Antibiotic Sensitivity

1. Antibiotic Sensitivity 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 Antibiotic Dispenser Microbe Samples Loops Pencil Bunsen burner

3. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C Antibiotic Sensitivity Antibiotics are antimicrobial agents produced by microorganisms that kill or inhibit other microorganisms. This is the microbiologist's definition. A more broadened definition of an antibiotic includes any chemical of natural origin (from any type of cell) which has the effect to kill or inhibit the growth of other types cells. Since most clinically-useful antibiotics are produced by microorganisms and are used to kill or inhibit infectious Bacteria, we will follow the classic definition. Pharmacologists refer to any antimicrobial chemical used in the treatment of infectious disease as as antibiotic. Antibiotics are low molecular-weight (non-protein) molecules produced as secondary metabolites, mainly by microorganisms that live in the soil.  Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

4. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C Among the molds, the notable antibiotic producers are Penicillium and Cephalosporium, which are the main source of penicillin and its relatives. In the Bacteria, the Actinomycetes, notably Streptomyces species, produce a variety of types of antibiotics including streptomycin, erythromycin, and the tetracyclines. Some Bacillus species produce antibiotics such as polymyxin and bacitracin.Semi-synthetic antibiotics are molecules produced my a microbe that are subsequently modified by an organic chemist to enhance their antimicrobial properties or to render them unique for a pharmaceutical patent. The modern era of antimicrobial chemotherapy began following Alexander Fleming's discovery in 1929 of the powerful bactericidal substance penicillin, and Domagk's discovery in 1935 of synthetic chemicals (sulfonamides) with broad antimicrobial activity. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

5. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C In the early 1940's, spurred partially by the need for antibacterial agents in WW II, penicillin was isolated, purified and injected into experimental animals, where it was found to not only cure infections but also to possess incredibly low toxicity for the animals. This fact ushered into being the age of antibiotic chemotherapy and an intense search for similar antimicrobial agents of low toxicity to animals that might prove useful in the treatment of infectious disease. The rapid isolation of streptomycin, chloramphenicol and tetracycline soon followed, and by the 1950's, these and several other antibiotics were in clinical usage. The most important property of a clinically-useful antimicrobial agent, especially from the patient's point of view, is its selective toxicity, i.e., the agent acts in some way that inhibits or kills bacterial pathogens but has little or no toxic effect on the animal taking the drug. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

6. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C This implies that the biochemical processes in the bacteria are in some way different from those in the animal cells, and that the advantage of this difference can be taken in chemotherapy. Antibiotics may have a cidal (killing) effect or a static (inhibitory) effect on a range of microbes. The range of bacteria or other microorganisms that are affected by a certain antibiotic is expressed as its spectrum of action. Antibiotics which kill or inhibit a wide range of Gram-positive and Gram-negative bacteria are said to be broad spectrum. If effective mainly against Gram-positive or Gram-negative bacteria, they are narrow spectrum. If effective against a single organism or disease, they are referred to as limited spectrum. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

7. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C Natural penicillins, such as Penicillin G or Penicillin V, are produced by Penicillium chrysogenum. They are effective against streptococcus, gonococcus and staphylococcus, except where resistance has developed. They are considered narrow spectrum since they are not effective against Gram-negative rods. Semisynthetic penicillins first appeared in 1959. A mold produces the main part of the molecule which can be modified chemically. Many of these compounds have been developed to have distinct benefits or advantages over penicillin G, such as increased spectrum of activity (e.g.  effectiveness against Gram-negative rods), or  effectiveness when administered orally. Amoxycillin and Ampicillin have broadened spectra against Gram-negatives and are effective orally. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

8. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C Although nontoxic, penicillins occasionally cause death when administered to persons who are allergic to them. In the U.S. there are 300 - 500 deaths annually due to penicillin allergy. In allergic individuals a part of the penicillin molecule  attaches to a blood protein which initiates an IgE-mediated inflammatory response. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

9. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C Find below a table that lists some common antibiotics, their source and mode of action. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

10. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C The Kirby-Bauer test for antibiotic susceptibility, called the disc diffusion test, is a standard that has been used for years.  It has been replaced in clinical labs by automated tests.  But the K-B is still used in some labs. The bacterium is swabbed on the agar and the antibiotic discs are placed on top.  The antibiotic diffuses from the disc into the agar in decreasing amounts the further it is away from the disc.  If the organism is killed or inhibited by the concentration of the antibiotic, there will be NO growth in the immediate area around the disc: This is called the Zone of Inhibition. The zone sizes are looked up on a standardized chart to give a result of sensitive, resistant, or intermediate. Many charts have a corresponding column that also gives the MIC (minimal inhibitory concentration) for that drug. In this experiment we will determine the susceptibility of Staph species to various antibiotics. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

11. Refrigerator Freezer Incubator Incubator Incubator 00C -100C 500C 350C 1000C Antibiotic Sensitivity In this lab you will be performing two experiments. The first experiment will investigate various antibiotic sensitivity of bacteria cultures from the nose. The second experiment will investigate various antibiotic sensitivity of bacteria cultures from the ear.Click on the experiment below that you want to perform.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 Antibiotic SensitivityVirtual Lab Print Table 1AntibioticSensitivity END LAB

12. Antibiotic Sensitivity

13. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C • We will be testing two different types of bacterial cultures for sensitivity to six different antibiotics. The bacterial cultures we will be using will be collected from the nose and the ear. The bacterial cultures produced from these collections will be Staph isoloates. • The types of antibiotics used will be: • AmoxicillinTetracyclinePenicillinAmpicillinStreptomycinErythromycin 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 Staph cultures to survive and reproduce. In order to introduce the bacteria to the sterile agar plates we will use sterile cotton tip swabs which can transfer the bacteria from the pure culture tubes to the agar plates. Once the plates are inoculated we will introduce the discs containing the antibiotics 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 locations listed below to collect bacteria and introduce to the broth culture. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Nose Culture Ear Culture END LAB

16. Nose Culture

17. In this experiment we will be swabbing the internal lining of the nose to collect bacteria, which we will introduce to a broth culture. 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 Nose Ear Agar Plates Swabs Loops Antibiotic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

18. Click on the cotton tip swabs to sample the lining of the interior of the nose and mix with the broth culture. Click on NEXT when you have inoculated the culture. Incubator 370C Nose Ear Agar Plates Swabs Loops Antibiotic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

19. 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 from the nose. Click on NEXT when the plates have been inoculated. Incubator 370C Nose Ear Agar Plates Swabs Loops Antibiotic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

20. Click on the Antibiotic Dispenser to introduce the antibiotic discs onto the surface of the inoculated agar plate. Each of the discs has been labeled indicating the antibiotic present in the disc. Next Click on the agar plate to place it into the incubator. ClickNEXT when finished. Incubator 370C Nose Ear Agar Plates Swabs Loops Antibiotic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

21. After 24 hours of incubation we can view the results of growth of bacteria while exposed to the antibiotic 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 Nose Ear Agar Plates Swabs Loops Antibiotic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

22. Click on the agar plate on the table to view the plate. Observe the size of the areas around the antibiotic 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 antibiotic type in Table 1. ClickNEXT when finished. AMC Nose Culture E TE S P Incubator 370C AM 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 Antibiotic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner Amoxicillin = AMCTetracyclin = TEPenicillin = PAmpicillin = AMStreptomycin = SErythromycin = E How do I measurethe Zone of Inhibition? Table 1

23. 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 antibiotic tested resistant, intermediate or susceptible for the culture isolate. Produce a sketch of the agar plate with the indicated clear zones for each of the antibiotics. ClickNEXT when finished. Incubator 370C Nose Ear Agar Plates Swabs Loops Antibiotic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner Table 1 Sketch Questions

