rh well, but not in the B well; what is their blood type?

3. If someone observes clumping (hemagglutination) in the Rh well only; what is their blood type?

181 Name ______Section______Date______Background:

People have different antigens on the surface of their red blood cells (RBCs, erythrocytes). There are 35 recognized (known) blood group systems. Obviously when you have 35 independent variables, things can get very complicated very quickly, so we are going to limit our experiments and discussion to the two most important: the ABO and Rh systems.

Blood types are determined by the presence of specific proteins on the surface of RBCs. People with type A blood have A antigen. People with type B blood have B antigen. People with type AB blood have A and B antigens. People with type O blood have neither A nor B antigens.

In the absence of autoimmune disease, the immune system does not routinely attack the body’s own cells. But the immune system may launch an attack on improperly matched blood because it recognizes the transfused blood as a foreign invader. For example, if a patient with type A blood were given type B blood, the patient will have an immune reaction to the type B blood because it identifies the transfused blood as something that should not be in the body. This is because the person has anti-B antibodies which bind to the type B blood. This reaction can occur very quickly (within minutes). As you recall, antibodies are not produced this fast. So where did these antibodies come from? The immune system produces antibodies against bacteria (intestinal bacteria, in this case). These antibodies may cross-react with the antigens of foreign blood. That is to say, antibodies that bind to intestinal bacteria may also bind to antigens on foreign RBCs. Remember, the immune system has many checks to ensure that it does not produce antibodies against its own RBCs.

Below is a table explaining what antigens and antibodies are present in the different ABO types of blood.

Blood Antigens Antibodies Can Donate To Blood Can Receive From Blood Type Present Present Types Types A A anti-B A and AB A and O B B anti-A B and AB B and O AB A and B AB A, B, AB, and O O anti-A and anti-B A, B, AB, and O O

182 You may be wondering, for example, why a patient with type AB blood can receive a transfusion of type O blood when type O blood contains both anti-A antibodies and anti-B antibodies. Wouldn’t the presence of these antibodies cause problems with the patient’s type AB blood? If a person is needing a emergency transfusion for something such as life-threatening blood loss, they really only need RBCs to transport oxygen and saline fluid. They don’t have an immediate need to replace the antibodies that they’ve lost. So they are given what are called O- packed RBCs. The potentially hazardous anti-A and anti-B antibodies are removed from whole O- blood by centrifuging the whole blood and then removing the antibody-containing plasma.

If time permits, testing is done to determine blood type and a person is given their own blood type (e.g. a patient with type AB blood is given type AB blood). If even more time permits, the patient’s blood type is determined and the donor and recipient bloods are crosshatched to ensure that there are not potentially hazardous cross-reactions with other, non-ABO antigen systems.

The Rh blood group system is named after the rhesus monkey in which it was discovered. It’s pretty complicated, but for our purposes, if a person has the Rh antigen (D antigen) on the surface they are said to be Rh+. If they do not, they are Rh-. People with Rh- blood produce anti-Rh antibodies. So, people who are Rh- can only receive from people who are Rh-. Rh+ people can receive from Rh+ or Rh- blood.

Unlike anti-A and anti-B antibodies, the immune system doesn’t normally produce anti-Rh antibodies. Patients will only develop anti-Rh antibodies if they are Rh- and are exposed to Rh+ blood (e.g. if Rh- mother is exposed to Rh+ fetal blood during delivery).

Blood Antigens Antibodies Can Donate To Blood Can Receive From Blood Type Present Present (after Types Types exposure) Rh+ Rh Rh+ Rh- and Rh+ Rh- anti-Rh Rh- and Rh+ Rh-

The ABO and Rh groups are combined. For example, if someone doesn’t have A or B antigens, but do have Rh antigens, then they are O+. Below is a table summarizing.

Blood Antigens Antibodies Can Donate To Blood Can Receive From Blood Type Present Present Types Types A+ A, Rh anti-B A+, AB+ A+, A-, O+, O- A- A anti-B, anti-Rh A+, A-, AB+, AB- A-, O- B+ B, Rh anti-A B+, AB+ B+, B-, O+, O- B- B anti-A, anti-Rh B+, B-, AB+. AB- B-, O- AB+ A, B, Rh AB+ AB+, AB-, A+, A-, B+, B-, O+, O- AB- A, B anti-Rh AB+, AB- AB-, A-, B-, O- O+ Rh anti-A, anti-B AB+, A+, B+, O+ O+, O- O- anti-A, anti-B, AB+, AB-, A+, A-, B+, B-, O- anti-Rh O+, O-

183 Note: there is not a preponderance of evidence supporting the claim that people with type A+ blood earn higher grades .

When antibodies bind to antigens on the surface of multiple RBCs, visible clumping occurs. This is called hemagglutination. We will utilize this phenomenon to quickly determine blood type.

Above is an example of A blood experiencing hemagglutination after being exposed to anti-A antibodies. From this test alone, you can conclude that the blood has A antigens.

