2011 Peabody Museum of Natural History, Yale University. All rights reserved.

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1 REMEMBER - FOLLOW THE LAB PROCEDURE EXACTLY AND HAVE STUDENTS POUR THE EASYGEL BEFORE GETTING THEIR SPICES!! YOU MUST DO THIS FIRST OR THE GEL WILL NOT HARDEN IN TIME TO ADD SPICES THAT STAY IN PLACE! 2011 Peabody Museum of Natural History, Yale University. All rights reserved.

2 *Order ciprofloxacin antibiotic discs and a Sensi-disc dispenser from *EasyGel can be ordered from the manufacturer,

3 Spicy Inhibitors Background Plants have been used as medicine from the beginning of human history. Human beings coevolved with other species including plants. Many chemicals in plants will react in some way with our bodies. A person can have a harmful reaction like the rash caused by touching poison ivy. A helpful reaction might be pain relief from the medical use of aspirin. Did you know that aspirin comes from the bark of the willow tree? Plants can also have an effect on other living things. For example, pyrethrin (say, pie-ree-thrun) is made from chrysanthemum flowers. It is used as an insect repellent for garden pests and won t harm the environment. In many countries people still use medicines that are not made by a drug company. These are called traditional or folk medicines. Most of them are made with plants or plant parts. But even modern medicines from drug companies rely on plants. Almost one in four prescription medicines uses chemicals from plants. These include heart medicines, pain relievers, and antibiotics (drugs that kill bacteria). Other medicines from plants fight cancer and inflammation (swelling and fever). Many of these medicines were discovered by first studying their uses in traditional medicine. Today, we are still finding uses for plants. The bark of the Pacific yew tree was recently found to be the best medicine to fight breast cancer, a disease that kills thousands of women each year. People also use plants for nutrition or to add flavor to foods. Corn, wheat, carrots, potatoes, spinach, and many other plants are eaten as food. Oregano, basil, garlic, and other flavorful plants called spices are used to add flavor to foods. People living in warm tropical regions tend to use more of these spices than people living in cooler regions. Why is that? Since bacteria grow faster at high temperatures, food spoilage is more of a problem in the tropics. Spices not only have strong odors and flavors, some of them can stop bacteria from growing and protect food from spoiling. Trial and error over many centuries has shown which spices are best at that. Garlic is one plant that has been shown to have strong antibacterial action. Because of that, garlic has been used for many centuries and by many cultures as food, spice, and even as medicine. Procedure You are an ethnobotanist. You study how people use plants as food and medicine. Lyme disease is caused by bacteria. This means that it can be treated with antibiotic medicine. The XYZ Pharmaceuticals Company wants to make a new antibiotic to treat Lyme disease. They have asked you to test plants that can control bacterial growth. Creating a new medicine usually takes 10 to 15 years of research. It often costs hundreds of millions of dollars. You will conduct an experiment as a first step in the process.

4 You have decided that your first step is to examine the effects of some spices on bacteria. You will grow bacteria on a Petri dish and add spices to see what happens. You and your team will test garlic, a common spice. Your team will also choose a second spice to compare with the garlic. You may also choose to include an antibiotic disk. This will compare a prescription medicine to a spice. Your lab supervisor (your teacher) will help you decide what is needed. Materials (per group) 1 EasyGel nutrient bottle and 1 EasyGel Petri dish Sterile swabs Wax pencil or marker Alcohol wipe Rubber gloves Ruler Forceps o.o5 ml Measuring scoop Shared Materials: Escherichia coli (E. coli) in nutrient broth Garlic powder Ground cinnamon Whole cloves Ground red pepper Optional materials: Antibiotic disks and disk dispenser Incubator or insulated cooler Lab Procedure: NOTE: Ethnobotanists do not usually work with bacteria. You must review the Sterile Technique instructions on page Wipe your work area with an alcohol wipe and let it dry. 2. Hold your Petri dish closed, with both parts together. Flip it upside down so you see the bottom (the smaller part). You will only mark the bottom of the dish. 3. Divide your dish into sections for each spice. You may also use an antibiotic disk in the same dish. Remember to have a section for your control area. 4. Mark the bottom of the Petri dish with wax pencil to show: o The spices used and where they will be placed G = Garlic, Cn = Cinnamon, Cl = Clove, P = Pepper o The control area C = Control o The group name or initials G C 5. Take the bottle of EasyGel nutrient and loosen the top. This is important: DO NOT remove the top until you are ready to pour it out. When you open the bottle, you might add other bacteria that can ruin your experiment.

