Pre- Lab Discussion: Controlling Bacterial Growth Most bacteria (and other microorganisms) are harmless. In fact, many bacteria are beneficial. Cheesemaking, decay, and soil building are a few of the important processes that depend on the action of decomposing bacteria, which thrive on decaying organic matter. Bacteria are also important in the process of digestion for many organisms, including humans. Bacteria in termites and ruminants (such as cows) help break down the cellulose in the food they eat so it can be used for energy and nutrients. Bacteria in the human digestive system help with the synthesis of vitamin K, which is essential for blood clotting. However, some bacteria are pathogens (disease causers). Tuberculosis, tetanus, strep, diphtheria, anthrax, syphilis, and some pneumonias are a few of the serious diseases caused by bacteria. Chemical substances that either kill bacteria or inhibit bacterial growth are called antimicrobial agents. Alcohol and some mouthwashes are antiseptics, and are used on cuts or wounds to inhibit bacterial infection. Others, like chlorine bleach, are too concentrated or toxic for use on living tissue. These are called disinfectants and are used on clothes, surfaces, or other non- living objects. These agents generally work either by disrupting the cell membrane, causing the bacterium to lyse, or binding to the bacterium enzymes, which inhibit its activity. Even though antiseptics and disinfectants are very useful in helping to prevent infections, we cannot use them internally to treat an infection. If bacteria enter our bodies, we rely on another class of chemicals called antibiotics to kill them. Although their use is now commonplace, antibiotics were only discovered about 85 years ago. Before then, more people died from infections than from all the wars in history combined. Antibiotics were the first of the miracle drugs and they have permanently altered the course of history. The effectiveness of each type of antimicrobial agent is influenced by many factors. Some of these factors include the environmental conditions in which the agent is applied, the chemical properties of the agent, how long the agent has been stored, and the rate of deterioration of the agent. The procedure of placing bacteria on agar plates is called inoculation. Organisms so small that they can only be seen with a microscope are living all around, on, and in us. They include bacteria, viruses, molds, and yeasts. In the laboratory, you will test the effectiveness of antibiotics in inhibiting the growth of bacteria. You will grow samples of bacteria until they form colonies so big that you can see them with the naked eye. You will grow your samples on sterile nutrient agar in a sterile petri dish. Sterile means that there is nothing alive in the agar or on the dish. Nutrient agar supplies the nutrients that microorganisms (not Renooy 1
just bacteria) need to live and reproduce. When you allow bacteria to grow, they will grow uniformly wherever their growth is not effectively inhibited by a bactericide. This uniform growth is called a bacterial lawn and the regions where no growth occurs are zones of inhibition. A large zone of inhibition is created by a bactericide that is more effective at inhibiting the strain s growth than a bactericide that creates a smaller zone of inhibition. Purpose: In part I of this this investigation, you will test the effectiveness of both commercial and natural disinfectants and antibiotics in inhibiting the growth of bacteria. Materials: Glass- marking pencil Bunsen burner Flint striker or matches 1 sterile cotton swab Inoculating loop Beaker of water Test tube rack Sterile filter- paper disks Sterile forceps Distilled water Metric ruler Transparent tape Culture of Escherichia coli 3 sterile nutrient agar plates 4 natural products chosen from the following: tea tree oil, honey, vinegar. 4 commerial antibiotics chosen from the following: aureomycin, chloromycetin, penicillin, streptomycin, tetracycline, terramycin Safety: Put on laboratory apron if one is available. Put on safetly goggles. Handle all glasswear carefully. Tie back loose hair and clothing. Always use special caution when working with laboratory chemicals and bacterial cultures. Procedure: 1. Obtain two sterile nutrient agar plates. Carefully turn each plate over and lay it on your worktable. CAUTION: be very carful not to open the petri dishes of sterile agar when handling them 2. With a glass marking pencil mark the bottom of each petri dish shown as shown. Draw two lines at right angles to each other so that the dish is divided into Renooy 2