184

Above is an example of AB blood experiencing hemagglutination after being exposed to anti-A antibodies. From this test alone, you can conclude that the blood is at least has A antigens. Note that from this test alone, A and AB blood will clump identically.

Above is an example of B blood not experiencing humagglutination after being exposed to anti-A antibodies. From this test alone, you can conclude that the blood doesn’t have A antigens.

In this lab, we will be identifying the blood types of several patients (we will be working with synthetic blood). To do this, we will be adding anti-A, anti-B, and anti-Rh antibodies to the blood to observe if clumping will occur. From this information, we should be able to deduce the patient’s blood type.

185 Name ______Section______Date______

Materials:

Synthetic blood

A, B, Rh antisera

Toothpicks

Blood mixing trays

Laboratory Exercise: Blood Typing:

1. Add one drop of the patient’s blood to each well in your blood mixing tray (A, B, and Rh).

2. Add one drop of the corresponding antisera to each well. 3. Mix the blood and antisera with toothpicks for 30 seconds. Use a clean toothpick for each well to prevent misleading results!

4. Look for clumping and record it in the data table below.

Patient 1 Patient 2 Patient 3 Patient 4 Anti-A Anti-B Rh Blood Type

Optional For those interested in Cancer Pre-Made Samples:

1. Use your microscope to observe pre-made slides of blood cancers and blood-borne infections. I would encourage you to do independent research to learn more about these diseases.

186

Name ______Section______Date______Post-Lab Questions:

1. What is your patient’s blood type? How do you know?

2. Your patient needs a transfusion. What blood types can they receive?

3. If a donor and a patient both have B- blood, why would a technician ideally still mix their blood in a test tube prior to transfusion to test for hemagglutination? Are there other factors that could cause hemagglutination besides ABO?

4. Why are potential blood donors not allowed to donate blood if they have recently engaged in HIV- risky behaviors even if blood banks test for the presence of anti-HIV antibodies?

5. Why/how do we have antibodies in our blood that will bind to non-self blood types when in theory we should have never been exposed to these antigens before?

187

Name ______Section______Date______

6. The primary two classes of antibodies produced which can bind to antigens on foreign RBCs are IgM and IgG. Both are present in the blood, but IgM is much larger and cannot cross the placenta. IgG, however, is much smaller and can cross the placenta and reach the fetus. During the course of a normal pregnancy, maternal and fetal blood do not come into contact. Consequently, maternal B cells do not produce antibodies against antigens in the fetal blood. During delivery, however, the blood supplies can mix. If mom is Rh- and baby is Rh+, then mom’s immune system may detect the Rh antigen in baby’s blood during delivery and then produce antibodies against it. To prevent this, RhoGAM (Rho immune globulin) is injected into the mother. RhoGAM is given at 26-28 weeks and within 72 hours of delivery. Additional injections of RhoGAM are needed if there is any trauma or problem with placental integrity. RhoGAM is an antibody that binds to and shields the perinatal Rh antigen thereby preventing it from sensitizing mom’s immune system. This prevents an immune response to Rh antigen which could be problematic in future pregnancies since IgG can cross the placenta leading to a disease called hemolytic disease of the newborn (HDN, erythroblastosis fetalis). Why do mothers who are Rh positive not receive RhoGAM?

188

White Blood Cell Identification

189 Name ______Section______Date______Pre-Lab Questions:

1. What is a phagocyte?

2. Green phlegm often indicates a bacterial infection due to a high concentration of heme-containing myeloperoxidase found in which granulocyte?

3. How will we get the samples that we are going to look at for this lab?

190 Name ______Section______Date______Background:

White blood cells (WBCs, leukocytes) are immune cells that defend local tissues and circulate in the blood and lymph. WBC attack pathogens, cancer cells, and anything recognized as “non-self” inside the body. What are the different types of WBCs? What do they do? How can we identify them? These are all excellent questions and this lab will be brief introduction to WBC. I would like to not that aside from the brain the immune system is the most complicated thing on the planet. It is a biological recording of everything that our body comes in contact with. Entire courses are dedicated to the study of the immune system and understanding how it works is still very much a work in progress.

We can identify WBCs by looking at a sample of our blood using Wright’s stain. The cells we will be looking at today in lab will be premounted and will be pretreated with stain. There are two major classes of WBCs: granulocytes and agranulocytes. Granulocytes have visible granules in their cytoplasm and agranulocytes do not. Furthermore, there are three types of granulocytes: neutrophils, eosinophils, and basophils. There are two types of agranulocytes: monocytes and lymphocytes (each have many different subtypes but you will not need to know about these for this class).

Cell Cytoplasm Nucleus Simplified Function % of WBCs Morphology Neutrophil Granular, 2-5 Kill bacteria and fungi 65 pinkish connected lobes Eosinophil Granular, 2 lobes Kill parasites, allergies 4 to 5 orange Basophil Granular, 2-3 lobes Allergies <1 purple Monocyte Agranular, Kidney Differentiate in tissues to phagocytic 2 to 4 light blue shaped cells (e.g. macrophages) Lymphocyte Agranular, Large, most Adaptive immune response, kill 25 blue of cell cancer and virally infected cells

Materials:

Compound light microscope

Pre-made slides

191 Name ______Section______Date______Laboratory Exercise:

1. Use your microscope to analyze pre-made slides of white blood cells and white blood cell cancer. You should be able to see these cells well on 40x magnification so you will not need oil.