5 6. Take a culture tube of bacteria and loosen the top. DO NOT remove it until you are ready to use it. 7. Carefully open a sterile swab at the stick end. Do not touch the cotton tip. Dip the cotton tip into a culture tube of bacteria. Carefully remove the swab and place it gently into the bottle of EasyGel nutrient. Replace the top of the EasyGel bottle and gently swirl it around to mix the bacteria. Be very gentle to avoid creating bubbles in the nutrient. 8. Loosen the top of the EasyGel nutrient again. Hold the top (the bigger part) of the Petri dish, but do not open it yet. Lift the top just enough to pour the EasyGel bottle into the bottom (the smaller part). Do not get too close and do not breathe into the open dish. This can contaminate the sterile field and ruin the experiment. 9. Pour all of the liquid from the bottle into the bottom of the dish (the smaller part). Swirl it gently so the entire surface is covered without any empty spaces. Do not touch or move the dish while the nutrient is hardening. The dish must stay flat and untouched for at least 15 minutes. 10. While you are waiting for the nutrient to harden you will get your spices. Remember to use sterile technique! Use a scoop to remove powdered spices from a jar. Fill the scoop completely with powdered spice. Do not make the scoop too full. Use a sterile swab (the stick part) to scrape off extra spice. The scoop must be level for an accurate measurement. WARNING: DO NOT TOUCH THE SPICES DIRECTLY! WEAR RUBBER GLOVES AND BE SURE TO TURN THE GLOVES INSIDE OUT AS YOU REMOVE THEM. THROW THEM AWAY IMMEDIATELY. IF YOU GET SPICE IN YOUR EYES, GO AT ONCE TO THE EYEWASH SINK. 11. Once the nutrient is almost solid, place spices or antibiotic disks on the dish. Do your best to place each one in the center of its section. This will give you the best way to see the antibiotic effects. As soon as everything is in place, cover the dish again. 12. Your lab supervisor will collect the dishes. For fastest results they can be placed into an incubator at 36 o C or an insulated cooler placed in a warm spot. Several dishes can be stacked on top of each other. ***They must be stored upside down, with the bottom (smaller part) facing up.

6 13. Check your dish after hours. Hold it up to the light for best viewing. DO NOT OPEN THE DISH. Are there small dots on the surface? Is there a color change? Do you see any clear spaces? These are zones of inhibition, where the bacteria were inhibited from growing. Refer to the sheet on Zones of Inhibition on page Record your findings in the data chart from XYZ Pharmaceuticals. Measure the zone of inhibition - the clear space that has no bacteria. Refer to the sheet on Zone of Inhibition for measuring instructions. You may also record what you observe in your notebooks. You and the lab supervisor will determine which data to record. 15. Prepare a brief report of your findings for XYZ Pharmaceuticals Peabody Museum of Natural History, Yale University. All rights reserved.

7 Discovery File Sterile Technique Have you ever used an antiseptic spray or ointment on a cut? Did you know that the word sepsis is a medical word for an infection caused by pathogens or the toxins they can make? Anti- means against, so using an antiseptic means you are fighting those pathogens. When scientists grow bacteria on Petri dishes, they only grow the bacteria they want to study. They are careful to keep the Petri dish sterile. This means that the dish is free from any microorganisms. The scientist will then place the bacteria to be studied onto the Petri dish, so it can grow by itself. Because bacteria are all around us, this can be a bit tricky. Luckily, scientists and doctors have figured out a method to keep most bacteria and other pathogens from getting into places they aren t wanted. This is called sterile technique, because it is a way to keeps things as sterile as possible. It is also the method nurses and doctors follow during surgery, so another name for it is surgical asepsis. Surgical asepsis is used in the operating room, delivery room, during surgical procedures, and when bandages are changed. When you follow sterile technique, you must pay close attention to the way you handle items. These rules will keep an area free from all microorganisms. An object or area is described as sterile or not sterile. Basic rules of sterile technique include: Only a sterile object can touch another sterile object. Open sterile packages carefully. Be sure to avoid the possibility of the inner surface of a sterile wrapper touching a non-sterile area. Avoid spilling any solution on a cloth or paper used as a field for a sterile set-up. Hold sterile objects above the level of the waist. If you can t see it, you might contaminate it. Avoid talking, coughing, sneezing, or reaching over a sterile field or object. Use dry, sterile forceps when necessary. Consider the edge (outer 1 inch) of a sterile field to be contaminated. Consider an object contaminated if you have any doubt whether it is sterile.