24. You have now finished this experiment. There are two experiments in this lab involving two separate bacterial cultures. If you have performed both of the experiments you can click on END LAB below. If you still need to perform another experiment click on the appropriate link given. Incubator 370C Nose Ear Agar Plates Swabs Loops Antibiotic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner Nose Culture Ear Culture END LAB

25. Ear Culture

26. In this experiment we will be swabbing the internal lining of the ear to collect bacteria, which we will introduce to a broth culture. 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 Nose Ear Agar Plates Swabs Loops Antibiotic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

27. Click on the cotton tip swabs to sample the lining of the interior of the ear and mix with the broth culture. Click on NEXT when you have inoculated the culture. Incubator 370C Nose Ear Agar Plates Swabs Loops Antibiotic 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 bacteria culture from the ear. Click on NEXT when the plates have been inoculated. Incubator 370C Nose Ear Agar Plates Swabs Loops Antibiotic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

29. Click on the Antibiotic Dispenser to introduce the antibiotic discs onto the surface of the inoculated agar plate. Each of the discs has been labeled indicating the antibiotic present in the disc. Next Click on the agar plate to place it into the incubator. ClickNEXT when finished. Incubator 370C Nose Ear Agar Plates Swabs Loops Antibiotic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

30. After 24 hours of incubation we can view the results of growth of bacteria while exposed to the antibiotic 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 Nose Ear Agar Plates Swabs Loops Antibiotic 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 antibiotic 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 antibiotic type in Table 1. ClickNEXT when finished. Ear Culture AMC E TE S P Incubator 370C AM 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 Antibiotic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner Amoxicillin = AMCTetracyclin = TEPenicillin = PAmpicillin = AMStreptomycin = SErythromycin = E How do I measurethe Zone of Inhibition? Table 1

32. 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 antibiotic tested resistant, intermediate or susceptible for the culture isolate. Produce a sketch of the agar plate with the indicated clear zones for each of the antibiotics. ClickNEXT when finished. Incubator 370C Nose Ear Agar Plates Swabs Loops Antibiotic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner Table 1 Sketch Questions

33. You have now finished this experiment. There are two experiments in this lab involving two separate bacterial cultures. If you have performed both of the experiments you can click on END LAB below. If you still need to perform another experiment click on the appropriate link given. Incubator 370C Nose Ear Agar Plates Swabs Loops Antibiotic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner Nose Culture Ear Culture END LAB

34. Measurement Instructions

35. At this point you should. ClickNEXT when finished. Incubator 370C Nose Ear Measure this distance Agar Plates Swabs Loops Antibiotic Dispenser Broth Culture Tubes Loops Pencil E Bunsen burner Each mark = 1 mm Measure the diameter of the zone indicated by the clear area of growth around the disc Click here to returnto the experiment

36. archive Click here to returnto the experiment

37. AMC Nose Culture E TE S P AM Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm VESTIGES

38. Saccharomyes cerevisiae

39. On the shelf to the left you will find numerous tools and equipment that we will require for testing the microbes. Click on the Agar Plates to bring the plates to the table for inoculation with the different bacteria. Incubator Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Loops Microbe Samples Loops Pencil Bunsen burner 300C Click on Next

40. Click on the Bunson Burner to bring it to the table. We will use the burner to sterilize the inoculating loops. Next click on the loops to bring one to be sterilized. Incubator Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Loops Loops Bunsen burner 300C Click on Next

41. While we are sterilizing the loop, click on the test tube rack to bring the microbe samples to the table. The test tubes each have a different microbe according to the number chart shown here. 5 4 3 2 1 Incubator Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Loops Microbe Samples Loops Bunsen burner 1-Bacillus coagulans2-Micrococcus luteus3-Penicillium chrysogenum4-Pseudomonas fluorescens5-Saccharomyes cerevisiae 300C Click on Next

