Oxygen Requirements and Spice Tolerance

1. Oxygen Requirements and Spice Tolerance IF YOU CAN SEE THIS MESSAGE YOU ARE NOT IN “SLIDE SHOW” MODE. PERFOMING THE LAB IN THIS MODE WILL NOT ALLOW FOR THE ANIMATIONS AND INTERACTIVITY OF THE EXERCISE TO WORK PROPERLY. TO CHANGE TO “SLIDE SHOW” MODE YOU CAN CLICK ON “VIEW” AT THE TOP OF THE PAGE AND SELECT “SLIDE SHOW” FROM THE PULL DOWN MENU. YOU CAN ALSO JUST HIT THE “F5” KEY. Instructor Terry Wiseth

2. Click on the blackboard to view a larger board for discussion. Incubator 370C Agar Plates Swabs Loops Antiseptic Dispenser Microbe Samples Loops Pencil Bunsen burner

3. Refrigerator Freezer Incubator Incubator Incubator 00C -100C 350C 500C 1000C SPICE TOLERANCE AND OXYGEN REQUIREMENTS IN MICROBES There are two parts to this lab. Part one will investigate the tolerance different microbes have to various spices. Part two will investigate the oxygen requirements various microbes exhibit. Using the links given below choose the lab you want to perform by Clicking on the link. If you have performed both of the labs you can Click on “END LAB”. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Spice SensitivityVirtual Lab OxygenRequirementsVirtual Lab END LAB

4. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C SPICE TOLERANCE IN MICROBES In contemporary western culture, we have come to rely on chemical preservatives to prevent food spoilage and antibiotics to treat bacterial diseases. While the use of these has helped to diminish the impact of many common diseases, many pathogens have evolved resistance to some or all of the known antibiotics. The source for our arsenal of antibacterial drugs has traditionally been the natural world of fungi, plants, and bacteria. As we see more resistant strains of bacteria evolve, we must turn to that source again for potential new drugs. Food poisoning affects millions of people each year. Spices have been used for centuries to flavor food while killing selective bacteria. For example, spices used in making sausages will inhibit bacteria that cause spoilage while not harming the bacteria that add flavor to the sausage. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

5. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C Long before the Crusaders introduced the fragrance and taste of the East to the unsophisticated European cuisine, people in India, Asia, and the Far East were trading spices. One of Christopher Columbus's goals in setting sail for India was to bring back pepper, cinnamon, and other flavorful dried herbs, for Europe did not supply the need of its populations for them. Not only do spices enhance the taste of food, the aroma of them arouses the appetite. Spices themselves provide many trace minerals which some populations, especially impoverished or vegetarian ones, depend on to maintain health. History tells us that the European hunt for pepper, ginger, and other "hot" spices grew out of a need to preserve meat, or to disguise the taste of meat which had gotten a little to old to be pleasant by itself. Many herbs and spices MAY have antimicrobial activity:  thyme, clove, cinnamon, garlic, ginger, chamomile, oregano, sage,  echinacea, wasabi, etc.  We will be testing clove oil and various antimicrobial chemicals against different bacteria. Many of the medicines used to treat a variety of ailments are produced naturally by a diverse assortment of organisms including many plants, fungi, bacteria and animals. The use of naturally occurring medicines is not new. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

6. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C Traditional cultures throughout human history have used naturally occurring medicines. There is even some evidence that non-human animals use plants medicinally. It is estimated that in many developing countries, 85% of health problems are treated with plant-derived medicines. In the United States, roughly 25% of all prescription drugs contain compounds extracted directly from plants. Currently, we have the technology and knowledge necessary to synthesize many chemical compounds in our search for new medicines. However, there are many chemical compounds found in nature that we cannot synthesize. Moreover, a vast number of plant species (250,000+) have never been screened for potential medicinal value. Many have never even been described. Much of the planet’s untapped reserve of biological diversity is in the tropics. However, most of the world’s money for collecting and testing species for useful chemical compounds is in the hands of corporations in North America, Europe, and Australia. Hence, many pharmaceutical companies and tropical countries have forged agreements to put these two resources together.Needless to say, testing the many biological samples collected for bioactive compounds is a time and resource consuming process. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

7. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C In order to narrow the search, chemical prospectors use various criteria to select the species, which are most likely to have useful compounds. Fans of hot, spicy cuisine can thank nasty bacteria and other food-borne pathogens for the recipes that come, not so coincidentally, from countries with hot climates. Cornell University biologists have demonstrated humans' use of antimicrobial spices developed in parallel with food-spoilage microorganisms. The same chemical compounds that protect the spiciest spice plants from their natural enemies are at work today in foods from parts of the world where, before refrigeration, food-spoilage microbes were an even more serious threat to human health and survival than they are today. "The proximate reason for spice use obviously is to enhance food palatability," says Sherman, an evolutionary biologist and professor of neurobiology and behavior at Cornell. "But why do spices taste good? Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

8. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C Traits that are beneficial are transmitted both culturally and genetically, and that includes taste receptors in our mouths and our taste for certain flavors. People who enjoyed food with antibacterial spices probably were healthier, especially in hot climates. They lived longer and left more offspring. And they taught their offspring and others: 'This is how to cook a mastodon.' We believe the ultimate reason for using spices is to kill food-borne bacteria and fungi.“ Sherman credits Billing, a Cornell undergraduate student of biology at the time of the research, with compiling many of the data required to make the microbe-spice connection: More than 4,570 recipes from 93 cookbooks representing traditional, meat-based cuisines of 36 countries; the temperature and precipitation levels of each country; the horticultural ranges of 43 spice plants; and the antibacterial properties of each spice. Garlic, onion, allspice and oregano, for example, were found to be the best all-around bacteria killers (they kill everything), followed by thyme, cinnamon, tarragon and cumin (any of which kill up to 80 percent of bacteria). Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

9. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C Capsicums, including chilies and other hot peppers, are in the middle of the antimicrobial pack (killing or inhibiting up to 75 percent of bacteria), while pepper of the white or black variety inhibits 25 percent of bacteria, as do ginger, anise seed, celery seed and the juices of lemons and limes. The Cornell researchers report in the article, "Countries with hotter climates used spices more frequently than countries with cooler climates. Indeed, in hot countries nearly every meat-based recipe calls for at least one spice, and most include many spices, especially the potent spices, whereas in cooler counties substantial fractions of dishes are prepared without spices, or with just a few." As a result, the estimated fraction of food-spoilage bacteria inhibited by the spices in each recipe is greater in hot than in cold climates. Accordingly, countries like Thailand, the Philippines, India and Malaysia are at the top of the hot climate-hot food list, while Sweden, Finland and Norway are at the bottom. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

10. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C The United States and China are somewhere in the middle, although the Cornell researchers studied these two countries' cuisines by region and found significant latitude-related correlations. Which helps explain why crawfish etoufée is spicier than New England clam chowder. The biologists did consider several alternative explanations for spice use and discounted all but one. The problem with the "eat-to-sweat" hypothesis, that people in steamy places eat spicy food to cool down with perspiration, is that not all spices make people sweat, Sherman says, "and there are better ways to cool down -- like moving into the shade." The idea that people use spices to disguise the taste of spoiled food, he says, "ignores the health dangers of ingesting spoiled food." And people probably aren't eating spices for their nutritive value, the biologist says, because the same macronutrients are available in similar amounts in common vegetables, which are eaten in much greater quantities. However the micronutrient hypothesis, that spices provide trace amounts of anti-oxidants or other chemicals to aid digestion, could be true and still not exclude the antimicrobial explanation, Sherman says. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

11. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C However, this hypothesis does not explain why people in hot climates need more micro-nutrients, he adds. The antimicrobial hypothesis does explain this. Sherman maintains, eating is definitely one of the more social behaviors of humans, and it's a good way to see the interaction between cultural evolution and biological function. "I believe that recipes are a record of the history of the co-evolutionary race between us and our parasites. The microbes are competing with us for the same food," Sherman says. "Everything we do with food; drying, cooking, smoking, salting or adding spices, is an attempt to keep from being poisoned by our microscopic competitors. They're constantly mutating and evolving to stay ahead of us. One way we reduce food-borne illnesses is to add another spice to the recipe. Of course that makes the food taste different, and the people who learn to like the new taste are healthier for it." Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

12. Refrigerator Freezer Incubator Incubator Incubator 00C -100C 500C 350C 1000C Spice Sensitivity in Microbes In this lab you will be performing two experiments. The first experiment will investigate various spice tolerances of a bacteria cultures. The second experiment will investigate various spice tolerances of a yeast culture.Use Table 1 Spice Sensitivity to record your results.Click on the Spice Sensitivity Virtual Lab link below.If you have performed the experiment you can Click on “END LAB” below. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Spice SensitivityVirtual Lab Print Table 1SpiceSensitivity END LAB

13. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C • We will be testing two different types of microbial cultures for sensitivity to six different spices. The cultures we will be using will be a yeast (Saccromyces) culture and a bacterial (Salmonella) cultures. • The types of spices used will be: • Garlic • OreganoThyme Cinnamon • MintPepper Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

14. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C In this experiment we will utilize two agar plates which contain the necessary nutrients and moisture necessary for the collected cultures to survive and reproduce. In order to introduce the microbes to the sterile agar plates we will transfer the bacteria from the pure culture tubes to the agar plates. Once the plates are inoculated we will introduce the discs containing the various spices to the surface of the agar. Next, we will incubate the plates at 37 OC for 24 hours. An incubator will be used to house these plates so the temperature and moisture will remain stable during the culture time. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

15. Refrigerator Freezer Incubator Incubator Incubator 00C -100C 350C 500C 1000C Click on one of the sample links listed below to introduce the microbes from the broth culture to the agar plates. If you are finished with both experiments you can click on “END LAB” Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Yeast Culture Bacterial Culture END LAB

16. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C OXYGEN REQUIREMENTS FOR MICROBES Anaerobic bacteria occur both in the environment and as normal inhabitants of humans and other animals, and can cause disease in 2 ways. A person can become ill either by ingesting toxins produced by anaerobic bacteria (as occurs in botulism food poisoning), or by infection with the organism itself. Infection, which causes most disease, occurs in 3 ways. First, anaerobic infections can occur when an anatomic barrier is compromised, thus allowing normal anaerobic flora to enter a sterile site. This can happen either when a physical barrier is broken during surgery or other trauma, or when other host defenses are weakened by malignancy, diabetes, burns, immunosuppressive therapy, or aspiration. Infections caused in this manner are termed endogenous infections. Second, infections can result when anaerobic bacteria native to the environment contaminate wounds; these are termed exogenous infections. Clostridium species cause most exogenous infections. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

17. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C Finally, infections by anaerobic bacteria can spread through person-to-person contact. For example, nosocomial infections caused by Clostridium difficile are transmitted person-to-person. An anaerobic bacteria culture is a method used to grow anaerobes from a clinical specimen. Obligate anaerobes are bacteria that can live only in the absence of oxygen. Obligate anaerobes are destroyed when exposed to the atmosphere for as briefly as 10 minutes. Anaerobes that are tolerant to small amounts of oxygen are called microaerophilic. Facultative anaerobes are those organisms that will grow with or without oxygen. Anaerobic bacterial cultures are performed to identify bacteria that grow only in the absence of oxygen and which may cause human infection. If overlooked, anaerobic infections result in such serious consequences as amputation, organ failure, sepsis, meningitis, and death. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

18. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C Some types of specimens should always be cultured for anaerobes if an infection is suspected. These include abscesses, bites, blood, cerebrospinal fluid and exudative body fluids, deep wounds, and dead tissues. The collected specimen must be protected from oxygen during collection and transport. Anaerobes are normally found within certain areas of the body but result in serious infection when they have access to a normally sterile body fluid or deep tissue that is poorly oxygenated. Some anaerobes normally live in the crevices of the skin, in the nose, mouth, throat, intestine, and vagina. Injury to these tissues (i.e., cuts, puncture wounds, or trauma) especially at or adjacent to the mucous membranes allows anaerobes entry into otherwise sterile areas of the body and is the primary cause of anaerobic infection. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

19. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C A second source of anaerobic infection occurs from the introduction of spores into a normally sterile site. Spore-producing anaerobes live in the soil and water, and spores may be introduced via wounds, especially punctures. Anaerobic infections are most likely to be found in persons who are immunosuppressed, those treated recently with broad-spectrum antibiotics, and persons who have a decaying tissue injury on or near a mucous membrane. Some bacteria have an absolute requirement for oxygen. These are the obligate aerobes. Others, the facultative anaerobes can survive in the absence as well as the presence of oxygen. The obligate anaerobes are killed by traces of oxygen. A small group of bacteria are killed by normal atmospheric levels of oxygen, but yet require traces of oxygen to grow. These are the referred to as microaerophiles. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

20. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C Agar Plates The culture on the far left is an obligate aerobe that can only grow at the surface of the culture medium. Next is a microaerophile. It can only grow where the oxygen concentration is low. Then follows a facultative anaerobe, which is indifferent to the presence of oxygen. The culture on the far right is an obligate anaerobe, unable to grow in the presence of oxygen. pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

21. Refrigerator Freezer Incubator Incubator Incubator 00C -100C 350C 500C 1000C We will be using 4 different types of bacteria in performing this experiment. Our goal will be to determine what type of oxygen requirements each of the bacteria types exhibit. In order to find the requirements we will be using test tubes which have been filled with agar (the same material found in agar plates). When these tubes are warmed (above 45 degrees Celsius) the agar becomes a fluid. In this fluid state the agar will mix readily with a sample of broth culture containing the bacteria culture. Upon cooling the agar will solidify into a gel. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 The agar gel once solidified (semi-gel) becomes less permeable to oxygen. Oxygen will only permeate into the top 10% of the agar (the portion exposed to the air). The portion of the agar gel below this 10% will exhibit an area which contains no oxygen. Bacteria growing in the top 10% and the surface would be classified as aerobic while bacteria culturing below the 10% zone would be classified as anaerobic.

22. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C • The types of bacteria used will be as follows: • Clostridium botulinumCorynebacterium diphtheriaeNeisseria gonorrhoeaeHelicobacter pylori • Depending on where we find these cultures growing in the tubes after inoculation and incubation, we will be able to classify each bacteria as: Obligate anaerobe, Facultative anaerobe, Obligate aerobe, Microaerophile. Use the descriptions given on the previous page to determine what oxygen requirements are exhibited by each bacteria. Enter your analysis in Table 2. Add any comments for each of the bacteria cultures in Table 2 as well. You will also need to produce a sketch of each of the bacteria cultures indicating the locations of the zones of growth for each culture. Also answer the questions on oxygen requirements. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

23. Refrigerator Freezer Incubator Incubator Incubator 00C -100C 350C 500C 1000C Click on one of the link below to introduce the microbes from the broth culture to the agar tubes. If you are finished with this experiment you can click on “END LAB” Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Oxygen RequirementsExperiment Table 2 Agar TubesSketches Oxygen RequirementsQuestions END LAB

24. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C QUESTIONS OXYGEN REQUIREMENTS AND SPICE TOLERANCE IN MICROBES . Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

25. Spice Sensitivity

26. Yeast Culture

27. In this experiment we will be introducing the Yeast culture to the agar plates. Click on the Broth Culture Tubes to bring the rack to the table. Click on NEXT when the test tube rack is placed on the table. Incubator 370C Yeast Bacteria Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

28. Click on the Agar Plates to bring a plate to the table. When the agar plate has arrived to the table Click on the blue eyedroppers on the shelf to inoculate the plate with the Yeast culture. Click on NEXT when the plates have been inoculated. Incubator 370C Yeast Bacteria Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

29. Click on the Antiseptic Dispenser containing the various spices to introduce the spice laden discs onto the surface of the inoculated agar plate. Each of the discs has been labeled indicating the spice present in the disc. Next Click on the agar plate to place it into the incubator. ClickNEXT when finished. Incubator 370C Yeast Bacteria Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

30. After 24 hours of incubation we can view the results of growth of yeast while exposed to the spice soaked discs.We will assume at this point that the plate has been incubated for 24 hours. Click on the incubator to bring the agar plate to the table. ClickNEXT when finished. Incubator 370C Yeast Bacteria Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

31. Click on the agar plate on the table to view the plate. Observe the size of the areas around the spice discs in which there is no yeast growth evident. These areas are called “Zones of Inhibition”. Measure the size of each of these zones by clicking on each of the labeled discs. Record your data for each spice type in Table 1. ClickNEXT when finished. G Yeast Culture P O M T Incubator 370C C Nose Ear Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner Garlic = GOregano = OThyme = TCinnamon = CMint = MPepper = P How do I measurethe Zone of Inhibition? Table 1

32. At this point you should have entered your measurement values in Table 1. Using the Interpretive Zone Chart given to the left, indicate in Table 1 whether the spice tested Resistant or Susceptible for the culture isolate. Produce a sketch of the agar plate with the indicated clear zones for each of the tested spices. ClickNEXT when finished. Incubator 370C Nose Ear Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner Table 1 Sketch Questions