2. Draw what you see under the microscope and take note of the differences between the different types of white blood cells.

192 Name ______Section______Date______

Cell Type ______Ce ll Type ______

Magnification ______Magnification ______

Cell Type ______Ce ll Type ______

Magnification ______Magnification ______

193 Name ______Section______Date______Post-Lab Questions:

1. Which are larger, RBCs or WBCs?

2. A patient has neutrophilia (high concentration of neutrophils in the blood) and a fever. What is the most likely explanation?

3. What are the possible health consequences if you have too few white blood cells?

4. What are the possible health consequences if you have too many white blood cells?

5. A survivalist has been living off of the land and has not had access to clean water or well cooked food for the past month. He comes to the clinic because he feels “a little under the weather” and complains of diarrhea. Blood tests indicate elevated eosinophils. What is the likely cause of this elevation? He has no history of seasonal allergies.

194

Rapid Antigen Detection Test for Group A Streptococcus (Streptococcus pyogenes)

https://en.wikipedia.org/wiki/Streptococcal_pharyngitis

195 Name ______Section______Date______Pre-Lab Questions:

1. What is an antigen?

2. How long should I leave the swab in the testing vial for after swirling it ten times?

3. What are the results if I don’t see the test line after 5 minutes?

196 Name ______Section______Date______Background:

Group A Streptococcus infection (strep throat) is usually caused by Streptococcus pyogenes. There are millions of cases of this throughout the world each year. Its incidence is highest in the winter and early spring. Group A Streptococcus is the causative agent of roughly 15-30% of acute pharyngitis (sore throat) in children and roughly 5-20% the cause in adults. When not caused by Group A Streptococcus, acute pharyngitis is almost always caused by viruses and since are not susceptible to antibiotics. Remember unnecessary use of antibiotics can create an environment which is conducive to the development of antibiotic-resistant bacteria, but if streptococcal pharyngitis is left untreated, the immune system may mount a response including the production of antibodies which cross-react with the person’s own connective tissues causing rheumatic fever. As such, it is imperative that we are able to quickly determine what microbe is causing a throat infection.

So when should clinicians prescribe antibiotics for pharyngitis? To provide guidelines for the practical answer of this question, the CENTOR Criteria were developed. In these diagnostic guidelines, patients are scored based off five criteria that can either add or deduct points.

Cough—add 1 point if there is an absence of cough

Exudates—add 1 point if pus is exuding from tonsils

Nodes—add 1 point if there are swollen and tender cervical (neck) lymph nodes

Temperature—add 1 point if history of fever; particularly rapid onset

OR—young OR old. If under 15 years old, add 1 point. If over 44, deduct 1 point.

If patients have 4-5 points, they likely have streptococcal pharyngitis and should undergo rapid antigen- based diagnostic testing. If this is positive, the patient should be prescribed antibiotics. Many clinicians will treat even without a positive test with a score of 4-5 if they don’t suspect another infectious agent is the cause such as the Epstein-Barr virus in infectious mononucleosis. If the rapid test is negative, a culture should be taken. If the culture is positive, they should be prescribed antibiotics.

If patients have 2-3 points, they may have streptococcal pharyngitis and should be cultured (many clinicians will perform a rapid test prior and will only culture if the rapid is negative).

If patients have -1-1 points, they likely do not have streptococcal pharyngitis and should not be treated with antibiotics. They should be reevaluated if symptoms persist for longer than 7-10 days (the typical duration of viral infections).

These guidelines are based off of probabilities and are by no means absolutes. For example, a patient can have a streptococcus pharyngitis and another concurrent infection which causes cough. In addition, patients can present with conditions abnormally or misremember/misreport their symptoms. In these instances, clinicians need to rely on their clinical acumen and experience.

197 Name ______Section______Date______

In this lab, we will be practicing running rapid antigen based diagnostic tests for Group A Streptococcus. Our kit will determine if Group A strep-specific antigens are present in our samples. Antigens are molecules that can elicit an immune response. If there are antigens, an antibody in the test kit will bind to the antigens, causing a confirmation change in the antibody leading to a visible color change (a red line to appear on the test strip). Many diagnostic tests today operate on this same principle. The exact mechanisms by which these tests work are trade secrets.

We are testing for Group A Strep in this lab, but you may be wondering, what is Group B Strep? Group B Strep (GBS, Streptococcus agalactiae) is part of the normal vaginal and rectal flora of roughly 25% of women. Normally this isn’t a problem, except during labor when the neonate can be exposed, potentially causing meningitis, pneumonia, and/or sepsis. Women are generally swabbed at 35 and 37 weeks for GBS. Those who test positive are treated with antibiotics to reduce the risk of transmission.