8 Zone of Inhibition Petri dish Zone of Inhibition Antibiotic spice Bacteria This is a drawing of a Petri dish with bacteria growing on it. Bacteria are shown as the circular pattern of dots in the dish. A small black circle in the upper right section of the Petri dish shows the antibiotic spice or medicine. Notice the space around the antibiotic. It is different. No bacteria are growing there. This area is called the zone of inhibition sometimes written as ZOI. A zone of inhibition is seen wherever bacteria are inhibited from growing. A ZOI is measured by placing a ruler across the empty circle at its widest point. Below are ZOI drawings showing incorrect and correct measurements. Incorrect 2.8 cm Correct 3.3 cm

9 XYZ Pharmaceuticals To: Subject: Data: Ethnobotany research groups Report on using spices to inhibit bacteria Zones of Inhibition measurements Please complete this data table. Include Zone of Inhibition measurements, and any qualitative observations you have made about your results. hrs. growth hrs. growth hrs. growth Area 1 Spice: Antibiotic: Area 2 Spice: Antibiotic: Area 3 Spice: Antibiotic: Area 4 Spice: Antibiotic: Additional Notes and observations: XYZ Pharmaceuticals Official Laboratory Report Form #2837 (Revised 8/2007)

10 Discovery File Antibiotics Antibiotics are chemicals that slow down or sometimes stop the growth of bacteria. They are called antibiotics because they fight against (anti-) living (-biotic) organisms. Antibiotics are not effective against viruses. How do they do it? Different antibiotics stop the growth of bacteria in different ways. Some: kill bacteria by destroying their cell walls or cell membranes stop bacteria from reproducing interfere with protein making Where do antibiotics come from? The chemicals we call antibiotics are released by molds and some other living things. Some ancient cultures Chinese, Egyptian, and Greek used molds and plants to treat infections as long as 2,500 years ago. But our story starts in the 1920s when a world-changing drug was discovered by accident! In 1928, Sir Alexander Fleming was studying a bacterium that causes a common form of food poisoning. After returning from a vacation, Fleming noticed that mold was growing on some of his culture plates. He was ready to throw the moldy plates away but then he stopped. Around the mold the plate was a clear area where the mold had stopped the bacteria from growing. Scientists call this a zone of inhibition (see Zone of Inhibition on page 87). If mold was able to stop bacteria on a plate, maybe it could do the same in the body. Fleming worked on this problem until he found the chemical that was stopping the bacteria from growing. He called it penicillin, after the Latin name of the mold. Before penicillin, many people died from all kinds of infections. After its discovery countless lives were saved. And many scientists thought that antibiotics would get rid of infectious disease forever. They were wrong! How do we know? See the Discovery File: Bacteria Fight Back on page 90.

11 Discovery File Bacteria Fight Back After years of working well, some antibiotic medications are failing. Their regular doses no longer cure the diseases they once cured. Are antibiotics losing their strength? We know the answer bacteria are fighting back. They are adapting. In every species there is variation. Humans differ in height, eye color, skin color, and many other ways. Bacteria of the same species differ, too. One way is in their sensitivity to antibiotics. When a patient with a bacterial disease takes an antibiotic, the bacteria stopped first are the ones most sensitive to it. If the patient doesn t take the antibiotic long enough or in a strong enough dose, the less sensitive bacteria survive. One bacterial cell can divide into two new cells every 20 minutes. At that rate, one cell can become millions in a few hours (see chart). Soon the bacteria that are hardest to stop take over. Over time, the whole population becomes antibiotic resistant. By 1984, half of the people with active tuberculosis in the United States had a strain that resisted at least one antibiotic. Scientists are always searching for new antibiotics and new mixtures of antibiotics. This battle will probably never end but you can help. Hours Cells ,024 2, ,096 8,192 16, ,768 65, , , ,288 1,048, ,097,152 4,194,304 8,388, ,777,216 33,554,432 67,108, ,217, ,435, ,870, ,073,741,824 Always take your antibiotics exactly in the dosage prescribed. And just because you start to feel better, don t stop taking them.