33. You have now finished this exercise. There are two exercises in this lab involving two separate microbial cultures. If you have performed both of the exercises you can Click on “END LAB” below. If you still need to perform another exercise Click on the appropriate link given. If you have performed both the Yeast and Bacterial exercises, Click on the link for Oxygen Requirements to perform this portion of the lab. Incubator 370C Yeast Bacteria Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner Yeast Culture Bacterial Culture OxygenRequirements END LAB

34. Bacterial Culture

35. In this experiment we will be using bacteria (Salmonella) to test spice tolerance. Click on the Broth Culture Tubes to bring the rack to the table. Click on NEXT when the test tube rack is placed on the table. Incubator 370C Yeast Bacteria Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Pencil Bunsen burner

36. Click on the Agar Plates to bring a plate to the table. When the agar plate has arrived to the table Click on the blue eyedroppers on the shelf to inoculate the plate with the bacteria culture. Click on NEXT when the plates have been inoculated. Incubator 370C Yeast Bacteria Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

37. Click on the Antiseptic Dispenser to introduce the spice laden discs onto the surface of the inoculated agar plate. Each of the discs has been labeled indicating the spice present in the disc. Next Click on the agar plate to place it into the incubator. ClickNEXT when finished. Incubator 370C Yeast Bacteria Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

38. After 24 hours of incubation we can view the results of growth of bacteria while exposed to the spice soaked discs.We will assume at this point that the plate has been incubated for 24 hours. Click on the incubator to bring the agar plate to the table. ClickNEXT when finished. Incubator 370C Yeast Bacteria Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

39. Click on the agar plate on the table to view the plate. Observe the size of the areas around the spice discs in which there is no bacterial growth evident. These areas are called “Zones of Inhibition”. Measure the size of each of these zones by clicking on each of the labeled discs. Record your data for each spice type in Table 1. ClickNEXT when finished. G Bacteria Culture P O M T Incubator 370C C Nose Ear Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner Garlic = GOregano = OThyme = TCinnamon = CMint = MPepper = P How do I measurethe Zone of Inhibition? Table 1

40. At this point you should entered your measurement values in Table 1. Using the Interpretive Zone Chart given to the left, indicate in Table 1 whether the antiseptic tested resistant or susceptible for the culture isolate. Produce a sketch of the agar plate with the indicated clear zones for each of the antiseptics. ClickNEXT when finished. Incubator 370C Mouth Fecal Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner Table 1 Sketch Questions

41. You have now finished this exercise. There are two exercises in this lab involving two separate microbial cultures. If you have performed both of the exercises you can Click on “END LAB” below. If you still need to perform another exercise Click on the appropriate link given. If you have performed both the Yeast and Bacterial exercises, Click on the link for Oxygen Requirements to perform this portion of the lab. Incubator 370C Yeast Bacteria Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner Yeast Culture Bacterial Culture OxygenRequirements END LAB

42. Oxygen Requirements

43. In this experiment we will be using four different bacterial cultures to test for oxygen requirements for growth. Click on the Agar Culture Tubes to bring the agar tubes to the table. Click on the Bacterial Culture Tubes to bring the bacteria cultures to the table. Click on NEXT when the test tube rack is placed on the table. Incubator 370C 4 4 3 3 2 2 1 1 Agar Plates Swabs Loops Antiseptic Dispenser Agar Culture Tubes Bacterial Culture Tubes Pencil Bunsen burner

44. The four different bacteria types and their codes indicated on the broth test tubes are shown in the yellow tag below. The agar in the tubes on the left is still warm and in a liquid form. We will need to transfer a portion of each of the broth cultures to their respective agar tubes before the agar solidifies. Click on the blue eye-droppers to transfer the bacteria cultures. Click on Next when finished. Incubator 370C 4 4 3 3 2 2 1 1 Agar Plates Swabs Loops Antiseptic Dispenser Agar Culture Tubes Bacterial Culture Tubes Pencil Bunsen burner 1-Clostridium botulinum2-Corynebacterium diphtheriae3-Neisseria gonorrhoeae4-Helicobacter pylori