Laboratory Exercise: Working in Groups of 4

1. Drop 4 drops of Reagent A and 4 drops of Reagent B into the testing vial.

2. Collect a small amount of one of the bacteria samples (1-6) using a cotton tipped applicator. Each group should use a different sample this way all of the bacterial cultures can be tested.

3. Twirl the applicator in the vial ten times.

4. Leaving the cotton tipped applicator in the vial, wait 1 minute.

5. After waiting one minute, pull the cotton tipped applicator out of the vial while squeezing the sides of the vial so as to remove as much fluid from the cotton as possible.

6. Place the testing strip in the vial and wait 5 minutes. You will see the mobile phase of the fluid move up the testing strip via capillary action.

7. After 5 minutes, remove the testing strip and analyze. The top pink line is a control. The line beneath the control is the test (it may or may not be there). If the test line is there, the test is positive for Group A strep. Post your results on the board.

198

Name ______Section______Date______Post-Lab Questions:

1. Which samples were positive for Streptococcus A.

2. Why do doctors order strep tests? Why don’t they just prescribe antibiotics (e.g. amoxicillin) for everyone who has a sore throat?

3. Why might this test not work in practice?

4. What class of infectious agent is the most common cause of sore throats?

5. Is penicillin effective against viruses, why or why not?

199

Name ______Section______Date______

6. What is the most common species name for Group A streptococcus?

7. A young child comes into the clinic with a cough and pus exuding from their tonsils. They have a fever and slightly swollen lymph nodes. Should a Strep test be ordered according to the Centor criteria?

200 Public Service Announcement (PSA): Communicating Science to the Public

“This is drugs, this is your brain on drugs, any questions?”

Partnership for a drug free America PSA from when I was a child. https://www.youtube.com/watch?v=3FtNm9CgA6U

201 The quote on the cover of this lab is a PSA from when I was a kid. It is probably the most famous PSA of all time. If you are my age you absolutely remember it and many of you that are much younger than may have ever heard about it or seen the add. They have also made follow ups to this add. I bring this up as an example of how powerful communication can be. As a scientifically literate citizen and future health care professional, you will be expected to communicate scientific information regularly. In some cases, this may be as informal as discussing a scientific question or news story with friends, colleagues, or family. In other cases, you may be asked to evaluate scientific information and provide an expert opinion that will inform others and potentially impact personal and/or policy-level decisions. The best scientific communicators are able to immediately read a person they are talking to and tailor their message appropriately such that the audience comes away informed but not confused. While this sounds easy very few people can do this consistently.

For your PSA you will work with a partner to develop a product that communicates scientific information about a specific disease or pathogen to a particular public audience. The disease will be randomly drawn though you can decide who you want to be your target audience (e.g., elementary school children; high school; elderly; health care practitioners; etc). The media that you will use for your project is up to you. Think about attributes that make some PSAs particularly effective. Your goal is to effectively communicate the SCIENCE that underlies the disease/pathogen you have been assigned. You should focus on clearly communicating aspects of your disease such as causative agent, diagnosis, prevention, and treatment strategies for your disease. Are you expected to come up with an add that stays in the human conversation for decades like the one listed on the cover, of course not. You are however expected to give a good faith effort to educate your target audience about the risks associated with your disease.

Learning Objectives:

1. Explore in depth a contemporary, relevant public health issue caused by a microorganism

2. Synthesize information about the issue from a variety of sources

3. Interpret and communicate your findings for a specific audience

4. Work in pairs to enhance project quality

Deadlines: TBA

202 Tips For an Effective PSA

1) Aligning your topic to a target audience In this project, you must clearly articulate to whom your communication is directed and what you're trying to accomplish. Is it reasonable to see how your topic connects to the target audience you've defined? Or, does it appear as though you're making fairly big leap in assuming their interest? Choose the most relevant target audience for your PSA and tailor your communication to their interests.

For example, let’s say hypothetically the state of Indiana had been actively pursuing initiatives to development renewable energies. Evaluating the environmental impacts of this venture qualifies as an interesting and locally relevant topic that has broad appeal. However, if you've decided that your target audience is kindergarteners, then a website with a position paper that evaluates the impacts of wind turbines on migrating bird populations might not be very effective. However, creating a web-based, interactive game that teaches 5 year-olds how wind is just one of many sources of energy that can be used to make power for your house might be more appropriate.

2) Deciding what you want to communicate and how you can make it most effective Spend some time thinking about your goals for this project. Is the media you propose to use going to be effective? Again, consider your population. Suppose your group has the goal of promoting awareness about the environmental impacts of disposable plastic water bottles. You could design an informative poster that you display in your residence hall - depending on the nature of the poster it may be impactful. Alternatively, you could organize a collection of all the plastic water bottles used by dorm residents over the course of a week and arrange them into a "sculpture" that creates a strong visual representation of the quantity of plastic that gets discarded each week by choosing bottled water over tap. This kind of visual may capture their attention more effectively. The options are endless - don't be afraid to be creative!