42. Click on the sterile inoculating loop to add the Saccharomyes cerevisiae microbe to the five agar plates on the table using a simple single streak technique. Be sure to sterilize your loop for the next inoculation. 5 4 3 2 1 Incubator Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Loops Microbe Samples Loops Bunsen burner 1-Bacillus coagulans2-Micrococcus luteus3-Penicillium chrysogenum4-Pseudomonas fluorescens5-Saccharomyes cerevisiae 300C Click on Next

43. Click on each of the beakers on the shelf to expose the inoculated agar plates to each of the different pH solutions. Click on NEXT when you have added the solution from all of the beakers. 5 4 3 2 1 Incubator Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Loops Microbe Samples Loops Bunsen burner 1-Bacillus coagulans2-Micrococcus luteus3-Penicillium chrysogenum4-Pseudomonas fluorescens5-Saccharomyes cerevisiae 300C

44. Place the covers on the agar plates by clicking on them on the top of the bench. Click on the Pencil to label each of the agar plates as #5 to indicate the microbe type and each of the various pH solutions used. 5 4 3 2 1 Incubator Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Loops Microbe Samples Loops Pencil Bunsen burner 1-Bacillus coagulans2-Micrococcus luteus3-Penicillium chrysogenum4-Pseudomonas fluorescens5-Saccharomyes cerevisiae Covers 300C #5 pH=7 #5 pH=9 Click on Next #5 pH=11 #5 pH=3 #5 pH=5

45. Click on the Incubator to open the door. Next click on the inoculated agar plates to insert them into the incubator. 5 4 3 2 1 Incubator Incubator #1 pH=7 #5 pH=7 #1 pH=9 #5 pH=9 #5 pH=11 #5 pH=11 #5 pH=3 #1 pH=3 #1 pH=5 #5 pH=5 Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Loops Microbe Samples Loops Pencil Bunsen burner Covers 300C Click on Next

46. Click on the Thermostat located on the door of the incubator to set the temperature to 30 degrees Celsius. 5 4 3 2 1 Incubator Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Loops Microbe Samples Loops Pencil Bunsen burner Covers 300C Click on Next

47. Now all we have to do is wait for 48 hours to view our cultures. If you do not want to wait this long, click on the convenient “Time Machine” button below. 5 4 3 2 1 Incubator Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Loops Microbe Samples Loops Pencil Bunsen burner Covers 300C Press Button To Turn Time Machine On It has now been 48 hours. Press NEXT to view results

48. Click on the Incubator door to bring the agar plates to the table for observations. 5 4 3 2 1 Incubator #1 pH=7 #5 pH=7 #1 pH=9 #5 pH=9 #5 pH=11 #5 pH=11 #1 pH=3 #5 pH=3 #1 pH=5 #5 pH=5 Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Loops Click on Next Microbe Samples Loops Pencil Bunsen burner Covers 300C

49. Click on each of the agar plates to observe the magnitude of growth, growth patterns and colors of the colonies for each pH environment. Record this information on Table 1. Click on NEXT when you have observed and recorded all of the plates. 5 4 3 2 1 Incubator #5 pH=7 #5 pH=9 #5 pH=11 #5 pH=3 #5 pH=5 Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Loops Microbe Samples Loops Pencil Bunsen burner 1-Bacillus coagulans2-Micrococcus luteus3-Penicillium chrysogenum4-Pseudomonas fluorescens5-Saccharomyes cerevisiae Covers 300C

50. Click on each of the agar plates to observe the magnitude of growth, growth patterns and colors of the colonies for each pH environment. Record this information on Table 1. Click on NEXT when you have observed and recorded all of the plates. 5 4 3 2 1 Incubator #5 pH=7 #5 pH=9 #5 pH=11 #5 pH=3 #5 pH=5 #5 pH = 7 Agar Plates Click here if you have viewed all of the agar plates and are ready to prepare a different culture pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Loops Microbe Samples Loops Pencil Bunsen burner 1-Bacillus coagulans2-Micrococcus luteus3-Penicillium chrysogenum4-Pseudomonas fluorescens5-Saccharomyes cerevisiae Covers 300C Click here if you need help in figuring out how to interpret the results of the agar plate growth