45. The inoculated agar tubes will need to be incubated for 48 hours at 37 degrees Celsius. Click on the agar tubes to place them in the incubator. Click on NEXT when the agar tubes have been placed in the incubator. Incubator Incubator 370C 4 4 3 3 2 2 1 1 Agar Plates Swabs Loops Antiseptic Dispenser Agar Culture Tubes Bacterial Culture Tubes Pencil Bunsen burner 1-Clostridium botulinum2-Corynebacterium diphtheriae3-Neisseria gonorrhoeae4-Helicobacter pylori

46. We will assume that 48 hours has elapsed. Click on the incubator to bring the agar tubes to the table. Once the tubes are on the table Click on each of the tubes to view in more detail the growth of the cultures in each agar tube. Record your data and sketches in Table 2 and sketch each of the tubes indicating the growth patterns exhibited for each bacteria. Click on END LAB when you have viewed each of the agar tubes. Incubator Incubator 370C 4 3 2 1 Agar Plates Swabs Loops Antiseptic Dispenser Agar Culture Tubes Bacterial Culture Tubes Pencil 1 Bunsen burner 1-Clostridium botulinum2-Corynebacterium diphtheriae3-Neisseria gonorrhoeae4-Helicobacter pylori Click Here if you haveviewed all of the Agar tubes END LAB

47. You are viewing Agar tube #1 which contains Clostridium botulinum. Construct a sketch of the tube indicating where the bacteria is showing growth in the tube. The bacterial growth is the dark colonies, in this case at the bottom of the tube. You will also need to classify the bacteria as an Obligate anaerobe, Facultative anaerobe, Obligate aerobe, Microaerophile. Enter this data in Table 2. Click on another tube to view bacterial growth and analyze. Incubator Incubator 370C 4 3 2 1 1 Agar Plates Swabs Loops Antiseptic Dispenser Agar Culture Tubes Bacterial Culture Tubes Pencil 1 Bunsen burner 1-Clostridium botulinum2-Corynebacterium diphtheriae3-Neisseria gonorrhoeae4-Helicobacter pylori Click Here if you haveviewed all of the Agar tubes END LAB

48. You are viewing Agar tube #2 which contains Corynebacterium diphtheriae. Construct a sketch of the tube indicating where the bacteria is showing growth in the tube. The bacterial growth is the dark colonies, in this case through out the tube. You will also need to classify the bacteria as an Obligate anaerobe, Facultative anaerobe, Obligate aerobe, Microaerophile. Enter this data in Table 2. Click on another tube to view bacterial growth and analyze. Incubator 370C 4 3 2 2 1 Agar Plates Swabs Loops Antiseptic Dispenser Agar Culture Tubes Bacterial Culture Tubes Pencil 2 Bunsen burner 1-Clostridium botulinum2-Corynebacterium diphtheriae3-Neisseria gonorrhoeae4-Helicobacter pylori Click Here if you haveviewed all of the Agar tubes END LAB

49. You are viewing Agar tube #3 which contains Neisseria gonorrhoeae. Construct a sketch of the tube indicating where the bacteria is showing growth in the tube. The bacterial growth is the dark colonies, in this case at the very top of the tube. You will also need to classify the bacteria as an Obligate anaerobe, Facultative anaerobe, Obligate aerobe, Microaerophile. Enter this data in Table 2. Click on another tube to view bacterial growth and analyze. Incubator 370C 4 3 3 2 1 Agar Plates Swabs Loops Antiseptic Dispenser Agar Culture Tubes 3 Bacterial Culture Tubes Pencil Bunsen burner 1-Clostridium botulinum2-Corynebacterium diphtheriae3-Neisseria gonorrhoeae4-Helicobacter pylori Click Here if you haveviewed all of the Agar tubes END LAB

50. You are viewing Agar tube #4 which contains Helicobacter pylori. Construct a sketch of the tube indicating where the bacteria is showing growth in the tube. The bacterial growth is the dark colonies, in this case near the top of the tube. You will also need to classify the bacteria as an Obligate anaerobe, Facultative anaerobe, Obligate aerobe, Microaerophile. Enter this data in Table 2. Click on another tube to view bacterial growth and analyze. Incubator 370C 4 4 3 2 1 Agar Plates Swabs Loops Antiseptic Dispenser Agar Culture Tubes 4 Bacterial Culture Tubes Pencil Bunsen burner 1-Clostridium botulinum2-Corynebacterium diphtheriae3-Neisseria gonorrhoeae4-Helicobacter pylori Click Here if you haveviewed all of the Agar tubes END LAB