3) Remember the science! Use scientific evidence to support your position. A well-constructed response to a problem completely and concisely defines and models the scientific and health issues underlying the problem. A good PSA incorporates carefully chosen images and words to clarify and explain succinctly the relevant issues without being misleading. One bad thing that a PSA can do is overplay a hand based on personal bias not scientific evidence. In some cases, authors may choose to advocate for a particular side of a controversial issue but remember a convincing argument will include data and arguments from credible sources and a careful synthesis of the available evidence – including the evidence and arguments from the opposing side. A good debater anticipates the refutation of their argument and pre-empts them with their own rebuttal!

4) What makes a poster/video/pamphlet/etc effective? An effective PSA uses eye-catching, memorable images and/or phrases to establish a personal connection with the audience. The brain on drugs add is a very good example of this. Readers drawn to your PSA will be informed by the use of concise and relevant arguments based on

203 sound scientific principles and information derived from credible sources. Highly effective PSAs inspire readers to take action – especially when the action is simple, achievable, and rational. Lofty, ambiguous, or seemingly impossible goals (e.g., “stop global warming now!”) generally do not motivate individuals toward meaningful change. Getting people to change their behavior is very difficult so it should not be surprising that getting them to change their behavior drastically is even more challenging.

As you prepare your and your classmates PSAs, think about the following questions:

• What did I learn? Did the presenters provide content that taught you something you didn't already know? • How would you rate the quality and quantity of printed text (if present)? Was there so much text that you were discouraged from reading it, or so little that you didn't really grasp the essence of the problem? Too much text is bad in almost any presentation!!!! • Were the images effective? Eye-catching images are great in attracting the reader, but were they relevant to the topic, or possibly misleading? An effective image attracts and informs. • Did the authors present information that was supported with facts from credible sources? Did the authors rigorously research the topic and present coherent information from peer-reviewed journals and credible organizations, or did it seem as if they did a quick internet search and grabbed the first few hits? • Did the authors succeed in making a personal connection with you? Were they able to present a convincing argument that made you care about the topic? • Did the authors succeed in making you feel competent to critically assess the problem? Did they inspire you to want to take action? Did they direct you to resources that you can turn to if you want to learn more?

Each group will have ~8-12 minutes to present their project to the class in lab. Alternatively if you are going to make a video or some kind of performance piece you can videotape it and bring it in to show on the computer in lab. Just make sure we are able to play whatever format you use. Aside from being educational this should be fun!!!!!

Before turning in your project, review the rubric we will use for assessing your project. Ask yourself how you would score your own project in each of the categories.

PSA Grading Rubric (25 points total):

• Medium and content of PSA was appropriate for the target audience (5 points) • PSA employed creative techniques to engage the target audience (5 points) • PSA was carefully produced with no errors (e.g., spelling, video lapse, etc) (5 points) • PSA addressed the specific disease inclusive of diagnosis, treatment and prevention (5 points) • PSA employed accurate and applicable scientific information (5 points) • Note: Plagiarism in any portion of this assignment will result in a score of 0 and possible other academic repercussions. For example, it’s okay when composing a tweet to quote

204 a source, just make sure you give them credit by saying where the quote originated from. Ask your professor for clarification if needed.

205 BIO 113 Public Service Announcements List of Target Diseases/Causative Organism

1. Chickenpox 2. Meningitis 3. Mononucleosis 4. Malaria 5. Shistosomiasis 6. MRSA (Methicillin Resistant Staphylococcus aureus) 7. C – Diff (Clostridium difficile) 8. Tetanus 9. Measles 10. Hepatitis B 11. Tuberculosis 12. Typhoid fever 13. Pneumonia 14. Food poisoning 15. Gonorrhea 16. Syphillis 17. Chlamydia 18. Urinary tract infections 19. HIV/AIDS 20. Influenza 21. Pertussis 22. Lyme disease 23. Zika Virus 24. Ebola 25. Candidiasis

206

Universal Precautions in Action, Specimen Transport

I intend to live forever or die trying.

Groucho Marx

207 Name ______Section______Date______

Pre-Lab Questions:

1. What is the purpose of wearing nitrile exam gloves? Are nitrile exam gloves sterile?

2. What will your GA demonstrate today?

3. What bacteria will you use to demonstrate the importance of hand washing?

208 Background: Is it really important to wash your hands? Does it actually help? Does wearing exam gloves protect people from infections? These questions seem simple but it would probably surprise how difficult it is to consistently apply these concepts in large complicated healthcare facilities such as a hospital.

There is possibly no better place to get sick than a hospital. This statement was not made to be funny although the cleverness that it holds within is intentional. Every year thousands of people get sick from going to the hospital and many more healthcare workers are stricken with illness by being exposed to disease while treating patients. As such it is important that all healthcare works follow protocols that minimize the chance of these infections occurring.