12 Discovery File Human Clinical Trials Do you remember the last time you had to take medicine? How about the last time you were vaccinated or had a booster shot? Have you ever thought about how medicines and vaccines are made? Developing a new vaccine or a new medicine takes a long time. Scientists must follow many steps in a process to make sure they work and also do no harm. There are government agencies that check each step of the way before a new treatment can be approved. The last step is the biggest! After a new medicine or vaccine is proven safe and effective with animals, it s time for the last step in the process human clinical trials. During human clinical trials, a new vaccine or medicine is given to people for the first time. The research scientists who are developing the new medicine ask doctors to test it on volunteers. The IRB But it is not just up to the researchers and doctors to round up a group of volunteers. Before a human clinical trial also called a study can begin, a study plan must be approved. That approval is given by an Institutional Review Board, or IRB. An IRB can be set up by a hospital, a university, or even the health department of a town. Not everyone who is on the IRB is a doctor. In fact, an IRB is made up of people who work in all kinds of jobs: nurses, scientists, and pharmacists. There are even people who work outside of science, like social workers, ministers, rabbis, teachers, and other community members. The members of an IRB share their expertise and opinions about any clinical study that they are asked to review. They discuss many questions about the study plan and whether it keeps the volunteers safe. Sometimes, to have as much information as possible, the IRB may invite other experts to speak with them about how a human clinical trial will work, or why it is set up in a certain way. An IRB can even send a study plan back to the researchers and doctors, and ask them to make changes the IRB thinks are necessary to improve the study. The IRB has a lot of power. A study can only begin if the IRB is sure that the human volunteers will be protected. Informed Consent Before the volunteers are allowed to participate, they must sign a special document called an informed consent form. The name of the form tells us the two important things that it does:

13 1. It informs (tells) the person who is volunteering what the test is about, what will happen, and whether there are any risks 2. More importantly, by signing the form the volunteer gives consent. That means that he or she agrees to take part in the study and that no one is forcing him or her to participate. It is the IRB s job to make sure the informed consent form clearly describes the study and lists all of its risks and benefits. The informed consent form must be written in language that most people can understand. It also must include a section that tells volunteers that they can leave the study at any time. The IRB must make sure that no one feels forced to be in or stay in a study. Once an IRB has looked at all of these things, the Phase I trial of the medicine or vaccine can begin. How much should a person take? In Phase I, researchers observe a small group of volunteers to see whether the vaccine or medicine is safe and how large the dose should be. To do this, a few patients are given a very small dose. If all goes well, the next group gets a slightly higher dose. The dose is gradually increased from group to group with the researchers monitoring everything. They are looking for the dosage that produces the best results with the fewest side effects. When researchers think they have enough data for Phase I, the study goes back to the IRB for approval of Phase II. Side effects are the negative actions of a medicine or vaccine. The term usually refers to undesired or negative effects, such as headache, rashes, nausea, or liver damage. There can be both immediate and longterm side effects. The IRB examines the data collected in Phase I and decides whether the study can continue. The IRB may tell the researchers they need more data. They may decide to stop the study altogether because there are too many side effects. Sometimes the IRB will tell researchers that the informed consent form must be changed to list new side effects or risks found in Phase I. Once the IRB gives approval for Phase II, researchers test and observe a larger group of volunteers who take a full dose of the medicine. During Phase II some of the volunteers are given a placebo (say pluh-see-bo). Phase II studies lead to Phase III. All along the way the IRB monitors everything. A placebo is a medical treatment that has no value and no side effects. It is given to volunteers as a comparison to make sure that the new medicine or vaccine being tested does a better job than nothing at all. Finally, after years of successful human clinical trials, a new medicine or vaccine can be approved by the U.S. Food and Drug Administration (FDA). Then it can be used by people everywhere in the country. Phase III Trials clinical trials usually involve several hundred human volunteers. In Phase III a vaccine or medicine is compared with the standard treatment. The purpose is to gather evidence that may lead to a change in the recommended treatment Peabody Museum of Natural History, Yale University. All rights reserved.