four equal quadrants. Number the quadrant on each disk 1 thru 4. Note: Place the numbers near the edges of the dishes. Please write you initials along the edge as well. Carefully turn the petri dishes right side up. 3. Obtain a test tube containing a bacterial culture of Esherichia coli. Place the test tube in a test tube rack. CAUTION: Use extreme care when working with bacterial cultures. Avoid spills. If a spill does occur, immediately call your teacher for assistance. 4. Light the Bunsen burner. CAUTION: Use extreme caution when working with an open flame. 5. Pick up the test tube (or plate) of E. coli. Remove the cap (or cover), under the Bunsen burner. If it is a test tube pass the mouth of the tube back and forth through the burner flame. 6. Insert the inoculating loop into the culture. Pass the mouth of the test tube back and forth through the flame, then recap and put in test tube rack. NOTE do not put the inoculating loop down! 7. Slightly open a sterile nutrient agar plate, under the flame. Place the broad side of the inoculating at the top center of the agar. Streak the agar as shown in the diagram. Lift the loop and turn the petri dish 90 degrees. Streak the agar again. Place the cover on immediately. 8. Hold the inoculating loop in the flame to re- sterilize. 9. Repeat steps 5-8 for ONE other plate. 10. Take one inoculated plate of E. Coli that you have prepared. Selected 3 commercial antibiotics and record these in your data table. 11. With sterile forceps, pick up a disk of filter paper. Insert this paper into the antibiotic of your choice (call this commercial antibiotic 1) shake off access liquid. 12. Slightly open the agar plate (under the flame). Position the filter- paper disk in the center of quadrant 1. With the tips of the forceps, gentle press the disk against the agar until it sticks. Renooy 3
13. Pass the forceps back and forth through the flame of the Bunsen burner several times. This procedure sterilizes the forceps. 14. Repeat steps 11-13 with the remaining commercial antibiotics in quadrants 2 and 3. (Remember to sterilize the forceps between applications). 15. In quadrant 4, place the filter- paper disk soaked with distilled water. 16. Repeat steps 11-15 with the other inoculated agar plate using natural antibiotics. 17. With transparent tape, tape the petri dishes closed. Turn the dishes upside down. Incubate the dished for 48 hours at 37 degrees. 18. Observe the petri dishes after 48 hours. White or cloudy areas of the agar indicate bacterial growth. Notice any clear areas, called zones of inhibition, surrounding the filter- paper disks. A clear area indicates that the disinfectant or antibiotic inhibited bacterial growth. A lightly cloudy area surrounding a disk indicates that bacterial growth was slowed down. 19. With a ruler, measure to the nearest millimeter the size of the zone of inhibition. Record your measurement in the data table. Renooy 4
Observation: Data Table 1: Effects of Antibiotics on Growth of E. Coli Commercial Antibiotic 1 2 3 4 Distilled Water Natural Antibiotic 1 2 3 4 Distilled Water Zone of Inhibition (mm) Zone of Inhibition (mm) Analysis and Conclusions 1. Why is it important not to open the sterile nutrient agar plates? 2. Why do you think it is so important to write the numbers and initials near the edges of the petri dish? 3. Why is it important to use sterile techniques while inoculating the agar plate? Renooy 5
4. What is the purpose of the disks soaked in distilled water in each inoculated petri dish? 5. What is the purpose of taping closed the lids of the petri dishes? 6. Which commercial antibiotic was most effective in preventing the growth of E. Coli? 7. Which natural antibiotic was most effective in preventing the growth of E. Coli? 8. How do you know that any inhibition you have observed is due to the antibiotic disks? Critical Thinking and Application: 1. Pretend that a serious staphylococcus infection has developed in the locker room of your school s gym. Assume that you are responsible for getting rid of the staph contamination. How would you do so? 2. Why are the different antibiotics not equally effective against all species of bacteria? Renooy 6
3. Suppose you doctor diagnoses your condition as a bacterial infection and prescribes an antibiotic. Your doctor cautions you to take the antibiotic for 10 days even though you may feel fine after a few days. Explain why you should follow your doctor s orders? Conclusion: Compare the 3 commercial antibiotics you used and the 3 natural antibiotics you used to control the growth of E. Coli. Research: Scientists have observed that an antibiotic seems to lose its effectiveness against a particular population of bacteria after a prolonged period of time. What do you think is responsible for this phenomenon? Explain in detail Renooy 7
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