The techniques used to keep a patient’s body fluids from coming into contact with healthcare workers is called body substance isolation (BSI). The use of this and other universal precautions minimize the risk of transmission of disease in hospitals and other healthcare settings. For the sake of your own protection as healthcare workers, when working with a patient’s bodily fluids, it is to be assumed that the patient is infected with hepatitis C, HIV, and influenza. This should encourage you to practice techniques such as washing your hands before and after entering a patient’s room, wear gloves when handling bodily fluids, washing your hands before putting on gloves and after taking them off, and wearing a face shield when there is a potential for aerosolization of body fluids (e.g. when draining an abscess).

Additional protective equipment (e.g. yellow isolation gowns or a respirator) should be worn when the patient is known or suspected to be infected with certain microbes (e.g. MRSA or TB). Additional precautions such as bleaching anything that comes into contact with patients may also need to be adopted if they are known or suspected of having a C. difficile infection. Since needles are capable of puncturing the skin and are often contaminated with bodily fluids, vigilant care should be taken to avoid needle pricks. Needles should be disposed of in biohazard sharps boxes as soon as they have served their purpose.

Nitrile exam gloves protect the wearer from getting possible infectious microbes on their skin. Nitrile gloves are not sterile and therefore do little to protect the patient from acquiring microbes from the caregiver. For this reason, it is critical that caregivers wash their hands prior to working with patients. Although you greatly reduce your risk of getting microbes on your skin when wearing gloves, it is important to be vigilant. Body fluids leak under the gloves at the wrists and gloves can also lose their integrity—especially when working with sharps. After removing your gloves, it is important to wash your hands once more, since microbes may accidently come into contact with your skin.

Surgical gloves are worn over nitrile exam gloves. Unlike nitrile exam gloves, surgical gloves are sterile and come individually wrapped. Only the inside of surgical gloves should be touched to prevent contaminating the outside (which will be touching the patient). The use of surgical gloves and the other tools in a sterile field, helps prevent post-operative infections dramatically.

It is particularly important to maintain BSI precautions when handling body fluids prior, during, and after testing. There are many different types of containers that are used for chemical and microbial tests. When you begin your careers, make sure you familiarize yourself thoroughly with the proper handling of

209 these containers. In this lab we will demonstrate how hand washing can prevent further contamination when working with microbial samples. Materials: MSA plates

Nitrile gloves

Surgical gloves

Biohazard bag

Biohazard sharps container

BSI facemask

Isolation gown

Collection and transport devices

Micrococcus luteus

TSA plates

Test tubes

Cotton tipped applicators

Laboratory Exercise: Use of Nitrile Exam Gloves and Surgical Gloves: 1. Your instructor or lab GA will demonstrate how to properly put on and take off nitrile exam gloves and surgical gloves.

During this lab, you may wear gloves if you wish but do not need to as the microbes we will be working with are harmless.

Other Universal Precautions: 1. Your instructor or lab GA will demonstrate how to handle and properly dispose of sharps (e.g. needs and disposable blades) and other biohazardous materials. 2. Your instructor will demonstrate how to put on a body isolation gown and face mask.

210 Name ______Section______Date______

Devices Used in the Collection and Transport of Clinical Samples: 1. Your instructor or lab GA will demonstrate the proper use of several devices used in the collection and transport of clinical samples.

Handwashing after Handling Samples: Working in Groups of 2 1. Use a cotton tipped applicator to get a sample of Micrococcus luteus from a TSA plate.

2. Wipe this against the side of a test tube.

3. Wait a few minutes to allow the test tube to dry.

4. Handle the test tube with your bare hands. (Micrococcus luteus is a normal inhabitant of the human skin and does not pose a significant health threat to healthy individuals. If you do not want to touch the test tube with your bare hand or you feel this would be dangerous to you, wear a glove during the experiment. To reiterate, wearing gloves is not necessary, but you can if you wish.)

5. Place your fingertips on the TSA plate. (You can divide a plate into four quadrants and work in pairs so each person will get a quadrant for dirty and a quadrant for clean.)

6. Wash your hands thoroughly. (Sing Happy Birthday or the Alphabet twice at a normal pace)

7. Place your fingertips on the clean section of the TSA plate.

8. Be sure to appropriately label your plates.

9. Incubate your plates for 24-48 hours and record the growth on each plate.

211 Name ______Section______Date______Post-Lab Questions:

1. Where did you see bacterial growth?

2. Is handwashing effective? If handwashing was not effective why do you think that this is?

3. Your friend tells you that if you wear sterile surgical gloves and don and doff your gloves appropriately, you don’t need to wash your hands before or after a procedure. Are they wrong? Explain.

4. There was an epidemic of HIV in 2015 in rural Scott County, Indiana. Many of those infected reported heroin addictions. What route of transmission do you suspect spread this virus so quickly?

5. What is the purpose of BSI?

212

Dipstick Urinalysis for the Detection of Urinary Tract Infections (UTIs)

213 Name ______Section______Date______Pre-Lab Questions:

1. Which microbe is the most common cause of UTIs?

2. What types of agar will we be culturing on?

3. What two chemicals will we be analyzing to determine the presence of an infection?

214 Name ______Section______Date______

Background:

You may have heard that urine is sterile and for the most part this is true. In the healthy state, urine should be sterile in the bladder. Urine however, can become contaminated by a small number of bacteria which reside in the urethra. Urination expels most of these bacteria, however when significant numbers of bacteria travel up through the urethra and colonize the bladder they can cause an infection.

More than 3 million urinary tract infections are diagnosed in the United States each year. These infections happen primarily in females but males are susceptible to a lesser degree as well. Symptoms of a UTI in the bladder (cystitis) include pain with urination (dysuria), sudden feeling of need to urinate (urgency), increased urinary frequency, and feeling of incomplete emptying of bladder (vesical tenesmus). When the kidneys are infected (pyelonephritis), symptoms include those of cystitis plus fever, nausea/vomiting, flank pain radiating to the low back pain, and low back tenderness (costovertebral angle [CVA] tenderness). For otherwise healthy patients, UTIs are usually very painful but luckily these infections are usually easily treatable with antibiotics.

Most urinary tract infections are caused by E. coli, a gram negative bacteria. Many Gram negative bacteria are capable of reducing the nitrates normally present in urine into nitrites (see the simplified reaction below). Some Gram positive bacteria, however, are also capable of this. Nitrites are not normally present in urine, so when detected, nitrites in the urine usually indicate a UTI (usually due to E. coli).

- - NO3  NO2 Nitrate reductase Nitrate Nitrite

When other signs/symptoms of a UTI are present, alkaline urine often indicates that the causative agent is a urea-splitting bacterium which is usually Proteus mirabilis or Proteus vulgaris (see the reaction below). Urea-splitting bacteria synthesize urease which converts urea into ammonia (a base). Urea is found in urine because it is a breakdown product of proteins. Alkaline urine can contribute to kidney stone (renal calculi) formation. Alternatively, alkaline urine can be the result of several factors such as alkalosis or a diet high in citrate.

+ H20  2NH3 + CO2 Urease Urea Water Ammonia Carbon Dioxide

215 Name ______Section______Date______Note: the presence of leukocyte esterase (an enzyme produced by neutrophils) in urine can indicate an infection and can be detected by certain urinalysis dipsticks. We will not, however, be testing for leukocyte esterase in this lab.

In this lab, we will be analyzing the urine of a patient who has already provided a sample (this is synthetic urine). After dipstick urinalysis, we will be culturing the urine to confirm a tentative diagnosis relying on dipstick urinalysis.

Materials:

Synthetic urine

McKesson Urine Reagent Strip

Dipstick spectrophotometer

EMB plates

MSA plates

Glass beads

Micropipettes and tips Procedure: Dipstick Urinalysis: 1. Insert a dipstick into each urine sample, ensuring that each testing strip is wetted.

2. Promptly remove the dipstick, wiping the side of the dipstick on the mouth of the cup to remove excess liquid.

3. Place the side of the dipstick on a paper towel to remove excess liquid.

4. Wait the 1-2 minutes for each strip and then analyze the color change of the pads. You can do this by comparing the color of the strips to the side of the dipstick stock bottle.

Culturing:

1. Add 100uL of the urine sample to an EMB and a MSA plate and use beads to spread.

2. Incubate for 24 hours or until next lab period and observe the results.

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Name ______Section______Date______

Post-Lab Questions:

1. Fill out the chart below.

Group pH Nitrites present Results of culture 1 2 3 4 5 6

2. How do you interpret the results of the dipstick urinalysis for each sample? Explain.

3. Are the results from the dipstick urinalysis consistent with the results from each culture? Explain.

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Name ______Section______Date______

4. A patient presents with reoccurring UTIs. She has taken Bactrim and Keflex (two antibiotics), but these don’t seem to be effective. Design an experiment to determine what antibiotics this particular infection is susceptible to.

5. It’s recommended that parents wipe children in diapers from front to back. Why is this?

6. When a person’s blood sugar raises above approximately 160-180mg/dL, the kidneys are unable to remove all of the sugar from the filtrate resulting in glycosuria (sugar in the urine). Why might uncontrolled diabetics be at a higher risk for UTIs?

218

Bacteria of the Gastrointestinal (GI) Tract

219 Name ______Section______Date______

Pre-Lab Questions:

1. Which media will we be using in this lab?

2. What happens to the pH of a media when a bacterium ferments? What happens when it oxidizes amino acids? What color will phenol red be in these cases.

3. What type of bacteria will grow on MacConkey agar?

220 Name ______Section______Date______

Introduction:

Bacteria in our food, mouths, and mucus travel down the esophagus into the stomach. The vast majority of these bacteria are killed by the sudden drop in pH due to the hydrochloric acid in the stomach. Partially digested food then passes from the stomach into the small intestine where the pH suddenly becomes very alkaline. The small intestine is relatively narrow and so food moves through it at a relatively rapid pace. The small intestine then feeds into the large intestine which has a much larger diameter and more favorable environment for microbes. The many commensal bacteria in the large intestine compete with would-be pathogens and even provide us with vitamins like vitamin K. There are a dizzying number of bacterial species that can reside in the human intestines. Different people have unique compositions of bacteria and these compositions vary throughout the lifespan depending on numerous known and unknown factors.

There are many ways to determine the presence and biochemical properties of fecal bacteria in a sample. We will be utilizing MacConkey agar, eosin-methylene blue, and triple sugar iron agar.

As you may recall, MacConkey agar (MA) is both a selective and differential media. MA contains crystal violet which inhibits Gram positive bacteria, but allows for the growth of Gram negative bacteria (selective). MA contains the pH indicator neutral red which is red under acidic conditions and yellow under alkaline conditions. MA also contains lactose. Coliform bacteria (lactose fermenting, Gram negative bacilli), such as E. coli, Citrobacter and Enterobacter, produce acid as a byproduct of lactose fermentation. Non-coliform enteric bacteria, such as Salmonella typhimurium, can grow on MA, but do not ferment lactose. As a result, coliform bacteria will change the media red and non-coliform enteric bacteria will not change the color of the media (differential). Remember, Gram positive bacteria will not grow on MA (selective).

As you may recall, eosin-methylene blue agar (EMB) is also both a selective and differential media. EMB contains eosin Y and methylene blue pH indicator dyes which inhibit the growth of Gram positive bacteria, but permit the growth of Gram negative bacteria (selective). Eosin Y and methylene blue pH indicator dyes are dark purple under acidic conditions and are red under neutral and alkaline conditions. Non-lactose fermenters will appear red. Lactose fermenters will appear purple. E. coli will have dark metallic-green growth.

Triple sugar iron (TSI) agar is poured at a slant in test tubes and is a differential media. TSI contains phenol red, lactose, sucrose, a small amount of glucose, amino acids, and iron. Phenol red is a pH indicator that turns yellow under acidic conditions (indicating fermentation of sugars) and is red under neutral or alkaline conditions. If the organism is capable of fermenting lactose or sucrose, the bottom and slant of the agar will be yellow. If the organism is capable of fermenting glucose only, the bottom and the slant will be yellow for a time, but since there is a limited amount of glucose, the bacteria will have to turn to amino acid oxidation for energy. Amino acid oxidation makes the pH alkaline and can only occur where there is oxidation (on the slant but not on the bottom). So, if a bacteria can ferment glucose only, the slant will remain yellow at the bottom, but will be red on the slant. Some bacteria can

221 desulfurate (remove the sulfur groups from two amino acids and use it for energy) amino acids which produces hydrogen sulfide (H2S). Hydrogen sulfide reacts with iron forming a black compound. So if the agar appears black, the bacteria is capable of desulfuration and forms hydrogen sulfide. If the bacteria produces a gas, there will be bubbles in the agar and the bottom of the agar will be pushed up from the glass.

In this lab, we will collect single colonies from a fecal sample and then utilize EMB, MacConkey, and TSI to help identify some bacteria within it. Our fecal sample will come from a dog or a baby. The week prior to lab, your instructor or lab GA will provide more instructions on who will provide the samples.

Materials: Fecal sample MacConkey agar Eosin methylene blue agar Triple sugar iron agar Inoculating loop Inoculating needle

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Laboratory Exercise: In Groups of 4

Be sure to wear gloves and wash your hands after this lab. Be mindful of things you are touching. Day 1: Use the streak plate technique to isolate single colonies of bacteria from the fecal sample on MacConkey agar. (You shouldn’t need very much sample. Remember there are billions of bacteria in feces and we are looking for single colonies.)

label First Streak

Second Streak label label Third Streak

label Fourth Streak

Incubate over night or until next lab period at 37 degrees.

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Name ______Section______Date______Day 2: After incubation, hopefully we should have single colonies.

1. Divide your EMB and MacConkey plates into four quadrants.

2. Use your inoculating loop to gather bacteria from single colonies.

3. Next, streak these onto a quadrant on MacConkey agar and EMB. Be sure to flame your loop in between inoculations. Repeat this, using a new single colony for each quadrant.

4. Collect single colonies on the tip of your inoculating probe. Stab your inoculating probe straight down into your TSI agar.

5. Be sure to label your samples.

6. Incubate for 24-48 hours. Post-Lab Questions:

1. What results did you obtain from your MacConkey tests? What can you determine about the bacteria you tested from this? (Feel free to draw your results if appropriate)

2. What results did you obtain from your EMB tests? What can you determine about the bacteria you tested from this? (Feel free to draw your results if appropriate)

224

Name ______Section______Date______

3. What results did you obtain from your TSI tests? What can you determine about the bacteria you tested from this?

4. Do you believe we cultured all of the different types of bacteria present in the fecal sample? Why or why not?

5. Why doesn’t amino acid oxidation occur at the bottom of the TSI tube?

225

Name ______Section______Date______

6. Why wouldn’t we be able to determine if an organism were capable of fermenting glucose if we poured TSI into a petri dish?

7. If a bacteria produces hydrogen sulfide, will you be able to accurately determine if it can ferment lactose or sucrose? Explain.

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Have a happy healthy break!

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